Regulation Of MiR-101 On Let-7a-1 Maturation In Cervical Cancer Cells

ObjectiveMicroRNAs(miRNAs)participate in different biological processes,including embryonic development,metabolism,viral infections and human malignancies.These small non-coding RNAs belong to a class of novel gene regulators and control gene expression by binding to complementary sequences in the 3 UTR of the target mRNA.Let-7,the first to determine tumor suppressor miRNA,plays an important role in the regulation of Ras and high-mobility histone AT-hook 2(HMGA2)in the pathogenesis of cancer.MiRNA regulation occurs at the level of transcription and transcription.For example,the transcription of let-7 was directly inhibited by c-Myc,which was a nucleoprotein that regulates cell proliferation and differentiation.When the cells undergo differentiation,the pluripotency factor Lin28 inhibits the biogenesis of let-7 by binding to its primary and precursor transcripts.In general,miRNAs inhibit the translation of target mRNA by its base pairing interaction with its 3 UTR.Interestingly,in Drosophila,it has been assumed that miRNAs can target other miRNAs.This possibility prompted us to assume that miRNAs can regulate non-mRNA targets,such as miRNAs.Through this study,we have tried to elucidate the regulation of miRNAs and the important role of precursor loop structures during miRNA maturation.MethodFirst,bioinformatics predicts miRNAs that interact with the pre-let-7a-1 loop structure.RT-qPCR was used to identify miR-101 that inhibited the expression of let-7a-1 matures and had no effect on the expression of pri-let-7a-1 and pre-let-7a-1.The inhibitory effect of miR-101 on let-7a-1 was confirmed by EGFP fluorescence reporter system,but the inhibitory effect disappeared when the interaction site mutation.Western blot was used to verify the regulation of miR-101 on direct target gene of let-7a-1.RIP experiments demonstrated that the regulation of miR-101 on the loop structure of let-7a-1 depends on the Ago protein.ResultBioinformatics predicts three miRNAs that can interact with loop structures with pre-let-7a-1.In endogenous level.RT-qPCR showed that miR-101 could inhibited the expression of let-7a-1,and had no effect on the expression level of pre-let-7a-1.EGFP Fluorescence Reporting Vector System found that miR-101 inhibited EGFP expression.Western blot demonstrated that miR-101 up-regulates the direct target gene Aurora of let-7a-1.RIP experiment demonstrated that the regulation of the loop structure of miR-101 to let-7a-1 depends on the Ago protein and does not depend on the Dicer protein.ConclusionThis study had demonstrated that miR-101 inhibits the maturation of let-7a-1 by binding the pre-let-7a-1 loop structure and this binding requires Ago2 protein.