N6-Methyladenosine Modification of circRNA Mediates Silica-Induced Pulmonary Fibrosis

RATIONALE: In the past 10 years, researches on RNA methylation have made rapid progress. As the most common type of RNA methylation, N6-adenosine methylation(m 6 A) is involved in the occurrence and development of a variety of diseases. It has been found that circRNA may be an important target for diagnosis and treatment of silica-induced pulmonary fibrosis. CircRNA can be modified by m 6 A, indicating m 6 A modification of circRNA may regulate the occurrence and development of the disease. Therefore, whether circRNA methylation is involved in silica-induced pulmonary fibrosis deserves to be studied. METHODS: Human pulmonary fibroblasts from adults (HPF-a) were used in current study. M 6 A RNA methylation quantification kit was used to determine the total RNA m 6 A modification level in HPF-a and mouse tissuse. M 6 A-meRIP was applied for determinationof target circRNA m 6 A modification level. The expression of multiple proteins and gene were measured by Western blotting, RT-qPCR and immunofluorescence-staining after SiO 2 exposure. RNA interference technology was used to clarify the function of circRNA and protein. Then the co-localization of circRNA and METTL3 was detected by in situ hybridization. RESULTS: Total RNA m 6 A level and METTL3 expression increased in mouse model of silicosis and HPF-a cells after SiO 2 exposure, indicating METTL3-mediated m 6 A participated in pulmonary fibrosis. M 6 A-circRNA epigenetic transcriptome microarray assay showed difference in circRNA methylation in lung tissue from mice. Finally, two circRNA (hsa_circ_0000672 and hsa_circ_0005654) were selected based upon the high exppression level. Interestingly, knock-down of single circRNA did not show significant effect on migration and proliferation in HPF-a, which only showed up after knock-down of both two circRNA. CONCLUSION: Our results suggested that the m 6 A methylation of circRNA was involved in the pulmonary fibrosis process, which provided evidence for the correlation between circRNA m 6 A modification and disease, explore the possibility of circRNA as a therapeutic and diagnostic target, and provided theoretical support for clinical treatment.