The Effect Of Hsa-miR-MTCO3P38 On The Invasion And Migration Of Hepatocellular Carcinoma Cells

Part ? Hsa-miR-MTCO3P38 is a novel micro RNA derived from the pseudogene MTCO3P38 Objective To find the potential novel micro RNAs(miRNAs)in hepatocellular carcinoma(HCC)tissues and verify the sequence and authenticity of novel miRNAs.Methods The differentially expressed novel miRNA(Hsa-miR-MTCO3P38)was found by small RNA deep sequencing of postoperative liver tissues collected from patients with HCC and normal liver.Structure prediction of the novel miRNA was performed using miRdeep2 2.0.0.5 software and RNAstructure software.Northern blot and DNA sequencing confirmed the sequence and authenticity of the novel miRNA.Results The reads count of Hsa-miR-MTCO3P38(miR-MTCO3P38)is significantly down regulated in HCC tissues compared to adjacent non-tumor tissues(ANTs)(p<0.001,q<0.001)and normal liver tissues(p<0.001,q<0.001).miR-MTCO3P38 is located on the pseudogene MTCO3P38 and is predicted to have a stable precursor secondary structure(miRdeep2 score = 1.4,p < 0.05).The results of Northern blotting showed that the plasmids containing the sequence of pre-miR-MTCO3P38 can be transcribed and processed in hepatoma cells to produce mature miR-MTCO3P38.The RT-PCR product of miR-MTCO3P38 in HCC tissues was detected by DNA sequencing,and the results confirmed that the sequence of miR-MTCO3P38 was 5'-UAGGAGGGCUGAGAGGGC-3'.Conclusion miR-MTCO3P38(5'-UAGGAGGGCUGAGAGGGC-3')is a novel miRNA stably expressed in human liver cancer tissues and cells.Part ? Relationships between expression of miR-MTCO3P38 in HCC tissues and clinical data of HCC patients and its effect on invasion and migration hepatoma cells Objective To observe the expression of miR-MTCO3P38 in HCC tissues and to analyze the relationships between the expression of miR-MTCO3P38 and the prognosis of HCC patients.To explore the effect of miR-MTCO3P38 on biological behavior of hepatoma cells.Methods RT-PCR was used to detect the differential expression of miR-MTCO3P38 in HCC tissues,ANT tissues and portal vein tumor tissues(PVTT).At the same time,the expression of miR-MTCO3P38 in human normal liver cell line L-02 and various hepatoma cell lines was detected,either.Kaplan-Meier assays was performed to analyze the relationship between the expression of miR-MTCO3P38 and the overall survival of HCC patients.Single multi-factors combined with COX regression model were used to explore independent risk factors for HCC prognosis.The ROC curve was used to calculate the Youden index of miR-MTCO3P38 as a diagnostic markers in HCC.Transwell and cell scratch repair assays were performed to analyze the effect of miR-MTCO3P38 on the invasion and migration of hepatoma cells.Results The expression of miR-MTCO3P38 was significantly down-regulated in HCC tissues and liver cancer cells.The 3-year overall survival rate of HCC patients with high miR-MTCO3P38 expression was significantly higher than the patients with low miR-MTCO3P38 expression(p<0.01).miR-MTCO3P38 is an independent protective factor affecting prognosis of HCC patients(p<0.05),and its expression is closely related to HBV infection,tumor number,PVTT and BCLC staging in HCC patients(p<0.05).In vitro experiments showed that miR-MTCO3P38 can inhibit the invasion and migration of liver cancer cells.Conclusion miR-MTCO3P38 acts as a inhibitor in the development and metastasis of HCC,and it has the potential to be a warning indicator for prognosis of HCC.Part ? miR-MTCO3P38 inhibits invasion and migration of hepatoma cells via down-regulating TMOD1/MMP13 pathway by binding to TMOD1 m RNA 3'UTR Objective To found the targeted gene of miR-MTCO3P38.To investigate the molecular mechanism of miR-MTCO3P38 acts as a inhibitor in invasion and migration of HCC cells.Methods Gene expression profiles and targeted gene prediction software were used to find the potential targeted gene of miR-MTCO3P38(TMOD1);RT-PCR was performed to detect the expression of TMOD1 in miR-MTCO3P38 over expression hepatoma cells;luciferase reporter assay was used to verify the binding sites between miR-MTCO3P38 and TMOD1.Western blotting was performed to detect the expression of MMPs family in miR-MTCO3P38 over expression hepatoma cells.Rescue experiments confirmed whether miR-MTCO3P38 regulates downstream pathways via TMOD1.Results TMOD1 was significantly down-regulated in miR-MTCO3P38 over expression hepatoma cells detected by the gene expression profiles(FDR<0.05)and RT-PCR(p<0.01).TMOD1 is a downstream targeting gene of miR-MTCO3P38.miR-MTCO3P38 directly targets on TMOD1 m RNA 3'UTR.Over expression of TMOD1 can rescue the inhibition of invasion and migration in liver cancer cells and down-regulation of MMP13 protein caused by miR-MTCO3P38 over expression.Conclusion miR-MTCO3P38 inhibits the migration and invasion of hepatocellular carcinoma cells via the TMOD1/MMP13 pathway.miR-MTCO3P38 is a potential therapeutic target for against HCC metastasis.