Post-transcriptional regulation of tumor suppressor gene lncRNA CARMN via m6A modification and miRNA regulation in cervical cancer

PurposeThe abnormal regulation of lncRNA CARMN has been proved to be a tumor suppressor gene of cervical cancer (CC). However, its role in CC is still elusive. The regulation of CARMN post-transcriptional level by m(6)A modification and miRNA has not been studied. This study aims to analyze the molecular mechanism of m(6)A modification and miRNA on the abnormal expression of CARMN in CC cells, so as to provide a new theoretical basis for the diagnosis and treatment of CC.MethodsMeRIP-seq was used to identify the differential m(6)A-modified genes between tumor and normal cervical tissues. RT-qPCR assay was used to detect gene expression levels in tissues or cells. The m(6)A modification sites of CARMN was predicted by bioinformatics, and the modification of m(6)A and its regulatory effect on CARMN were analyzed by MeRIP-qPCR, Actinomycin D assay and RIP assay. RIP-microarray combined with bioinformatics methods to screen miRNAs that may target CARMN. The regulation mechanism between miRNA and CARMN was verified by RT-qPCR, nucleo-plasmic separation assay, mRNA stability assay, dual-luciferase reporter assay, and in vivo experiments.ResultsMeRIP-seq found that CARMN is a significant different gene in the abundance of m(6)A in CC, and the modification level of m(6)A in CC tissues was higher than that in normal cervical tissues. Further, this study verified that m(6)A reader YTHDF2 could recognize m(6)A-modified CARMN and promote its degradation in CC cells. miR-21-5p was proved to be the downstream target gene of CARMN, and miR-21-5p could negatively regulate the expression of CARMN. Further experiments showed that miR-21-5p could directly bind to CARMN and lead to the degradation of CARMN. The in vivo experimental results indicated that the level of miR-21-5p in the overexpressed CARMN group was significantly lower than that in the control group.Conclusionm(6)A modification and miR-21-5p play important roles in promoting the occurrence and development of tumors by regulating CARMN, provide new potential targets for the treatment of CC.