The Role Of MicroRNA-214/Caspase-1 Signaling Pathway In The Regulation Of Hepatocellular Carcinoma Growth And Metastasis

Objective: Liver cancer is one of the main causes of cancer-related death in the world.Hepatocellular carcinoma(HCC)is the most common type of liver cancer with high incidence rate and poor prognosis.In recent years,various methods have been applied to the clinical treatment of liver cancer with the development of medicine,but the overall survival rate of patients has not been significantly improved.Therefore,it is very important to investigate the molecular mechanism of HCC development and find new therapeutic targets.Caspase-1 is the central enzyme of inflammatory corpuscles.Active caspase-1 stimulates proinflammatory cytokines by inhibiting the expression of IL-18 and IL-1 β.This proinflammatory microenvironment eventually leads to tumor expansion,including induction,progression,deterioration,invasion and metastasis.MicroRNAs(miRNAs)are short and endogenous,non coding RNAs that control gene expression by recognizing and attaching to the 3 ’-untranslated region(3’-UTR)of the target genes,inducing m RNA degradation or inhibiting its protein translation.MiRNAs play key roles in many tumors,however,the role of miRNAs in liver cancer is not completely clear.In this study,we investigated the expression of miR-214 in HCC,and confirm the effect of miR-214 on HCC growth,invasion and metastasis.Furthermore,we investigate the molecular mechanism of mir-214 regulating the development of liver cancer.Methods: Firstly,the expression data of miRNAs in tumor and paracancer tissues were obtained from GEO and TCGA database,and the differential expression of miR-214 was analyzed.Secondly,the expression of miR-214 in human hepatocellular carcinoma cell lines SMMC-7721,Hep G2,Hu H-6 and human normal liver cell line L-02 was detected by real-time quantitative PCR(q RT-PCR).Thirdly,miR-214 mimics,miR-214 inhibitor and miR-214 control were transfected into human hepatocellular carcinoma cell line SMMC-7721 and the transfection efficiency was detected by q RT-PCR.The proliferation of hepatocellular carcinoma cells was detected by CCK8 assay and colony formation assay.Transwell invasion assay and Wound-healing assay were used to detect the metastasis of hepatocellular carcinoma cells.Then,we employed Target Scan to search for the potential targets of miR-214 and identified caspase-1 as a possible target of miR-214.Furthermore,the Dual-Luciferase assay was used to detect the effect of miR-214 on Caspase-1 3’-UTR reporter luciferase activity.The expression of Caspase-1 was detected by QRT PCR and Western blot.Finally,we investigate if miR-214 influence HCC development by controlling caspase-1 expression through Western blot assay,CCK8 assay,colony formation assay,Wound-healing assay,Transwell assay and xenograft model in vitro and in vivo.Results: Expression level of miR-214 is downregulated in HCC.The results of GEO and TCGA database analysis showed that the expression of mir-214 in liver cancer tissues was significantly decreased compared with normal liver tissues.Similarly,q RT-PCR results showed that the expression levels of mir-214 in human liver cancer cell lines(SMMC-7721,Hep G2 and huh-6)were significantly lower than those in normal liver cell lines(L-02).MiR-214 inhibits proliferation and metastasis of HCC cells.CCK8 assay and colony formation assay showed that the overexpression of mir-214 significantly inhibited the proliferation and cell cloning formation of HCC cells,while the specific inhibition of mir-214 showed the opposite effect.Transwell invasion assay and Wound-healing assay showed that the overexpression of mir-214 significantly inhibited the invasion and metastasis of HCC cells,while the specific inhibition of mir-214 showed the opposite effect.MiR-214 targets Caspase-1 and suppresses its expression.The results of Target Scan analysis showed that Caspase-1 was a possible target of miR-214.The Dual-Luciferase assay showed that relative luciferase activity of Caspase-1 3’-UTR reporter was significantly decreased by miR-214 overexpression.The results of q RT-PCR and Western blot assay showed that the overexpression of miR-214 significantly down-regulated the expression of Caspase-1,while the specific inhibition of miR-214 showed the opposite effect.Mi R-214 inhibits the proliferation and metastasis of HCC through Caspase-1 in vitro and in vivo.CCK8 assay and colony formation assay showed that the overexpression of mir-214 significantly inhibited the proliferation and cell cloning formation of HCC cells,while this effect was rescued by the overexpression of Caspase-1.Transwell invasion assay and Wound-healing assay showed that the overexpression of miR-214 significantly inhibited the invasion and metastasis of HCC cells,while this effect was rescued by the overexpression of Caspase-1.The results of xenograft model showed that miR-214 significantly inhibited the growth of HCC cells,while this effect was rescued by the overexpression of Caspase-1.And the Immunohistochemical assay showed that miR-214 down-regulated the expression of Ki67,while it was reversed by Caspase-1 overexpression.Conclusion: Expression level of miR-214 is downregulated in HCC.Mi R-214 inhibits the proliferation and metastasis of HCC through regulating Caspase-1 expression,indicating that miR-214 is a potential indicator of HCC treatment and prognosis,which provides a new theoretical basis for the clinical prevention and treatment of liver cancer.