MicroRNA-519d Targets Ki-67 And Suppresses Cell Growth In Hepatocellular Carcinoma Cell Line QGY-7703

[Background and Aims]MicroRNAs(miRNAs)are a class of small non-coding RNAs that include 22 nucleotides,which are the new family of small RNA molecules and which are proved to negatively regulate certain genes containing complementary sequences in 3’untranslated regions,which post-transcriptionally regulate gene expression.In several cases,miRNAs have shown to affect target genes involved in cell proliferation,apoptosis,differentiation,metabolism.Recent evidence indicates that some miRNAs function as oncogenes or tumor suppressors and play an important role in cancer initiation and progression.Ki-67 is a protein with higher levels detected in cancers,commonly used to detect and quantify proliferating cells.Although several studies have demonstrated the role of the Ki-67 in cancer cell proliferation,little is known about its regulation during cancer pathogenesis.Based on this,we focused on the effects of miR-519d which regulates Ki-67 on the phenotypes of Hepatocellular Carcinoma(HCC)cells,and reveal the mechanism through confirmation of Ki-67 in this study.So,we can exclusive the possible function of miR-519d in HCC.[Methods]At first,we combined bioinformatics and identified the candidate miRNA for Ki-67.And fluorescent reporter assay was performed to confirm the reliability of the direct regulative miRNA.Furthermore,in order to confirm the regulative role of miRNA on Ki-67 expression,the mRNA levels and protein levels of Ki-67 in tissues or miRNA overexpressed HCC cells were detected with Real-time PCR and Western blot.Subsequently,miRNA were overexpressed in HCC cell lines －QGY-7703 cell,and the changes of cell phenotypes were detected by MTT assay and colony formation assay.Then we detected the differencial expression of miRNA in HCC and adjacent normal tissues by Real-time PCR assay.Finally,the gene of Ki-67 was knocked down using RNA interference and changes of cell phenotypes were detected with MTT assay,colony formation assay as well as the flow cytometry.[Results]Firstly,we identified miR-519d as congenerous candidate miRNA for oncogene Ki-67.The Ki-67 mRNA 3’untranslated region(3’UTR)contains the potential binding site of miR-519d.The fluorescent reporter assay also confirmed that miR-519d can directly bind to the specific site of Ki-67 mRNA 3’UTR and negatively regulate the gene expression.When miR-519d function was overexpressed in HCC cells,mRNA level and protein level of Ki-67 were both depressed,and mRNA level of Ki-67 in HCC tissues were higher than those in adjacent normal tissues.Subsequently,Real-time PCR results showed that the expression of miR-519d in HCC tissues were lower than those in adjacent normal tissues.We report that after overexpressed miR-519d,the colony formation activity of HCC cells were suppressed.We also discovered that when the gene of Ki-67 was knocked down,the colony formation activity of HCC cells were inhibited apparently,consistent with the overexpression of miR-519d.Using the flow cytometry,Ki-67 is induced when quiescent arrested cells enter the G1-S phase transition was detected.With Pri-miR-519d in QGY-7703 cell which had knock-downed Ki-67,the cell cycle was rescued partly.[Conclusions]Our results confirmed that in Hepatocellular Carcinoma,the function of Ki-67 promotes cell proliferation and directly downregulates by miR-519d as a tumor gene.Based on our study,we can understand deeply the mechanism of Ki-67 which acts to tumors,and offer new clues to cancer diagnosis and therapy.