Function And Mechanism Of MiR-638 Regulating Cell Cycle By Targeting CCNA2

MiRNAs,a class of non-coding small RNAs,which participate in regulating gene expression at transcriptional or post-transcriptional level.As a primate specific miRNA,miR-638 has been reported to play a crucial role in some important biological processes such as early embryonic development,cell differentiation,the occurrence and progression of tumors,DNA damage repair and so on.In tumor-related research,we found that miR-638 regulates the development of various tumors and has different roles in different tumors.As it is usually accompanied by cell cycle disorders and abnormal cell proliferation during the occurrence and development process of tumors,exploring the molecular mechanism of miR-638 regulating cell cycle would provide clues to explain the complex function of miR-638 in tumor formation.In our study,we have discussed the relationship between miR-638 and cell cycle control,and we also studied the related biological function of miR-638.Our research further explained the important function of miR-638 in tumor formation,and it also laid a foundation to reveal the molecular mechanism of miR-638 regulating cell cycle.In our research,we analyzed and compared the expression level of miR-638 in three different cell cycle phases of HeLa and 293 T,and we found that the expression level of miR-638 was relatively higher in G2/M phase and lower in S phase.We further analyzed the impacts of miR-638 on cell cycle transition in 293 T and A549,as results showed by flow cytometry,overexpression of miR-638 could increase cell proportion of G2/M phase,and decrease the ratio of G0/G1 phase,which called G2/M phase retardation.At the same time,RT-qPCR results showed that overexpression of miR-638 regulated a variety of cell cycle gene in mRNA expression level,including target genes p53,CDK2,SMC1 A,some CDK inhibitors p15,p16,p21 and p27,p57,the key genes in regulating G2/M,such as CCNA2,CDK1,CCNB,and other related genes in cell cycle.These results indicated that the expression of miR-638 in cell cycle was regulated by cell cycle phases,and the overexpression of miR-638 inhibited the cell cycle transition from G2/M to G0/G1 phase,and affecting the expression of various cell cycle related molecules.MiRNAs usually function by regulating the expression of target genes.After a detection of large cell cycle genes,we noticed that overexpressing of miR-638 could up-regulate the expression of CCNA2 in 293 T,A549 and HeLa.Through the prediction of miRWalk website and the dual-luciferase reporting experiment,we confirmed that CCNA2 was one of the targets of miR-638,and the regulation pattern was targeting the 5’UTR of CCNA2 and up-regulating the mRNA and protein expression levels of CCNA2.Regulation manner of this kind is not a new finding but has been reported in the past.Lung cancer has a high morbidity and mortality around the world,and much more research should be devoted to improving the current means and effectiveness of the treatments.Therefore,we investigated the effect of miR-638 on cell proliferation and migration in A549.MTT experiments showed that miR-638 inhibited the proliferation of A549.However,both of the Trans Well experiments and the detection of EMT-related molecules showed that miR-638 could affect epithelial mesenchymal transformation of A549 by regulating CCNA2,which promoting the cell migration.In summary,our study showed that miR-638 is highly expressed in G2/M phase of HeLa and 293 T.Overexpression of miR-638 could inhibit cell cycle transition from G2/M to G0/G1 phase by regulating a variety of cell cycle related genes.CCNA2 is one of the target genes of miR-638,and it is accommodated by an up-regulated pattern.miR-638 inhibit cell proliferation,but promote migration by targeting CCNA2 in A549.