MiR-18a Promotes Cell Invasion And Migration Via Directly Targeting Dicer1in Hepatocellular Carcinoma

Background and ObjectiveMicroRNAs are non-coding RNAs of about22nucleotides in length that can bindto the complementary sequences in the3’UTR of multiple target mRNAs by regulatingmRNA stability and translation through the action of the RNA-induced silencingcomplex (RISC). MiRNAs have been a hotspo t in research for their involvement inbiological processes and tumor development. The tumorigenesis and development ofhepatocellular carcinoma (HCC) is a multistep process invo lving multiple genetic andenvironment factors. However, there has been limited research on microRNomics andthe interaction of miRNAs in HCC．A recent study showed HCC tissues miR-18a wassignificantly higher than that in the adjacent normal tissue. Our preliminary study alsoshowed that serum miR-18a was significantly higher in patients with hepatitis B virus(HBV) related HCC than that in healthy control. We designed a study to investigate theeffects of miR-18a on HCC cell invasion and migration potential, and to explore itsmechanism.Method1、HepG2and HepG2.2.15（later was transfected with HBV and stably expressingHBV）cells were transfected with miR-18a inhibitor, inhibitor negative control (inhibitorNC) by Lipofectamine. Cell invasion potential was evaluated by transwell invasionassay. Cell migrating ability was detected by transwell migration assay andwound-healing assay.2、Dicer1was predicted as the target of miR-18a by bioinformation softwares.Co-transfected HepG2and HepG2.2.15cells with miR-18a inhibitor and Dicer siRNAby Lipofectamine, Co-transfected with miR-18a inhibitor and negative control siRNA(siRNA NC) were cultured as negative controls. Transfected HepG2and HepG2.2.15 cells with miR-18a inhibitor, inhibitor negative control (inhibitor NC). Cell invasionpotential was evaluated by transwell invasion assay. Cell migrating ability was detectedby transwell migration assay and wound-healing assay. Western blot analysis was usedto monitor the expression levels of the Dicer1in each group. Observe whether miR-18ainhibitor HCC cell migration and invasion by directly targeting of Dicer1.Results1、Transwell inva sion and migration assay showed that compared with NC group,there was a significant reduction in cell invasion and migration capacity after HepG2and HepG2.2.15cells transfected with miR-18a inhibitor for72h. For HepG2cells, therates of invaded or migrated cells (20×10magnification) were0.410or0.446,respectively (P<0.01). For HepG2.2.15cells, the rates of invaded or migrated cells were0.565or0.602, respectively (P<0.01).Wound-healing assay showed that significant reduced cell migration was observedin miR-18a inhibitor transfectant compared with the NC transfectant when24h,48h and72h after the scratch in both HepG2and HepG2.2.15cells (P<0.05).2、Western blot analysis, transwell invasion and migration assay showed thatcompared with NC group, the protein level of Dicer1was significantly up-regulatedafter transfected HepG2and HepG2.2.15cells with miR-18a inhibitor48h(P<0.01).There was a significant reduction in cell invasion and migration capacity after HepG2and HepG2.2.15cells transfected with miR-18a inhibitor for72h. For HepG2cells, therates of invaded or migrated cells (20×10magnification) were0.462or0.460,respectively (P<0.01). For HepG2.2.15cells, the rates of invaded or migrated cells were0.574or0.579, respectively (P<0.01). Compared with the miR-18a inhibitor+siRNANC co-transfectant group, the protein level of Dicer1was significantly down-regulatedafter co-transfected HepG2and HepG2.2.15cells with Dicer siRNA+miR-18a inhibitor48h. Significant enhanced cell migration and invasion were observed after hepG2andhepG2.2.15cells co-transfected with miR-18a inhibitor+Dicer siRNA for72h (P<0.01).For HepG2cells, the rates of invaded or migrated cells (20×10magnification) were2.079or2.193, respectively (P<0.01). For HepG2.2.15cells, the rates of invaded ormigrated cells were1.619or1.777, respectively (P<0.01). Wound-healing a ssay showedthat significant increased cell migration was observed in Dicer siRNA+miR-18ainhibitor co-transfectant compared with the siRNA NC+miR-18a inhibitor transfectant when24h,48h and72h after the scratch in both HepG2and HepG2.2.15cells (P<0.05).Conclusion1、MiR-18a promotes HCC cell invasion and migration.2、MiR-18a promotes HCC cell invasion and migration via directly targetingDicer1.