Long Noncoding RNA MIR31HG Inhibits Hepatocellular Carcinoma Proliferation And Metastasis By Sponging Microrna-575 To Upregulate ST7L Expression

Primary liver cancer is one of the most common malignant tumors in humans.At present,hepatocellular carcinoma(HCC)is a kind of the most common liver cancer.Liver transplantation and hepatectomy are the main treatments for HCC,but the effect is still poor.Therefore,it is important to reveal the molecular mechanism of the occurrence and development of HCC and to find targets for early diagnosis or precise treatment of HCC.More and more studies have shown that long non-coding RNA(lncRNA)plays an important role in the pathogenesisand development of tumor.Current studies have shown that MIR31 HG has abnormal expression and played an important regulatory role in several cancers,but its expression and mechanism in HCC are still unknown.Therefore,the purpose of this study is to explore the expression of MIR31 HG in human HCC tissues and its relationship with clinical related indicators,and to reveal its potential significance in the diagnosis and treatment of HCC;to explore the effects of MIR31 HG on the biological function of HCC cells in vitro,and to study its specific molecular mechanism;finally,to further verify the function and molecular mechanism of MIR31 HG in HCC by in vivo study.This study includes three parts:Part ? Expression of MIR31 HG in human hepatocellular carcinoma cell line and hepatocellular carcinoma tissue samples and its effect on biological function of hepatocellular carcinoma cells Objective: To detect the expression of lncRNA-MIR31 HG in HCC cells and HCC tissues,and to study the relationship between MIR31 HG and clinicopathological features of HCC patients,so as to prove the effect of MIR31 HG on the biological function of HCC cells.Method: 1.The expression of MIR31 HG in human normal liver cell line and HCC cell line,as well as in adjacent normal liver tissue and HCC tissue was detected by quantitative real-time PCR(qRT-PCR).2.The relationship between the clinicopathological data of 42 patients with HCC and the expression of MIR31 HG in HCC was statistically analyzed.3.The localization of MIR31 HG in HCC cells was detected by RNA nucleoplasmic separation technique.4.The MTS assay and clone formation assay were used to detect the effect of MIR31 HG overexpression or interference on the proliferation of HCC cells.The wound-healing assay and transwell assay were used to detect the ability of migration and invasion respectively.Result: 1.The expression of MIR31 HG was downregulated in HCC cell lines and HCC tissues.2.In HCC tissues,the expression of MIR31 HG was correlated with tumor size(P=0.0268),number of nodules(P=0.0477),vascular invasion(P=0.0332)and TNM stage(P=0.0308),but not correlated with patient's age,sex,hepatitis B surface antigen and so on(P>0.05).3.The expression of MIR31 HG was mainly localized in the cytoplasm of HCC cells.4.Overexpression of MIR31 HG inhibited the proliferation,migration and invasion of HCC cells,whereas knockdown of MIR31 HG had the opposite effects.Conclusion: The expression of MIR31 HG was downregulated in both HCC cell lines and HCC tissues.The low expression of MIR31 HG in HCC patients indicated worse clinical prognosis,and MIR31 HG could inhibit the proliferation and metastasis of HCC cells.Part ? The mechanism of MIR31 HG on the biological function of hepatocellular carcinoma cells Objective: To investigate the molecular mechanism of MIR31 HG affecting the biological function of HCC cells.Method: 1.Bioinformatics analysis of microRNA(miRNA)was used to find which miRNA might bind to MIR31 HG.2.QRT-PCR was performed to detect the expression of miR-575 in HCC cells after overexpression or knockdown of MIR31 HG,and the expression of miR-575 in HCC cells after overexpression or interference of MIR31 HG.3.The luciferase reporter gene assay was used to detect the binding of MIR31 HG to miR-575.4.Further detection of the correlation between MIR31 HG and miR-575 by RIP experiment.5.QRT-PCR was used to detect the expression of miR-575 in human normal hepatocyte lines and HCC cell lines,as well as in HCC tissues and adjacent normal liver tissues,and the correlation between MIR31 HG and miR-575 expression in HCC tissues.6.The MTS assay and clone formation assay were used to detect the effects of overexpression or knockdown of miR-575 on the proliferation of HCC cells.The wound-healing assay and transwell assay were used to detect the effects of overexpression or knockdown of miR-575 on the migration and invasion of HCC cells.7.The bioinformatics analysis was used to find target genes that might bind to miRNAs.8.QRT-PCR and western blot were used to detect the expression of ST7 L in HCC cells after overexpression or knockdown of MIR31 HG,and the expression of ST7 L in cells after overexpression or knockdown of miR-575.9.Luciferase reporter gene assay was used to detect the binding of miR-575 to ST7 L 3'UTR.10.QRT-PCR and western blot were used to detect the difference of ST7 L expression in human normal hepatocyte lines and HCC cell lines,as well as in human HCC tissues and adjacent normal liver tissues,and the correlation between MIR31 HG and ST7 L,and between miR-575 and ST7 L expression in HCC tissues.11.The MTS assay and clone formation assay were used to detect the effects of MIR31 HG and miR-575 overexpression and interfering plasmid co-transfection on the proliferation of HCC cells.The wound-healing assay and transwell assay were used to detect the effects of MIR31 HG and miR-575 overexpression and interfering plasmid co-transfection on the migration and invasion of HCC cells.12.The expression of ST7 L was detected by qRT-PCR and western blot after co-transfection of MIR31 HG and miR-575 overexpression and interfering plasmid.Result: 1.The biosoftware predicted 10 miRNAs,of which miR-575 was most significantly downregulated after overexpression of MIR31 HG.2.MIR31 HG and miR-575 could negatively regulate the expression of each other.3.The luciferase assay indicated that MIR31 HG could directly regulate the expression of miR-575.4.The RIP results showed that the expression of MIR31 HG precipitated by Ago2 antibody group was significantly higher than that of IgG control group,which further demonstrated that MIR31 HG could directly target the binding of miR-575.5.The expression of miR-575 was upregulated in HCC lines and HCC tissues,and was negatively correlated with the expression of MIR31 HG in HCC tissues.6.Overexpression of miR-575 promoted the proliferation,migration and invasion of HCC cells,whereas knockdown of miR-575 had the opposite effects.7.The biosoftware predicted eight genes,of which ST7 L was most significantly upregulated after MIR31 HG overexpression.8.QRT-PCR and western blot showed that MIR31 HG could positively regulate the expression of ST7 L,while miR-575 could negatively regulate the expression of ST7 L.9.The luciferase assay confirmed that ST7 L was a target gene of miR-575.10.Expression of ST7 L was up-regulated in HCC cell lines and HCC tissues,and positively correlated with the expression of MIR31 HG in HCC tissues,and negatively correlated with the expression of miR-575 in HCC tissues.11.pcDNA3.1-MIR31 HG could antagonize the proliferation,migration and invasion of HCC cells promoted by pre-miR-575,and enhance the inhibition of anti-miR-575 on the proliferation,migration and invasion of HCC cells,while sh-MIR31 HG has the opposite effect.12.pcDNA3.1-MIR31 HG could antagonize the inhibition of pre-miR-575 on ST7 L and enhanced the promotion of anti-miR-575 on ST7 L,whereas sh-MIR31 HG showed the opposite effect.Conclusion: MIR31 HG could act as a molecular sponge of miR-575,downregulate the expression of miR-575,and upregulate the expression of ST7 L through miR-575,thus inhibiting the proliferation and metastasis of HCC cells.Part ? Effect of MIR31 HG on proliferation and metastasis of hepatocellular carcinoma cells in nude mice Objective: To further verify the effect of MIR31 HG on the biological function of HCC cells and its mechanism in vivo.Method: 1.Stable cells overexpressed and interfered with MIR31 HG were injected subcutaneously or into the liver to establish subcutaneous tumorigenesis and lung metastasis model of nude mice.2.In the subcutaneous tumor group,the volume and weight of subcutaneous tumor were recorded every week.After 35 days,the nude mice were killed and the expressions of miR-575,ST7 L and Ki-67 were detected by qRT-PCR,western blot and immunohistochemistry.3.56 days later,the nude mice in the metastasis group were killed.After the liver and lung tissues were obtained,the number of nodules on the surface of the nude mice were counted and analyzed by HE staining.Result: 1.The results of subcutaneous tumors experiment group showed that the volume and weight of the tumors in MIR31 HG overexpression group were significantly smaller than those in the normal control group,and the expressions of miR-575 and Ki-67 in the tumorigenesis of MIR31 HG overexpression group were significantly downregulated,while the expression of ST7 L was significantly upregulated,whereas,the MIR31 HG interference group showed the opposite results.2.The experimental group of lung metastasis showed that compared with the normal control group,the liver and lung metastases in the MIR31 HG overexpression group were significantly less than those in the control group,while the MIR31 HG interference group showed the opposite results.Conclusion: MIR31 HG could significantly inhibit the growth and metastasis of HCC cells in vivo.It is further confirmed that MIR31 HG could act as a molecular sponge of miR-575 to interfere with the expression of miR-575 and upregulate the expression of ST7 L after transcription.Our data provided a new evidence for HCC targeted therapy of MIR31 HG.