Instituto de Biociências - Seção Técnica de Pós-Graduação Distrito de Rubião Júnior s/n CEP 18618-970 Cx Postal 510 Botucatu-SP Brasil Tel (14) 3880-0780 posgraduacao@ibb.unesp.br Campus de Botucatu PG-BGA EFEITO DO LASER DE BAIXA INTENSIDADE SOBRE O PERFIL TRANSCRICIONAL DE RNAm EM MIOBLASTOS C2C12 JUAREZ HENRIQUE FERREIRA Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, como requisito para a defesa de Doutorado no Programa de Pós- Graduação em Biologia Geral e Aplicada, Área de concentração Biologia Celular, Estrutural e Funcional. Prof. Dr. Robson Francisco Carvalho BOTUCATU – SP 2018 Instituto de Biociências - Seção Técnica de Pós-Graduação Distrito de Rubião Júnior s/n CEP 18618-970 Cx Postal 510 Botucatu-SP Brasil Tel (14) 3880-0780 posgraduacao@ibb.unesp.br Campus de Botucatu PG-BGA UNIVERSIDADE ESTADUAL PAULISTA “Julio de Mesquita Filho” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU EFEITO DO LASER DE BAIXA INTENSIDADE SOBRE O PERFIL TRANSCRICIONAL DE RNAm EM MIOBLASTOS C2C12 ALUNO: JUAREZ HENRIQUE FERREIRA ORIENTADOR: PROF. DR. ROBSON FRANCISCO CARVALHO CO-ORIENTADOR: DR. IVAN JOSÉ VECHETTI JÚNIOR Tese apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, como requisito para a defesa de Doutorado no Programa de Pós-Graduação em Biologia Geral e Aplicada, Área de concentração Biologia Celular, Estrutural e Funcional. Prof. Dr. Robson Francisco Carvalho BOTUCATU – SP 2018 Palavras-chave: Ciclo celular; Desenvolvimento muscular; Músculo esquelético; Terapia com laser; Transcriptoma. Ferreira, Juarez Henrique. Efeito do laser de baixa intensidade sobre o perfil transcricional de RNAm em mioblastos C2C12 / Juarez Henrique Ferreira. - Botucatu, 2018 Tese (doutorado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Robson Francisco Carvalho Coorientador: Ivan José Vechetti-Júnior Capes: 20600003 1. Ciclo celular. 2. Desenvolvimento muscular. 3. Músculo esquelético. 4. Terapia com luz de baixa intensidade. 5. Transcriptoma. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSANGELA APARECIDA LOBO-CRB 8/7500 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. Dedicatória “Dedico este trabalho a minha mãe Maria Consolação Ferreira e ao meu pai Joaquim Juarez Ferreira, por terem sempre me incentivado desde a infância, a buscar o caminho do conhecimento. Este título de doutor é para vocês!” Agradecimentos Agradeço primeiramente a Deus por ter iluminado meu caminho e me dado sabedoria para conquistar mais esta etapa da vida. Agradeço ao meu orientador professor Dr. Robson Francisco Carvalho por ter acreditado em mim e me dado a oportunidade de desenvolver este trabalho sob a sua orientação e por sempre ter sido solícito e dar apoio nesta jornada. Professor obrigado pela grande contribuição na minha formação acadêmica e também pelas palavras amigas ao longo desses anos. Minha eterna gratidão. Agradeço ao meu co-orientador Doutor Ivan José Vechetti-Júnior por toda amizade e conhecimento compartilhado. Ivan, você muito além de um co-orientador e amigo, você se tornou um irmão e uma inspiração. Agradeço a professora Dra. Maeli Dal Pai pela contribuição constante durante a execução deste projeto e por todo apoio prestado ao longo desses anos. Agradeço a toda a equipe LBME: Ana Omoto, Bruna Zanella, Bruno Duran, Bruno Fantinatti, Carlos Augusto, Carlos Freitas, Edson Marreco, Geysson Fernandez, Grasieli Oliveira, Ivan Vechetti, Jéssica Valente, Leonardo Nazário, Paula Freire, Rafaela Nunes, Rodrigo Souza, Rondinele Salomão, Sarah Cury e Tassiana de Paula, que sempre contribuíram de uma forma direta ou indireta para a realização deste trabalho, compartilhando seu conhecimento e amizade. Agradeço de maneira especial a algumas pessoas que ao longo desta jornada tiveram um papel fundamental na realização deste trabalho e que sem elas não teria sido possível a sua realização. Obrigado Leonardo Nazário de Moraes, Geysson Javier Fernandez Garcia, Tassiana Gutierrez de Paula, Ana Carolina Mieko Omoto e Carlos Augusto Alves pela contribuição e amizade. Agradeço a minha esposa Talita Mariana Morata Raposo Ferreira pelo seu apoio incondicional, por sempre ter me dado forças para seguir em frente, mesmo diante das adversidades que surgiam, pela paciência e compreensão dos meus momentos de ausência. Agradeço ao meu cachorro Jack (em memória) por sempre ter me recebido com amor e carinho quando voltava para casa após o trabalho no laboratório. Agradeço a toda minha família pelo apoio e por sempre terem acreditado que essa conquista seria possível. Agradeço as agências de fomento CAPES, CNPq e FAPESP por financiarem este trabalho e acreditar no nosso potencial. v RESUMO A irradiação pelo laser de baixa intensidade (LBI) tem sido utilizada como um método não- invasivo para promover ou acelerar a capacidade de regeneração muscular. No entanto, os mecanismos moleculares regulatórios pelos quais o LBI exerce esses efeitos, permanecem em grande parte desconhecidos. Nosso objetivo foi realizar uma análise de sequenciamento de RNA (RNA-Seq) em mioblastos C2C12 após LBI. Foram realizadas as taxas de viabilidade, migração, proliferação e os dados de RNA-Seq dos mioblastos C2C12, identificando 514 genes diferencialmente expressos após LBI. Em seguida, uma análise de ontologia genética e das vias dos genes diferencialmente expressos revelaram transcritos relacionadas ao ciclo celular, biogênese ribossômica, resposta ao estresse, migração celular, estrutura morfológica e proliferação de células musculares. Após, cruzamos nossos dados de RNA-Seq com dados de transcriptomas disponíveis em base de dados públicas, com dados de diferenciação miogênica que mostraram um total de 42 transcritos sobrepostos (mioblastos vs miotubos). Este conjunto de transcritos compartilhados mostrou que os mioblastos irradiados pelo LBI, possuem um perfil transcricional semelhante ao de miotubo, agrupando-se distante do perfil transcricional dos mioblastos. Concluíndo, revelamos pela primeira vez que LBI, induz a uma expressão de um grande conjunto de RNAm, que codificam proteínas reguladoras do ciclo celular que podem controlar a proliferação e diferenciação de mioblastos em miotubos. Importantemente, esse conjunto de RNAm, revelou um perfil transcricional semelhante ao dos miotubos, fornencendo novos conhecimentos para a compreensão das alterações moleculares específicas subjacentes aos efeitos da irradiação por LBI em células do músculo esquelético. Palavras-chave: transcriptoma, terapia com laser, desenvolvimento muscular, músculo esquelético, crescimento muscular, ciclo celular. vi ABSTRACT Low-level laser irradiation (LLLT) has been used as a non-invasive method to promote or accelerate muscular regeneration capability. However, the regulatory molecular mechanisms by which LLLT exerts these effects remain largely unknown. Our goal was to perform a RNA- sequencing (RNA-Seq) analysis in C2C12 myoblasts after LLLT. C2C12 myoblasts viability, migration, proliferation and RNA-Seq were performed, identifying 514 differentially expressed genes after LLLT. Next, gene ontology and pathway analysis of the differentially expressed genes revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, morphological structure and muscle cell proliferation. After, we intersected our RNA-Seq data with transcriptomes publicly available myogenic differentiation data that showed a total of 42 overlapping transcripts (myoblasts vs myotube). This set of shared transcripts showed that the LLLT-myoblasts have a myotube-like profile, clustering away from the myoblast profile. In conclusion, we revealed for the first time that LLLT induces the expression a large set of mRNAs encoding for cell cycle regulatory proteins that may control myoblasts proliferation and differentiation into myotubes. Importantly, these set of mRNA revealed a myotube-like transcriptional profile and provided new insights to the understanding of the specific molecular changes underlying the effects of LLLT irradiation on skeletal muscle cells. Key-words: Transcriptome, Laser treatment, Muscular development, Skeletal muscle, Muscle growth, Cell cycle. vii Lista de Abrviações AsGa – Arseneto de Gálio AsGaAl – Arseneto de Gálio e Alumínio ATP – Adenosina trifosfato BrdU - 5-Bromo-2′-deoxyuridine C2C12 – Linhagem celular imortalizada de mioblastos de camundongo CT – Grupo controle DAPI – 4’,6-diamidino-2-fenilindol DEG – Differentially expressed genes DMEM – Dubelcco’s Modified Eagle Medium DMSO – Dimetilsulfóxido GO – Gene Ontology HCl – Ácido clorídrico HeNe – Hélio/Neônio IL-1β – Interleucina 1 beta INF-γ – Interferon gama KEGG - Kyoto Encyclopedia of Genes and Genomes Laser – Light amplification by stimulated emission radiation LBI – Laser de baixa intensidade Linc-YY1 - Long intervening noncoding RNA LLLT – Low level laser irradiation miRNA – Micro-RNAs MRFs – Myogenic regulatory factors mRNA – RNA mensageiro MTT - brometo 3-(4,5-dimetilazol-2-yl)-2,5-difeniltetrazol MyHC – Cadeia Pesada de Miosina (do inglês: myosin heavy chain) MyoD – Myogenic differentiation NF-κB – Fator Nuclear-Kappa B (do inglês: nuclear factor kappa b) NGS – Next generation sequencing PBS – Tampão fosfato-salina RNA-Seq – Sequenciamento de RNA RPM – Rotações por minuto RT – Transcrição Reversa viii RT-qPCR – Reação em cadeia da polimerase em tempo real após transcrição reversa Setdb1 – Histona SFB – Soro fetal bovino TEAD – Fator transcricional TGF-β – Fator de Crescimento Transformante Beta (do inglês: Transforming growth factor beta) TGF-β1 – Transforming growth factor beta 1 TNF-α – Fator de Necrose Tumoral- α (do inglês: Tumor necrosis fator alpha) TWEAK - TNF-like weak inducer of apoptosis ix Lista de Figuras Figura 1. Processo de regeneração Muscular. Fibra muscular normal com célula satélite quiescente e mionúcleo (A). Após um miotrauma (B), as células satélites quiescentes são ativadas, proliferam-se (C) e se diferenciam em mioblastos (E). No miotrauma adaptativo do exercício físico, os mioblastos migram para a região danificada e fundem-se à fibra muscular pré-existente para reparar o local da microlesão e/ou adicionar núcleos para ampliar a taxa síntese protéica (hipertrofia) (F). Porém, em situações de miotraumas severos que ocorra necrose das fibras (ação de toxinas e distrofia), os mioblastos poderão se alinhar e fundir-se entre si, para formar uma nova miofibra (G), e reparar o dano da fibra muscular (H). Durante o processo de regeneração, alguns mioblastos retornam ao estado quiescente e restabelecem a população de células satélites (D) (Adaptado de Chargé and Rudnick 2004). Figura 2. Representação esquemática das células satélites no crescimento muscular (adaptado de Zammit, Partridge, and Yablonka-Reuveni 2006). Figura 3. Esquema de grade utilizado para a irradiação do laser de baixa intensidade nas placas de 6-poços. Figura 4. Ensaio de migração/proliferação - Wound Healing. SUMÁRIO Resumo ................................................................................................................................. v Abstract ................................................................................................................................ vi Lista de Abreviações ........................................................................................................... vii Lista de Figuras ................................................................................................................... ix 1. INTRODUÇÃO ....................................................................................................... 11 1.1 Aspectos gerais da composição e emissão dos lasers ................................................ 11 1.2 Uso da irradiação laser em tecidos biológicos .......................................................... 12 1.3 Células C2C12 ........................................................................................................... 13 1.4 Perfil genômico global de mRNAs de células C2C12 .............................................. 15 2. OBJETIVO GERAL ............................................................................................... 18 2.1 Objetivos específicos ................................................................................................. 18 3. MATERIAL E MÉTODOS .................................................................................... 19 3.1 Cultura celular ........................................................................................................... 19 3.2 Irradiação pelo LBI .................................................................................................... 19 3.3 Migração celular ........................................................................................................ 20 3.4 Proliferação celular .................................................................................................... 20 3.5 Viabilidade celular ..................................................................................................... 21 3.6 Extração do RNA total .............................................................................................. 21 3.7 Sequenciamento do RNA .......................................................................................... 22 3.8 Análise de enriquecimento funcional de vias moleculares ........................................ 22 3.9 Análises estatísticas ................................................................................................... 22 4. CONSIDERAÇÕES FINAIS .................................................................................. 24 5. REFERÊNCIAS ...................................................................................................... 25 6. MANUSCRITO ....................................................................................................... 31 Anexo I - Table Supplementary 1 Genes differentially expressed after low-level laser irradiation .............................................................................................................................. 56 Anexo II - Table S2 - Enrichment analysis of differentially expressed genes in C2C12 myoblasts after LLLT ............................................................................................................ 75 11 1. INTRODUÇÃO 1.1. Aspectos gerais da composição e emissão dos lasers O termo Laser é uma abreviação para “Light Amplification by Stimulated Emission Radiation” que, em português, significa “amplificação da luz por emissão estimulada de radiação” 1,2. O primeiro feixe de laser produzido no espectro de faixa visível foi emitido por Maimam, em 1960, após excitar um cristal de rubi por intermédio de um flash fotográfico 1. O laser difere da luz comum devido suas propriedades particulares 2; suas ondas emitidas são sincronizadas em relação ao tempo e ao espaço, viajando ordenadamente e em amplitudes iguais 3. A colimação, nome que se dá para o processo de tornar as ondas paralelas, é obtida pela unidirecionalidade do laser, que possui um feixe de fótons paralelo ao eixo do tubo que o produz, possuindo uma divergência angular muito pequena, concentrando assim toda a energia emitida em um único ponto 4. Essas propriedades fazem com que o laser emitido possua uma elevada precisão e eficiência para transferir energia luminosa aos tecidos biológicos 5. Dentre as causas já descritas desse efeito bioestimulante, ou biomodulador do laser, destacam-se modificações no metabolismo celular, em especial, em reações bioquímicas nas mitocôndrias, estimulando a síntese de ATP 6–8. O laser pode ser classificado em duas categorias: lasers de alta intensidade (superam 1 W), utilizado para procedimentos mais agressivos como cirurgias, carbonização ou desnaturação de proteínas devido ao seu efeito fototérmico 9, e lasers de baixa intensidade (inferiores a 1 W), que interagem com os tecidos de forma indireta, produzindo efeitos para reparação tecidual, alívio de dor e a obtenção de efeitos anti-inflamatórios 10. Os lasers de baixa intensidade (LBI) podem apresentar configurações diversificadas dependentes do comprimento de onda, que pode variar entre um espectro visível do vermelho a um espectro invisível do infravermelho. Esses feixes de laser são produzidos devido a mistura de gazes diversos, sendo os mais utilizados o hélio neônio (HeNe), arseneto de gálio e alumínio (AsGaAl) e o arseneto de gálio (AsGa) 11. Os lasers que apresentam comprimento de onda na faixa de espectro infravermelho possuem maior poder de penetração nos tecidos. No entanto, o espectro de onda vermelho tem sido mais estudado 5,12. Para a obtenção de resultados satisfatórios com o uso do LBI deve-se considerar a escolha do conjunto dos parâmetros a serem adotados, dentre eles: 1) a potência (W) que, atualmente, é considerada um parâmetro fixo e invariável nos aparelhos terapêuticos de LBI, e indica a quantidade de energia (J) pelo tempo (s) que chegará ao tecido - essa energia corresponde à quantidade de energia que será utilizada durante a irradiação terapêutica (dose); 2) a área de secção transversa do aplicador (cm²), que corresponde à área a ser irradiada no tecido; 3) a densidade de 12 potência (W/cm²), definida pela potência de saída do laser pela área de secção transversa; e, 4) a densidade de energia (J/cm²), que corresponde à quantidade total da energia que foi entregue ao tecido através área de secção transversa do aplicador 5,13. Além do cuidado na escolha dos parâmetros citados anteriormente, o comprimento de onda (nm) do LBI é um parâmetro determinante para um efeito terapêutico satisfatório, pois esse parâmetro é que determina quais tipos de tecidos biológicos irão absorver a radiação incidente e sua capacidade de penetração nesses tecidos. Dessa forma, a densidade de energia a ser utilizada por aplicação, a densidade de energia cumulativa total, e a frequência do tratamento devem ser cuidadosamente avaliadas de acordo com cada situação 5. 1.2. Uso da irradiação laser em tecidos biológicos O efeito bioestimulante ou biomodulador do laser de baixa intensidade (LBI), vem sendo estudado desde os anos 60 1,14–18. No entanto, a era da terapia laser começou literalmente muito antes do desenvolvimento do próprio laser. As propriedades terapêuticas da luz “concentrada”, já eram conhecidas no século XIX, quando Finsen recebeu o Prêmio Nobel em 1903, "em reconhecimento à sua contribuição para o tratamento de doenças, especialmente, o Lupus vulgaris, com radiação de luz concentrada, pelo qual abriu um novo caminho para a ciência médica" 19. O uso do LBI como opção terapêutica vem sendo muito utilizado nas últimas décadas em diversas áreas médicas, objetivando promover ação ou efeito bioestimulador nas células e nos tecidos, para restabelecimento do equilíbrio celular e, consequentemente, da hemostasia dos tecidos 20. A terapia com LBI é um método terapêutico seguro e eficaz, devido ao uso de recurso à base de luz que possui propriedades particulares que, em contato com a célula ou tecido, promove reações biológicas, gerando como resultado efeitos de diminuição da inflamação, da dor e cicatrização 21. Diversos estudos vêm demonstrando a capacidade do LBI modular vários processos celulares em diferentes tecidos biológicos tais como estimulação na produção de colágeno pelo tecido conjuntivo, angiogênese, estimulação e diferenciação de osteoblastos, regeneração de tecido muscular esquelético, entre outros 22–38. Em específico, Passarela et al.34 e Karu et al.39, demonstraram que o LBI era capaz de gerar um potencial eletroquímico extra e aumentar a síntese de ATP no interior das mitocôndrias. No tecido muscular esquelético, o uso do LBI vem demonstrando efeitos benéficos no processo de reparação tecidual. Durante a fase de reparo do tecido muscular, o laser reduziu os níveis de IL-1β e o número de células inflamatórias 40–42. Na fase de reparo muscular, o LBI aumentou os níveis de expressão de importantes fatores transcricionais como a MyoD e a 13 miogenina 43,44, e diminui o processo de fibrose devido à redução dos níveis de TGF-β1, TGF-β e dos níveis de colágeno do músculo 35,41,43,45. O processo de inflamação no tecido muscular após lesão é caracterizado principalmente por necrose da fibra muscular, infiltrado inflamatório, aumento local de citocinas pró- inflamatórias, presença de fatores de crescimento e enzimas proteolíticas envolvidas na fagocitose de fragmentos celulares 42,46,47. Simultaneamente, células precursoras miogênicas, denominadas de células satélites, são ativadas, proliferam e se diferenciam podendo, posteriormente, fundir-se com fibras musculares para promover o reparo de fibras musculares lesionadas, ou então, formar uma nova fibra muscular funcional 48–50 (Figura 1). Figura 1 – Processo de regeneração Muscular. Fibra muscular normal com célula satélite quiescente e mionúcleo (A). Após um miotrauma (B), as células satélites quiescentes são ativadas, proliferam-se (C) e se diferenciam em mioblastos (E). No miotrauma adaptativo do exercício físico, os mioblastos migram para a região danificada e fundem- se à fibra muscular pré-existente para reparar o local da microlesão e/ou adicionar núcleos para ampliar a taxa síntese protéica (hipertrofia) (F). Porém, em situações de miotraumas severos que ocorra necrose das fibras (ação de toxinas e distrofia), os mioblastos poderão se alinhar e fundir-se entre si, para formar uma nova miofibra (G), e reparar o dano da fibra muscular (H). Durante o processo de regeneração, alguns mioblastos retornam ao estado quiescente e restabelecem a população de células satélites (D) (Adaptado de Chargé and Rudnick 2004 51). 1.3. Células C2C12 Os estudos avaliando os efeitos benéficos do LBI podem ser realizados diretamente em modelos animais ou através do uso de células de linhagem miogênicas que simulem a condição regeneração da células musculares 52,53. Dentre as linhagens miogênicas, a linhagem C2C12 é uma 14 das mais utilizadas, devido sua capacidade de mimetizar o processo de miogênese ou regeneração in vitro 52. As células C2C12 são mioblastos murinos derivados a partir de células satélites com o comportamento correspondente ao da linhagem progenitora 52. Essas células são subclones de mioblastos C2 54, que se diferenciam na cultura celular após a remoção do soro fetal bovino ou substituição pelo soro de cavalo 55,56. O ciclo celular dos mioblastos C2C12 são comparáveis à ativação das células satélites presentes nas fibras musculares 57, tanto no estado quiescente como no estado ativado, expressam marcadores miogênicos 48 (Figura 2), chamados de fatores de regulação miogênica (MRFs, do inglês myogenic regulatory factors), que controlam as fases de ativação, proliferação e diferenciação durante o processo de crescimento ou reparo muscular 58. Os mioblastos C2C12 são referência de modelo nos estudos para a compreensão dos mecanismos envolvidos no processo de reparo muscular devido ao fato de durante um processo de lesão muscular, a área afetada sofrer isquemia e consequentemente a perda de oxigênio e nutrientes 53, enquanto que, no modelo de estudo in vitro utilizando está linhagem celular, a diminuição dos níveis de soro fetal bovino pode mimetizar a situação de uma lesão muscular 59. Figura 2. Representação esquemática das células satélites no crescimento muscular (adaptado de Zammit, Partridge, and Yablonka-Reuveni 2006 60. 15 1.4. Perfil genômico global de mRNAs de células C2C12 Os recentes avanços de técnicas para análises globais utilizando RT-qPCR e de sequenciamento do transcriptoma, principalmente utilizando Next-Generation Sequencing (NGS) – permitem novos caminhos para análises de alta resolução do transcriptoma de tipos celulares específicos, o que revelou uma complexidade biológica anteriormente obscura nos estudos com tecidos inteiros 61–65. A composição da fibra de um músculo é determinada em parte por fatores genéticos. No entanto, as fibras musculares não são unidades fixas, mas células capazes de responder a demandas funcionais alterando muito o seu fenótipo. A plasticidade funcional envolve alterações metabólicas e a expressão diferencial de MyHC e outras proteínas miofibrilares, permitindo assim, um ajuste da performance muscular 66,67. Portanto, a real contribuição das células musculares para o fenótipo transcricional do músculo durante a regeneração muscular é ofuscada em estudos de expressão gênica com fragmentos musculares, devido a anatomia complexa do músculo esquelético e a heterogeneidade de suas fibras musculares 68. O problema é exacerbado em doenças musculares com infiltrado de células inflamatórias ou substituição de células contráteis por células do tecido conjuntivo, como nas distrofias ou regeneração muscular 69–71. Além disso, o perfil de expressão de uma população de fibras heterogêneas produz uma informação “média”, mesmo que a doença acometa mais drasticamente um determinado tipo de célula muscular em particular 72. O conhecimento das alterações na expressão gênica que ocorrem nas células musculares são de grande interesse para o estudo da plasticidade em relação à atividade, desuso e envelhecimento, e pode também ajudar no desenvolvimento de tratamentos para várias doenças musculares 73. Recentes pesquisas envolvendo perfil global de expressão de mRNAs em células C2C12 têm auxiliado na identificação de importantes vias moleculares envolvidos na regeneração músculo esquelético, como descrito na tabela 1. 16 Condição Experimental Pefil Genômico Plataforma Vias identificadas Referência Privação do meio de cultura (células C2C12) mRNA Microarray (22000*) A,C,F,I 74 Tratamento com TNF- α/INF-γ (células C2C12) mRNA Microaray (12000*) A,F,M,N 75 Tratamento com TNF-α (células C2C12) mRNA Microarray (25000*) A,I,M,N,Q 76 Tratmento com TWEAK (células C2C12) mRNA e miRNA Microarray (25000*) e TLDA(650*) A,C,F,GI,L,M ,O 77 Regulação da miogênese por Setdb1 (Células C2C12) mRNA RNA-Seq e Microarray Q 78 Regulação da miogênese por Linc-YY1 (Células C2C12) mRNA RNA-Seq Q 79 Organização espacial do genoma durante miogênese (células C2C12) mRNA RNA-Seq Q 80 Regulação da miogênese com TEAD (Células C2C12) mRNA RNA-Seq Q 81 Tabela 1. Estudos do perfil genômico global de mRNAs em células C2C12. As principais vias/genes identificados foram: A) Sistema Proteassomal Dependente de Ubiquitina, B) Chaperonas, C) Estresse Oxidativo, D) Calpaínas, E) Proteases Lisossomais, F) Myod, G) MyhC, H) Transportador de Glutamina, I) Citocinas Inflamatórias, J) Apoptose, K) Sistema de Splicing, L) Sinalização Wnt, M) Sinalização NFkB , N) Sistema iNOS, O) Rede MyomiR, P) miR-23, miR-221, miR-148b e miR-338, Q) Miogênese. *: número de sondas utilizadas nos experimentos; TLDA: TaqMan low density array (Life Technologies,EUA); DD: Differential Display. Embora os efeitos benéficos do LBI tenham sido demonstrados em diversos estudos, para nosso conhecimento, não há pesquisas relatando o perfil genômico global de mRNAs de células musculares tratadas com LBI para a elucidação dos mecanismos moleculares envolvidos na regeneração de células musculares induzida pelo tratamento com LBI. Nossa hipótese de trabalho é que a análise de dados de sequenciamento de alta performance de RNAm (RNA-Seq) permitirá 17 elucidar novas vias de regulação e mecanismos moleculares envolvidos na regeneração de células musculares tratadas com LBI. 18 2. OBJETIVO GERAL Identificar as vias moleculares alteradas de mioblastos C2C12 após irradiação com laser de baixa intensidade (LBI). 2.1. Objetivos específicos Avaliar em mioblastos C2C12 tratados com laser de baixa intensidade (LBI): • a capacidade de proliferação e migração; • a viabilidade celular; • o perfil global de expressão gênica; • o enriquecimento funcional do transcriptoma. 19 3. MATERIAL E MÉTODOS 3.1. Cultura celular Os mioblastos C2C12 foram mantidos na cultura em meio de crescimento Dulbecco’s Modified Eagle Medium (DMEM) suplementado com soro fetal bovino (SFB) (Thermo Scientific, USA) a uma concentração de 10%, penicilina 100 UI/mL e estreptomicina 100 µg/mL em uma incubadora umidificada a uma temperatura de 37ºC com uma concentração de CO2 5%. Após as células atingirem uma confluência em torno de 70-80%, elas foram lavadas três vezes com tampão fosfato-salina (PBS) para retirada de resquícios do meio de crescimento, tripsinizadas e, então, centrifugadas a uma rotação de 1500 rpm, durante 10 minutos, a 4ºC. Em seguida, as células foram resuspendidas em DMEM suplementado com SFB 10% e, uma alíquota de 10uL foi utilizada para determinar a concentração celular em uma câmara de Neubauer. Após esse processo, as células foram transferidas para placas de 6-poços ou placas de 96-poços (viabilidade celular) de acordo com cada experimento, a uma concentração de 1x105 células/poço e 5x103 células/poço, respectivamente. 3.2. Irradiação pelo LBI Os mioblastos C2C12 foram divididos em dois grupos experimentais: grupo controle (CT, n=3), constituído pelas células não irradiadas, e um grupo irradiado com o laser de baixa intensidade (LBI, n=3). O grupo LBI foi irradiado com um diodo laser de Gálio-Alumínio- Arseneto (GaAlAs), com comprimento de onda de 660 nm, energia de saída de 20 mW, área do feixe de 0,035 cm² e uma densidade de energia de 2 J/cm². O tempo de exposição foi de três segundos por ponto de aplicação. As placas de 6-poços foram irradiadas em 33 pontos (Figura 3), enquanto a placa de 96-poços foi irradiada em um único ponto. Dessa forma, pode-se obter uma cobertura total da área de cada poço da placa. O grupo CT foi submetido as mesmas condições experimentais que o grupo LBI, exceto o processo de irradiação pelo LBI. A irradiação foi realizada posicionando o feixe do laser perpendicularmente a uma distância de um centímetro da superfície inferior da placa. Todo o processo de irradiação foi realizado em ambiente escuro, para de evitar influência de outras fontes luminosas. 20 Figura 3 – Esquema de grade utilizada para a irradiação do laser de baixa intensidade nas placas de 6-poços. 3.3. Migração celular Os mioblastos C2C12 foram cultivados em placa de 6-poços até atingirem uma confluência em torno de 80-90%. Subsequentemente, foi utilizado uma ponteira estéril de 200 μL para realizar um corte retilíneo no maior diâmetro do poço, sobre a cultura celular, obtendo-se dessa forma uma condição que simulasse uma lesão. Posteriormente, foram realizadas três lavagens com tampão PBS para remover fragmentos celulares e adicionado 2 mL de meio de cultura DMEM suplementado com SFB 5%, em cada poço, quando, então, o grupo LBI foi irradiado (Figura 4). O processo de análise da taxa de migração foi realizado através de imagens digitais logo após a irradiação pelo LBI e 6, 12, 24 e 48 horas após a irradiação. Todas as imagens foram plotadas no software ImageJ (versão 1.6.0) para mensurar a área existente entre cada margem celular. Figura 4 – Ensaio de migração/proliferação - Wound Healing. 3.4. Proliferação celular Os mioblastos C2C12 foram submetidos a cultura sobre lamínulas de vidro inseridas em placas de 6-poços com meio de crescimento DMEM, suplementado com SFB 10%, até atingirem uma confluência de 70-80%, quando o meio de crescimento foi trocado por um meio suplementado com SFB 5% e as células foram irradiadas. Após 5 horas da irradiação, o meio foi substituído por um meio de DMEM contendo 5-Bromo-2′-deoxyuridine (BrdU; 10 µM) (Sigma-Aldrich). Após 1 hora, esse meio foi retirado e as células foram lavadas 3 vezes com tampão PBS, e então fixadas em paraformaldeído 4%, seguido de permeabilização com Triton X-100 0,1% em tampão PBS e 21 bloqueadas por uma hora com soro de cabra 10%, Triton X-100 1% em tampão PBS. Após o bloqueio, foi realizado a desnaturação com HCl 2 N, Triton X-100 0,5% em tampão PBS a uma temperatura de 37ºC durante 30 minutos. Após a desnaturação, foi realizado a neutralização com tampão de borato 0,1 M, pH 8,5, por 10 minutos. Subsequentemente, as células foram incubadas com anticorpo mouse anti-BrdU (Sigma-Aldrich) diluído em uma proporção de 1:1000, durante 1 hora e, posteriormente, overnight a 4ºC em uma solução de incubação composta por Triton X-100 0,3% e soro de cabra 5%. Finalmente, após a incubação overnight, a placa foi lavada 3 vezes com tampão PBS, e adicionado anticorpo goat anti-mouse IgG texas red-conjugated (Santa Cruz Biotechnology) em uma proporção 1:5000, durante 1 hora à temperatura ambiente. As lamínulas foram montadas em lâminas de vidro usando Vectashield (Vector Labs) e foram mantidas em ambiente privado de luz a uma temperatura de 4ºC até a leitura em microscópio de fluorescência invertido (BX61 Olympus). A análise foi realizada pela contagem dos núcleos que emitiram fluorescência, devido a incorporação do BrdU (corados em vermelho), e então foi calculado a taxa de incorporação do BrdU, em relação ao número total de núcleos celulares (corados em azul). 3.5. Viabilidade celular A viabilidade celular dos mioblastos C2C12 foi avaliada pela função mitocondrial utilizando-se o ensaio biológico do brometo 3-(4,5-dimetilazol-2-yl)-2,5-difeniltetrazol (MTT) (Sigma-Aldrich). Esse ensaio mensura a atividade das células viáveis, via atividade da desidrogenase mitocondrial, que reduz o MTT para cristais púrpuros de formazan. Os mioblastos C2C12 foram cultivados em placas de 96-poços contendo meio de crescimento DMEM suplementado com SFB 10% até atingirem uma confluência de 70-80%, quando foram irradiados pelo LBI, o meio de cultura foi substituído por DMEM suplementado com SFB 5% por um período de 6 horas. Após esse período, o meio foi trocado e adicionado DMEM com MTT a uma concentração final de 0,5 mg/mL, e incubado por 3 horas. Seguido a incubação, os poços foram lavados 3 vezes com tampão PBS e foi adicionado 100 µL de DMSO em cada poço, para dissolver os cristais de formazan. A absorbância da solução foi mensurada em um comprimento de onda de 620 nm utilizando um leitor de microplaca (Anthos2020, Anthos Labtec Instruments). 3.6. Extração do RNA total A extração do RNA dos mioblastos C2C12 foi realizada seis horas após a irradiação, utilizando TRIZOL (Thermo Fisher Scientific), de acordo com as instruções do fornecedor. O RNA total extraído foi quantificado por espectrofotometria usando NanoVue (GE Healthcare Life Sciences), e a qualidade do RNA foi obtida através do número de integridade do RNA por análises 22 baseadas no RNA ribossomal utilizando o 2100 Bioanalyzer system (Agilent, USA), todas as amostras apresentaram RIN > 9. 3.7. Sequenciamento do RNA Foram utilizados 5 µg de RNA total para construir as bibliotecas de RNA-seq para os grupos CT e LBI, os quais foram sequenciados em uma mesma flow cell, paired-end, 2 x 100 pb no equipamento Illumina HiScanSQ (Illumina, USA), segundo orientações do fornecedor. Foram gerados 25 milhões de paired-end reads por amostra. Os arquivos com as linhas de sequência (arquivos.fastq) foram submetidos a análise de controle de qualidade usando FastQC e os Phred quality score maior ou igual a 20 por posição de alinhamento. A linha de paired-end reads de fragmentos de DNAc foram alinhadas para o transcriptoma de camundongo (RefSeq, mm10) utilizando o software TopHat (versão 1.3.2) 82. O pacote Python HTSeq foi utilizado para contagem dos reads mapeadas de cada transcrito e a expressão diferencial dos grupos controle e LBI foi identificada utilizando o pacote DEseq (versão 1.22.1) e reportadas como Fold change associado ao valor de p. Os cutoffs para determinar alteração nos níveis de expressão foram Fold change > 1,2 e valor de p <0,05. As análises foram realizadas utilizando o software R (http://www.r- project.org). 3.8. Análise de enriquecimento funcional de vias moleculares Para uma melhor compreensão da relevância biológica dos genes que apresentaram alteração nos níveis de expressão, foi realizada uma análise de enriquecimento funcional no contexto das categorias do Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome e base de dados WikiPathways. As análises das vias foram realizadas utilizando o software Cytoscape (versão 3.4.0) 83 em conjunto com o pacote de dados ClueGO (versão 2.2.5) 84. Foram utilizados como cutoffs o valor de p<0,05 e um fold de enriquecimento maior que 10% para a identificação das categorias enriquecidas. O valor do kappa score foi calculado para refletir a base da relação dos termos na similaridade dos genes associados, com um threshold de 0,3 para fornecer uma visão da relevância das vias utilizando dados experimentais obtidos in silico e em base de dados de redes gênicas, interações proteína-proteína e interações funcionais 85,86. As redes obtidas foram visualizadas e analisadas pelo Cytoscape 83. 3.9. Análises estatísticas Todos os dados são apresentados como media ± desvio padrão (dp). As diferenças entre os grupos CT e LBI foram analisadas utilizando análises de teste t e Mann-Whitney U seguido de 23 comparações múltiplas utilizando o método proposto por Sidak-Bonferroni. Um valor de p<0,05 foi considerado significativamente estatístico. Os cálculos foram realizados através do software GraphPad Prism (versão 6.0.1). 24 4. CONSIDERAÇÕES FINAIS A irradiação pelo LBI sobre os mioblastos C2C12 promoveu uma diminuição das taxas de proliferação e migração, com base nos dados da expressão gênica obtida pelo RNA-Seq, nossa hipótese para essa diminuição é que o LBI exerce um papel sobre as células miogênicas, fazendo com que elas inicialmente parem o seu ciclo celular, diminuindo dessa forma a sua capacidade de proliferação e migração, e, induz as células irradiadas a iniciarem o processo de diferenciação celular. 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Carvalho1,* 1 Department of Morphology Institute of Biosciences, São Paulo State University (UNESP) Botucatu, SP, Brazil 2 Department of Physiotherapy University of Western Sao Paulo (UNOESTE) Presidente Prudente, SP, Brazil *rcarvalho@ibb.unesp.br Telephone: +55 14 3880 0473 Abstract Low-level laser therapy (LLLT) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLT exerts these effects remain largely unknown. We described global gene expression profiling analysis in C2C12 myoblasts after LLLT that identified 514 differentially expressed genes (DEG). Gene ontology and pathways analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLT with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on mailto:rcarvalho@ibb.unesp.br 32 myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLT. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLT-myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLT myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLT, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLT regulates a set of mRNAs that control myoblasts proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLT-myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLT on skeletal muscle cells. Key-words: Transcriptome, Laser treatment, Muscle Regeneration, Myogenesis, RNA Sequencing. Introduction Low-level laser therapy (LLLT) has been used as a non-invasive method to promote or accelerate skeletal muscle regeneration capability [1–5]. Skeletal muscle regeneration is mainly accomplished by the proliferation and differentiation of myogenic cells derived from satellite cells (reviewed in [6]). After trauma or injury, satellite cells are activated, and become immature muscle cells or myoblasts that proliferate, migrate, and fuse into existing muscle fibers, or form new myofibers during muscle repair [7]. Consequently, several studies have been conducted in satellite cells and myogenic cell lines to understand the biological effects of LLLT on cellular and molecular mechanisms 33 that contribute to muscle regeneration [8–12]. These studies clearly demonstrate that myogenic cells modulate proliferation and differentiation, and change the expression of myogenic regulatory factors and cell cycle regulatory proteins in response to LLLT. Although the increasing evidence of the beneficial effects of LLLT on myogenic cells has become widely accepted, the global regulatory molecular mechanisms by which LLLT exerts these effects remain largely unknown. Previous examinations of large scale mRNA expression generated by cDNA microarray analysis have generated insights on the molecular changes underlying the effects of LLLT in different cells types. For example, the expression levels of various genes involved in cell proliferation, apoptosis, and the cell cycle were affected by LLLT in mesenchymal stem cells [13]. Similarly, several differentially expressed genes (DEG) were identified in human fibroblasts after low-intensity red light; most of these genes either directly or indirectly participate in biologic processes related to cellular proliferation [14]. However, to the best of our knowledge, no other study has used a global mRNA expression profiling analysis by high-throughput RNA sequencing (RNA-Seq) method to study the effects of LLLT in skeletal muscle cells. This approach may provide meaningful insights into how LLLT exerts its regulatory effects in these cells and unravels laser-stimulated networks and molecular pathways. Therefore, our goal was to perform a global mRNA expression profiling analysis in C2C12 myoblasts after LLLT. Materials and Methods Cell culture C2C12 myoblasts were maintained in growth medium Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Scientific, USA), 100 IU/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator at 37°C 34 with 5% CO2. After reach 70-80% confluence, cells were washed in phosphate-buffered saline (PBS), trypsinized, centrifuged (1500 rpm, 10 min, 4°C), resuspended in DMEM medium with FBS 10%, and counted in Neubauer chamber. Subsequently, the cells were transferred and cultured into 6-well plates (1x105 cells/well) or 96-well plates (and 5x103 cells/well) in 96-well plates, according to the experiments. Laser irradiation Cells were divided into two experimental groups: non-irradiated control group (CT, n=3) and low-level laser irradiated group (LLL, n=3). The LLL cells were submitted to a Gallium-Aluminum-Arsenide (GaAlAs) diode laser, with 660 nm wavelength, output power of 20 mW, beam area of 0.035cm2, and an energy density of 2 J/cm2. The time of exposure was 3 seconds/point. Each well plate was irradiated in 33 points (6-well plate) or 1 point (96-well plate) to ensure the complete irradiation over the entire cell-culture plate. The CT group was subjected to the same experimental conditions as the LLL, except for the irradiation. Irradiation was performed in the dark to avoid the influence of other light sources and the beam incidence angle was positioned perpendicularly (90 degree) at 1 cm of lower surface plate to the irradiation. Cell viability assay The viability of the proliferating C2C12 myoblasts, after LLLT, was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, USA). This assay measures the activity of living cells via mitochondrial dehydrogenase activity that reduces MTT to purple formazan. The cells were seeded in a 96-well plate with DMEN, and at 0, 6, 12, 24, and 48 hours after LLLT, MTT (0.5 mg/mL) in phosphate- buffered saline (PBS) was added to each well. Following, 100 µL of DMSO was added to each well to dissolve formazan crystals. The solution absorbance 35 was measured at 570 nm using a microplate reader (2020, Anthos Labtec Instruments, Austria). Wound Healing Assay C2C12 myoblasts were plated in 6-well plate and cultured in DMEM until reach 80-90% confluence. Subsequently, a single-line scratch was mechanically generated in a cell monolayer by a 200-μl plastic tip. Cell debris were removed through two PBS washes, and 2 mL of DMEM supplemented with 5% fetal bovine serum was added to each well, when the LLL group was irradiated. The wound open area was photographed and analyzed at 0, 6, 12, 24, and 48 hours after LLLT, and the wound closure rate was determined by plotting the open wound area changes as a function of the time. 5-Bromo-2′-deoxyuridine (BrdU) incorporation C2C12 myoblasts were plated on a glass coverslip until reach 70-80 % confluence in DMEM and then were irradiated and incubated at 37ºC with 5% CO2 for 5 hours; subsequently, the medium was changed to a DMEM containing 10µM BrdU (Sigma- Aldrich, USA) for 60 minutes. Cells were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked for 1 hour in 10% goat serum, 1% Triton X-100 in PBS, denatured with 2 N HCl in PBS containing 0.5% Triton X-100 at 37ºC, for 30 min, and neutralized with 0.1 M borate buffer pH 8,5 for 10 minutes. Subsequently, the cells were incubated with mouse anti-BrdU antibody (diluted 1:1000; Sigma-Aldrich, USA), for 1 hour, and next, the cells were incubated overnight in incubation solution (5% goat serum, 0,3% Triton X-100) at 4ºC. Finally, goat anti–mouse IgG texas red-conjugated (1:5000; Santa Cruz Biotechnology USA) were added to the cells for 1 hour at room temperature, and coverslips were mounted using Vectashield (Vector Labs, USA). Cells were counted in a fluorescent inverted microscope (BX61 Olympus, Japan) to calculate the ratio of BrdU+ cells to the total cell number. 36 Total RNA Extraction Total RNA extraction was performed using TRIZOL kit (Thermo Fisher, USA) according to the manufacturer's instructions. The RNA was quantitated by spectrophotometry using NanoVue (GE Healthcare Life Sciences, USA) and the quality of RNA was obtained by the RNA integrity number (RIN) from the analysis of ribosomal RNAs based on microfluidics, using the 2100 Bioanalyzer system (Agilent, USA). RNA sequencing Total RNA (5 µg) was used to construct RNA-Seq libraries for CT (n=3) and LLL (n=3) groups that were sequenced in a same flow cell, as paired-end, 2 x 100 bp, on an Illumina HiScanSQ instrument (Illumina, USA), following manufacturer’s instructions, which generated an average of 25 million paired-end reads per sample. The raw sequence files (.fastq files) underwent quality control analysis using FastQC and average Phred quality scores of ≥ 20 per position were used for alignment. The raw paired-end reads of the cDNA fragments were aligned to the mouse transcriptome (RefSeq, mm10) using the TopHat (version 1.3.2) spliced junction discovery tool [15]. The Python package HTSeq was used to count mapped reads to each transcript and the differential expression across CT and LLL groups were identified using DEseq (version 1.22.1) and reported as Fold Change along with associated p-values. Cut-offs for significant changes were a fold- change > 1.2 and a p-value ≤ 0.05. The analyses were performed with software package R (http:// www.r-project.org). Pathway and Gene Ontology Enrichment Analysis To further understand the biological relevance of differential expressed genes, we performed functional enrichment analysis in the context of the Gene Ontology (GO) categories, Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways databases. Pathway analysis was performed using Cytoscape (v.3.4.0) [16] 37 with ClueGO (version 2.2.5) packages [17]. A p-value cut-off of 0.05 by group and fold enrichment greater 10% were used to identify the enriched categories. A kappa score was calculated to reflect the relationships between the terms based on the similarity of their associated genes, PSIQUIC web services with the threshold set at 0.3 was used to provide a comprehensive view on the relevant pathways using experimental and in silico data from gene networks, protein–protein interactions, and functional interactions [18, 19]. Identification of Transcriptionally Similar Muscle Gene Sets Transcriptionally similar Gene Sets to the differentially expressed genes in C2C12 myoblasts after LLLT were identified by the comparison with 154 Gene Sets derived from published in vitro muscle microarray studies, available at SysMyo Muscle Gene Sets (http://www.sys-myo.com/). The analysis of the SysMyo Muscle Gene Sets was carried out by using Enrichr, a comprehensive gene set enrichment analysis web tools (http://amp.pharm.mssm.edu/Enrichr/) [20, 21]. Statistical Analysis All data are presented as mean ± SD. Differences between treated and nontreated cells were analyzed using multiple-t test with Sidak-Bonferroni correction. p < 0.05 was considered statistically significant. The calculations and artwork were made using GraphPad software (version 6.01). Venn diagrams were created and analyzed with Venny web Server 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Results Low-Level Laser-Irradiation (LLLT) does not affect cell viability but reduces migration and proliferation of C2C12 myoblasts We found no significant effects on C2C12 myoblasts viability by MTT assay at 0h, 6h, 12h, 24h, and 48h after LLLT (Fig 1a). C2C12 myoblasts migration and http://www.sys-myo.com/ http://amp.pharm.mssm.edu/Enrichr/ http://bioinfogp.cnb.csic.es/tools/venny/index.html 38 proliferation were indirectly evaluated by wound healing assay at 0 h, 6 h, 12h, 24h, and 48h after LLLT treatment (Fig 1b). C2C12 myoblasts exposed to LLLT showed significantly decreased wound closure rate at 6 h, 12 h, and 24 h, when compared to the control group (p < 0.05) (Fig 1c). Considering these results, we selected the time-point 6 h after LLLT for the further analyses. Next, C2C12 myoblasts proliferation was measured by BrdU incorporation (Fig 1d), which showed a decreased C2C12 myoblasts proliferation ratio at 6 h after LLLT (p<0.05) (Fig 1e). 39 Fig 1 Low-Level Laser-Irradiation (LLLT) does not affect cell viability but reduces migration and proliferation of C2C12 myoblasts. A) Cellular viability rate analyzed by MTT assay after the LLLT of C2C12 myoblasts. Cellular viability rate was quantify considering the absorbance variation per hour between 6 h, 12 h, 24 h and 48 h related to the initial absorbance (time 0 h). B) Cellular migration and proliferation were indirectly measured by a wound-healing assay after LLLT of C2C12 myoblasts at 0 h, 6 h, 12 h, 24 h, and 48 h post-wound. C) The wound closure was quantified by measuring the remaining unmigrated area and is expressed as relative percentage (%). D) Cellular proliferation was analyzed by BrdU incorporation (red) detected 6 h after LLLT on the C2C12 myoblasts. Nuclei are counter-stained with 40 DAPI (blue). E) Number of BrdU+ cells per group after LLLT. Data represent mean of three independent experiments, and bars represent the standard deviation. Statistical significance was analyzed by the Student’s t-tests. *p < 0.05, **p < 0.002 LLLT promotes transcriptional changes in C2C12 myoblasts To understand the transcriptome changes associated with LLLT on C2C12 myoblasts, we performed a global mRNAs expression profiling that detected a total of 39,179 transcripts. Differential expressed genes between LLL and CT groups were selected and ranked by a combination of fold change ≥ 1.2 and adjusted p- value < 0.05 cutoff (Online Resource 1). LLLT affected the expression of 514 genes, out of which 263 and 251 were up- and down-regulated, respectively (Online Resource 1). Remarkably, the unsupervised hierarchical clustering analysis of the mRNAs expression data demonstrated biological triplicate clustering and a clear segregation between CT and LLL groups (Fig 2). All samples used in the RNA sequencing experiment presented a RIN ≥ 9.5. 41 Fig 2 Low-Level Laser-Irradiation (LLLT) promotes transcriptional changes in C2C12 myoblasts. Heatmap of mRNA expression levels for the differentially expressed genes (DEG) between LLL and CT groups (1, 2, and 3 represent independent biological replicates for each group). Unsupervised hierarchical clustering analysis was performed using DEG with p-value < 0.05 and fold change > 1.2 and are shown as a color scale LLLT induces changes in the expression of genes associated with different functional categories To determine the biological and functional implications of the expression changes induced by LLLT treatment in C2C12 myoblasts, we performed a functional enrichment 42 analysis on the set of DEG (Online Resource 2). This enrichment functional analysis in C2C12 myoblasts exposed to LLLT revealed categories related to cell cycle, cell migration, response to stress, muscle cell proliferation, ribosome biogenesis, anatomical structure, DNA metabolic process, cell death, and blood vessel development (Fig 3). To further understand the individual pathways, we also investigated the over- and under-expressed genes in each pathway. Interestingly, this analysis revealed that cell cycle pathway presented the higher enrichment (60%), and all the genes classified in this category were down-regulated (Fig 3). Fig 3 Low-Level Laser-Irradiation (LLLT) induces changes in the expression of genes associated with different functional categories. Pathway and Gene Ontology Enrichment Analysis of the differentially expressed genes (DEG) in C2C12 myoblasts after LLLT to identify top pathways and ontologies. Each vertical colored bars (y-axis) represent a major module; horizontal bars represent the percentage of genes presented in the data set compared to the total number of genes in each pathway/ontology. Fraction of the DEG in each pathway (red/down, blue/up; respectively) are shown in x-axis 43 LLLT induces distinct cell type-specific transcriptional changes Next, we evaluated whether the gene expression changes induced by LLLT treatment in C2C12 myoblasts were distinct from different cell types that were also irradiate with LLLT. For this purpose, we compared our RNA-Seq data with previous microarrays studies that have evaluated the effect of: 1) LLLT on mesenchymal stem cell (MSCs) [13], and 2) red light irradiation on human fibroblasts (HS27) [14]. The intersection of the three studies tested showed no overlapping transcripts (Fig 4a); however, LLLT-treated myoblast had six DEG (Ada, Ccnd1, Cfh, Creld1, Gfer, and Wdr12) in common with MSCs [13], and three (Ahcy, Ppih, and Serpine1) with the HS27 [14] (Fig 4a). We also performed functional enrichment analysis on the DEG in HS27 and MSCs cells to determine the functional categories and pathways associated with LLLT treatment in these cells and, finally, we compared these results with our data. This analysis showed no overlapping functional categories and pathways, indicating that that LLLT induces distinct functional categories that are cell type-specific (Online Resource 3). Moreover, comparing our RNA-Seq data with the transcriptome data generated by microarrays technique in HS27 and MSCs [14, 13], our data presented a higher number of DEG in C2C12 (Fig 4a) and, consequently, we were able to identify a higher number of functional categories after LLLT (Fig 4b). 44 Fig 4 Low-Level Laser-Irradiation (LLLT) induces cell type-specific transcriptional changes of distinct functional categories. Comparison of our RNA-Seq data (C2C12 myoblasts after LLLT) with previous microarrays studies that have evaluated the effect of: 1) LLLT on mesenchymal stem cell (MSCs) [13], and 2) red light irradiation on human fibroblasts (HS27) [14]. A) Veen diagram showing the overlap of genes change in C2C12 myoblasts, HS27, and MSC. B) Functional enrichment analysis on the differentially expressed genes in HS27 and MSCs cells was used to determine the functional categories and pathways associated with LLLT treatment in these cells, which were compared with our data (C2C12 cells). Each horizontal bar represents the quantity of enriched ontology terms presented in the data set LLLT induces a transcriptional myotube-like profile in C2C12 myoblasts Considering the number of previous studies that have indicated the potential effects of LLLT on myogenesis, we compared our RNA-Seq data with literature data and asked whether a sub-set of genes in LLLT-treated myoblasts overlaps with data from two myogenesis studies [22, 23] with datasets stored at Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/gds). These studies used global microarrays analysis to evaluate the transcriptome changes during myogenesis (myoblasts and myotubes), and the data are accessible at NCBI GEO database [22, 23] by the following accession numbers: GSE3243 (not published) and GEO990 [24]. Initially, using the dataset from https://www.ncbi.nlm.nih.gov/gds 45 these two myogenesis studies, we selected the set of DEG between myoblasts and myotubes to intersect with the genes that were changed in myoblasts after LLLT. This intersection showed a total of 42 transcripts in our data that overlaps with DEG in myotubes from GSE3243 and GEO990 (Fig 5a). Moreover, a total of 151 transcripts in our LLLT-treated myoblasts data overlaps with the DEG in myotubes from the GSE990 [24] dataset, and with 89 transcripts with the DEG in myotubes from the GSE3243 dataset (Fig 5a). We also compared these DEG that specifically overlap with the corresponding protein products from a study that evaluated the effect of LLLT on global protein expression in C2C12 myoblasts [10]. However, the proteomic data resulted in a lower number of overlapping to predict a transcriptional myotube-like profile. The intersection of all the four conditions tested revealed that only the protein Proliferation-associated protein 2G4 (Pa2g4), present in the proteomic study, overlaps with its corresponding transcript in the conditions that were tested (Fig 5a). Next, we performed a hierarchical cluster analysis with the 42 transcripts differentially expressed that overlap between GSE3243, GSE990 [24] and our data by using Euclidean distance similarity. This clustering analysis showed that the LLLT- treated myoblasts have a myotube-like profile, clustering away from the myoblast profile (Fig 5b). Importantly, the myotube-like transcriptional profile of the LLLT myoblasts was globally confirmed by a bi-dimensional clustering of the 1156 transcripts detected in our RNA-Seq data that specifically overlaps with the DEG from the GSE990 [24] dataset (Fig 5c). To further confirm the global myotube-like transcriptional profile of the LLLT myoblasts, we also compared the differentially expressed genes in C2C12 myoblasts after LLLT with 154 Gene Sets derived from published in vitro muscle microarray studies. This analysis revealed that, out of the 25 top-ranked most transcriptionally similar Gene Sets (overlapping genes) to the 514 DEG in myoblasts after 46 LLLT, 23 are derived from differentiated myotubes and one is from a cardiotoxin injury model for the study of muscle regeneration in mice [25] (Online Resource 4). Fig 5 Low-Level Laser-Irradiation (LLLT) LLLT induces a transcriptional myotube-like profile in C2C12 myoblasts. A) Venn Diagram showing the differentially expressed genes (DEG) of our RNA-Seq data from LLLT-treated myoblasts that overlaps with global microarrays data from two previously published studies that have evaluated the transcriptome changes during myogenesis (DEG between myoblasts and myotubes). These data are accessible at NCBI GEO database by the following accession numbers: GSE3243 (not published) and GEO990 [24]. The DEG from the three transcriptomics studies are also compared with the corresponding protein products from a study that evaluated the effect of LLLT on 47 global protein expression in C2C12 myoblasts [10]. B) Clustering analysis of the 42 DEG of our RNA-Seq data from LLLT-treated myoblasts that are shared with GSE3243 and GEO990 [22]. GSE990 myotubes: A = 10 days, B = 6 days, C = 4 days, and F = 2 days; GSE990 myoblasts: G = 0-day, H = -2 days, I = -1 day; GSE3243 myotube: D = 4 days; and GSE3243 myoblast: K = -2 days; LLLT myoblast: E = LLL group; and myoblasts: J = control group. C) Principal Component Analysis (PCA) for clustering the global gene expression data from LLLT myoblasts with the transcriptomic data from the in vitro myogenesis study (myoblasts and myotubes) conducted by Tomczak et al. [22] (GSE990). CT: control group; LLL: Low- level Laser group; MB: myoblast; and MT: myotube Discussion Several studies have indicated that LLLT affects skeletal muscle cell proliferation and differentiation in vivo and in vitro. Although the therapeutic value of LLLT has become widely accepted, the global regulatory molecular mechanisms by which it exerts these effects on skeletal muscle cells remain largely unknown. In a comprehensive examination of global mRNA expression levels, we identified a large set of mRNAs that respond to changes following C2C12 myoblast LLLT and appear to play an important role in myoblasts proliferation and differentiation into myotubes. Furthermore, we demonstrated that the LLLT effects on C2C12 myoblast differentially regulate mRNAs that reveal a myotube-like transcriptional profile, generating further insights on the specific molecular changes underlying the effects of LLLT on skeletal muscle cells. Initially, based on migration, proliferation, and cell viability parameters, we selected an appropriate time point, at 6 h post a single exposure of C2C12 cells to the laser radiation, to proceed to the transcriptomic analysis. These analyses showed that, after 6 h of LLLT, C2C12 myoblasts presented reduced migration and proliferation without affecting cell viability. These results are in accordance with a previous study performed in C2C12 cells, which also showed that laser treatment induced a decrease in the cell proliferation rate without affecting cell viability, while leading to the expression 48 of the early differentiation marker MyoD [10]. LLLT also has the potential to increase the survival of primary myogenic donor cells, promote their fusion with the host myofibers, and enhance their ability to recover [11]. Interestingly, LLLT in association with Bothrops jararacussu venom has been shown to promote C2C12 differentiation by up regulating myogenic factors [26]. On the other hand, Ben-Dov et al., [12] demonstrated that LLLT on primary rat satellite cell cultures induced cell cycle regulatory protein expression, increased satellite cell proliferation, and inhibited cell differentiation. LLLT also stimulated cell cycle entry and the accumulation of satellite cells around isolated single fibers [9]. Variable effects were achieved in a same study that used different LED light wavelengths, with blue (470 nm) or red (630 nm) LED light illumination decreasing or increasing C2C12 proliferation rates, respectively [8]. While there are no simple explanations to the apparent inconsistencies among these studies that have evaluated the effect of LLLT on proliferation and differentiation of skeletal muscle myogenic cells, it is clear that LLLT affect these processes and promotes or accelerates skeletal muscle regeneration [1–5]. To the best of our knowledge this is the first global transcriptome catalogue of skeletal muscle cells after LLLT. Our RNA-Seq analysis identified 514 DEG (p-value < 0.05 and fold change ≥ 1.2), of which 263 and 251 were up- or down-regulated, respectively. Importantly, the unsupervised hierarchical clustering analysis of the mRNAs expression data in C2C12 LLLT-treated myoblasts showed a biological replicate clustering and a segregation between CT and LLL groups. These data clearly indicate that the LLLT effects on myoblasts transcriptional regulation are not random, and that RNA- Seq is powerful tool to evaluated transcriptional changes promoted by LLLT in cultured cells. 49 Previously, Wu et al., [13] used a microarray analysis to identify transcriptome changes induced by LLLT in cultured mesenchymal stem cells, and identified 119 DEG (fold change ≥ 1.2). Zhang et al., [14] also used microarrays analysis to study the effect of LLLT on cultured human fibroblasts, and found 111 DEG by more than twofold. Noteworthy, the transcriptome changes between non-irradiated and irradiated in these previous studies show very few DEG in common. Our data from LLLT-treated myoblast had six DEG in common with one study [13], and three with the other [14]. Moreover, our RNA-Seq data identified a higher number of DEG when compared to these two previous microarray studies. Accordingly, our functional analysis also presented a higher number of categories that includes cell cycle, cell migration, response to stress, muscle cell proliferation, ribosome biogenesis, anatomical structure, DNA metabolic process, cell death, and blood vessel development. The comparison of these functional categories in which the DEG are classified may help to identify shared core molecular mechanisms underlying the effects of LLLT in different cell types. However, we did not identify functional categories affected by LLLT that overlap with C2C12 myoblasts when compared with mesenchymal stem cells [13] and fibroblasts [14]. These disparities in the number of the DEG and functional pathways among the studies may occur due to the different cells types or statistical cut-offs, or several laser-related factors such as wavelength of radiation, energy density, and time of irradiation. To further validate our RNA-Seq results and better understand the effects of LLLT on myoblasts, we asked whether a sub-set of DEG in LLLT-myoblasts overlaps with Geo Expression Omnibus datasets (GEO990 [24] and GSE3243) experiments that evaluated transcriptome changes during in vitro myogenesis (myoblasts vs myotubes). Our LLLT- myoblasts data had 151 transcripts in common with one dataset (GEO990), 89 transcripts with the other (GSE3243), and 42 transcripts that overlap with both datasets. Although 50 included in different functional categories, these 42 transcripts are useful to indicate alterations triggered by LLLT in myoblast. It is interesting to note that Hmga2 and Ccnd1 were among these deregulated genes after LLLT; both were down-regulated and associated with ontology groups such as cellular response to irradiation, DNA damage checkpoint, regeneration, and cell morphogenesis involved in differentiation. Importantly, several studies have demonstrated a key role for Ccnd1 in promoting myoblast cell cycle withdrawal and terminal differentiation into myotubes [27–32]. Hmga2 has been also directly the regulation of connected to myoblast proliferation and differentiation; Hmga2, increases is coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes [33]. Also in accordance with these literature data, our ontology analysis revealed that cell cycle pathway presented the higher enrichment (60%), and all the genes classified in this category were down-regulated. Together, these findings indicate that LLLT in C2C12 myoblast LLLT appear to play an important role in reducing myoblasts proliferation and inducing differentiation into myotubes. Next, we performed a hierarchical cluster analysis with those 42 transcripts differentially expressed that overlap between GSE3243, GSE990 [24] and our data by using Euclidean distance similarity. Noteworthy, this clustering analysis confirmed that the LLLT-myoblasts have a transcriptional myotube-like profile, clustering away from the myoblast profile. Monici et al., 2013 [10] previously showed that laser treatment decreased cell proliferation associated with changes of cell morphology and cytoskeletal architecture leading to the formation of tube-like structures. These authors also evaluated the effect of LLLT on global protein expression in C2C12 myoblasts and identified 42 differently expressed proteins in LLLT-myoblasts. However, when we analyzed the DEG (GSE3243, GSE990[24], and our data) that overlap with the corresponding protein 51 products from Monici et al., 2013 [10], the proteomic data resulted in a lower number of overlapping to predict a transcriptional myotube-like profile. The intersection of all the four conditions tested also revealed that only the protein Proliferation-associated protein 2G4 (Pa2g4) that overlaps in all conditions. Pa2g4, also known as Ebp1, is expressed during myogenesis in satellite cells; however, its knockdown inhibits both proliferation and differentiation of C2C12 myoblasts and satellite cells, which also present a reduced capacity of myotube formation [34]. We also compared the differentially expressed genes in C2C12 myoblasts after LLLT with 154 Gene Sets derived from published in vitro muscle microarray studies available at Gene Expression Omnibus to confirm a global myotube-like transcriptional profile of the LLLT myoblasts. This analyses further demonstrated that all 514 DEG in myoblasts after LLLT highly overlaps with genes sets of differentiated myotubes from the study of Tomczak et al., [35], but surprisingly, also overlaps specifically with additional 22 gene sets from differentiated myotubes, and with one cardiotoxin injury model used to study muscle regeneration in mice [25]. Although our study design has proven useful, there were also limitations in vivo study that must be considered. Specifically, there is a diversity of studies that analyze the effects of LLLT on C2C12 myoblasts and other cell types. These studies apply several different laser-related parameters such as wavelength of radiation, energy density, and time of irradiation, making it difficult to select specific parameters to become the results comparable. Thus, further studies are needed to better establish how these laser-related parameters globally affect cellular and molecular mechanism in different cell types. In summary, we demonstrate for the first time that LLLT regulates a set of mRNAs that control myoblasts proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLT-myoblasts and 52 provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLT on skeletal muscle cells. Funding: This study was supported by grants from the São Paulo Research Foundation Brazil (FAPESP n° 2012/13961-6) and National Council for Scientific and Technological Development (CNPq n° 476399/2013-0). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval: This article does not contain any studies with animals performed by any of the authors. 53 References 1. Vatansever F, Rodrigues NC, Assis LL, et al (2012) Low intensity laser therapy accelerates muscle regeneration in aged rats. Photonics Lasers Med 1:287–297 . doi: 10.1515/plm-2012-0035 2. 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Symbol Description ID fold change p value Serpinb2 serine (or cysteine) peptidase inhibitor, clade B, member 2 [Source:MGI Symbol;Acc:MGI:97609] ENSMUSG00000062345 -3.999398954 0.025955846 Gjb3 gap junction protein, beta 3 [Source:MGI Symbol;Acc:MGI:95721] ENSMUSG00000042367 -3.433498299 0.000582023 Lce1g late cornified envelope 1G [Source:MGI Symbol;Acc:MGI:1913445] ENSMUSG00000027919 -2.985445223 0.002520492 Prl2c2 prolactin family 2, subfamily c, member 2 [Source:MGI Symbol;Acc:MGI:97618] ENSMUSG00000079092 -2.467975714 0.000102734 Lrp2 low density lipoprotein receptor-related protein 2 [Source:MGI Symbol;Acc:MGI:95794] ENSMUSG00000027070 -2.173024881 0.013088201 Ctgf connective tissue growth factor [Source:MGI Symbol;Acc:MGI:95537] ENSMUSG00000019997 -2.131538871 9.70E-08 Ngf nerve growth factor [Source:MGI Symbol;Acc:MGI:97321] ENSMUSG00000027859 -2.01613754 4.15E-05 Shank2 SH3/ankyrin domain gene 2 [Source:MGI Symbol;Acc:MGI:2671987] ENSMUSG00000037541 -1.878258899 0.048516462 Hmga1 high mobility group AT-hook 1 [Source:MGI Symbol;Acc:MGI:96160] ENSMUSG00000046711 -1.728715071 9.70E-08 Etv4 ets variant 4 [Source:MGI Symbol;Acc:MGI:99423] ENSMUSG00000017724 -1.720927905 0.000668155 Siglecg sialic acid binding Ig-like lectin G [Source:MGI Symbol;Acc:MGI:2443630] ENSMUSG00000030468 -1.712048284 0.043672378 Hmga1-rs1 high mobility group AT-hook I, related sequence 1 [Source:MGI Symbol;Acc:MGI:96161] ENSMUSG00000078249 -1.701951271 5.45E-10 Pparg peroxisome proliferator activated receptor gamma [Source:MGI Symbol;Acc:MGI:97747] ENSMUSG00000000440 -1.701209887 0.013478123 Tagln transgelin [Source:MGI Symbol;Acc:MGI:106012] ENSMUSG00000032085 -1.686138608 0.014709073 Ccnd1 cyclin D1 [Source:MGI Symbol;Acc:MGI:88313] ENSMUSG00000070348 -1.685829908 7.36E-09 Tigit T cell immunoreceptor with Ig and ITIM domains [Source:MGI Symbol;Acc:MGI:3642260] ENSMUSG00000071552 -1.641660077 0.019177473 Hmga2 high mobility group AT-hook 2 [Source:MGI Symbol;Acc:MGI:101761] ENSMUSG00000056758 -1.637526088 0.000184456 Gm7008 predicted gene 7008 [Source:MGI Symbol;Acc:MGI:3647211] ENSMUSG00000035983 -1.626702086 0.001610793 Fosl1 fos-like antigen 1 [Source:MGI Symbol;Acc:MGI:107179] ENSMUSG00000024912 -1.626375009 0.000373325 Chek1 checkpoint kinase 1 [Source:MGI Symbol;Acc:MGI:1202065] ENSMUSG00000032113 -1.620200607 5.95E-05 Hbegf