Mir-103a-2-5p Modulates The Function Of MiR-34a Through Suppressing Its Maturation In Hepatoma Cells

Objective:MicroRNA(miRNA)regulates the expression of specific genes,typically by base pairing to the 3' untranslated regions(3' UTRs)of target messenger RNAs(mRNAs)to mediate repression of that target message,by transcript destabilization or translational inhibition.It influences many biological processes,including development,differentiation,proliferation,and apoptosis.Expression of miRNAs are abnormal in a variety of cancers,and miRNA may play a role in tumorigenesis.However,miRNA expression is often tissue specific and posttranscriptionally regulated.Although it has been postulated that miRNAs can regulate other miRNAs,this has never been shown experimentally to our knowledge.In this paper,we focus on looking for the mechanism of a miRNA post-transcriptionally regulated by other miRNA and the role of miR-103a-2-5p in HCC.Methods:First,the candidate miRNAs targeting pri-miR-34a were screened by bioinformatics,and further verified by EGFP report assay and RNA dot blot in QGY-7703 and HepG2 cell lines.Second,we designed several miR-34a mutation,including pri-miR-34a-M1,pri-miR-34a-M2,pri-miR-34a-M3,according to its loop structure characteristics.Then,combining with RNA dot blot and EGFP reporter assay,we explored the role of miR-103a-2-5p "seed" sequence and the loop of pri-miR-34a on the interaction of miR-103a-34a.qRT-PCR was used to analyze the expression of miR-103a-2-5p and miR-34a in many cell lines.Third,we enforced expression of miR-103a-2-5p or miR-34a in QGY-7703 and HepG2 cells,and evaluated the effects on HCC cell using a series of assays,including MTT,colony formation assays,Meantime,over-expression of miR-34a-M1 in experiment was used to further verify if the loop of pri-miR-34a was a target of miR-103a-2-5p.qRT-PCR and Western Blot was used to verity if miR-103a-2-5p could induce the SIRT1 though inhibiting miR-34a expression.Xenograft tumor formation assay was used to analyze the role of miR-103a-2-5p in vivo.Results:First,4 miRNAs were chosen as candidate miRNAs targeting pri-miR-34a by bioinformatics methods,including miR-103a-2-5p.Overexpression of miR-103a-2-5p in HEK-293T cells suppressed expression of miR-34a.Subsequently we validated miR-103a-2-5p directly target pri-miR-34a by EGFP reporter assay.Second,EGFP reporter assay and RNA dot blot were used to verify that miR-103a-2-5p could bind to the loop of pri-miR-34a.The "seed" of miR-103a-2-5p is critical for the interaction of miR-103a-34a.The negative correlation between miR-103a-2-5p and miR-34a exists in human cells.Third,after further exploring the cell function of miR-103a-2-5p,we found that overexpression of miR-103a-2-5p could enhance the cell activity and proliferation of QGY-7703 and HepG2 cells.Rescue experiment showed that miR-103a-2-5p could inhibit miR-34a expression to enhance the level of SIRT1.Additionally,the loop of pri-miR-34a is important for the activity.Finally,we found that miR-103a-2-5p can promote tumor formation in vivo.Conclusions:It is the first time we found that a miR-103a-2-5p could directly target miR-34a and inhibit its maturation.Meatime,the "seed" sequence of miR-103a-2-5p and the loop of pri-miR-34a are critical for the interaction between miR-103a-34a and pri-miR-34a.Additonally,miR-103a-2-5p can inhibit the production of miR-34a,thus promoting the expression of SIRT1,and eventually promote tumor formation in HCC.