Functional Characterization Of Long Non-coding RNA CRNDE In Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)occurs speedily,has high degree malignant and the patients have poor prognosis.Though,the technique of HCC treatment is advanced,there are about 750 thousands of people died each year.So,it is very necessary to reveal the potential pathogenesis of HCC for improving the treatment efficiency.In recent years,accumulating studies have found that a variety of long chain noncoding RNAs(lncRNAs)expression are imbalanced in HCC.LncRNA-CRNDE was found elevated in HCC.But the functional characterization of CRNDE is unclear at present.Objective:1.To investigate the function of CRNDE in HCC.2.To clarify the relationship between CRNDE and its adjacent gene IRX5 and its biological function.3.To clarify the mechanism of CRNDE regulating IRX5 to play the biology role.Methods:Part one: Explore the expression and biological function of CRNDE in HCC1.Fluorescence in situ hybridization was used to detect CRNDE localization.2.We testified CRNDE expression in HCC tissues and HCC cell lines by qRT-PCR.3.HCC cells were transfected with CRNDE plasmids and infected with sh-CRNDE virus.The proliferation of HCC cells was analyzed by colony-formation assay and MTS assay.The migration and invasion was evaluated by wound-healing and Transwell assay.4.Stable CRNDE overexpression and interference cells were constructed.Tumor xenograft models were used to verify the growth of HCC cells.5.Orthotopic tumor models and HE staining were used to verify the metastasis of HCC cells in the liver of mice.Part two: Investigate the relationship between CRNDE and its adjacent gene IRX5 and its biological function1.HCC cells were transfected with CRNDE plasmids and infected with sh-CRNDE virus.IRX5 mRNA expression was testified with qRT-PCR.IRX5 protein expression was testified by western blot.2.In the liver and lung tissues of nude mice metastasis model which was induced by CRNDE gene,immunohistochemical assay(IHC)was used to detect IRX5 protein.3.IRX5 mRNA in HCC tissues and HCC cell lines were testified by qRT-PCR.4.IRX5 protein in HCC tissues and HCC cell lines were testified by western blot.5.HCC cells were transfected with pcDNA3.1-IRX5 plasmids,infected with sh-IRX5 virus.The proliferations of HCC cells were analyzed by colony-formation assay and MTS assay.The migration and invasion were evaluated by transwell assay and wound-healing assay.Part three : Investigate the molecular mechanism of CRNDE regulating IRX51.MiRNAs combined with CRNDE and IRX5-3'UTR were predicted with biological software.HCC cells were transfected with pcDNA3.1-CRNDE plasmids.MiRNAs were tested by qRT-PCR.2.HCC cells were transfected with CRNDE plasmids and infected with sh-CRNDE virus.MiR-136-5p expression was detected by qRT-PCR.3.HCC cells were transfected with miR-136-5p mimic and mi R-136-5p-inhibitor.CRNDE expression was checked by qRT-PCR.4.The expressions of miR-136-5p in HCC tissues and HCC cell lines were detected by qRT-PCR.5.HCC cells were transfected with mi R-136-5p mimic and miR-136-5p inhibitor.The proliferations of HCC cells were analyzed by colony-formation assay and MTS assay.Migration and invasion were evaluated through transwell assay and wound-healing assay.6.HCC cells were co-transfected with miR-136-5p mimic and pcDNA3.1-CRNDE plasmids.The proliferations of HCC cells were analyzed by colony-formation assay and MTS assay.The migration and invasion were evaluated by wound-healing assay and Transwell assay.7.293 T and SMMC7721 cells were co-transfected with miR-136-5p mimic and PGL3-CRNDE.The luciferase activities were measured by Dual-Luciferase reporter assay.8.HCC cells were transfected with biotinylated miR-136-5p-inhibitor, the amount of CRNDE bound to AGO2 were detected through RIP.9.HCC cells were co-transfected with miR-136-5p mimic and mi R-136-5p-inhibitor.IRX5 mRNA was tested by qRT-PCR.IRX5 protein was tested by western blot.10.293 T and SMMC7721 cells were co-transfected with miR-136-5p mimic and PGL3-IRX5-3'UTR.The luciferase activities were measured by Dual-Luciferase reporter assay.11.HCC cells were co-transfected with pcDNA3.1-CRNDE and mi R-136-5p-inhibitor,or co-transfected with pcDNA3.1-CRNDE and mi R-136-5p mimic.IRX5 mRNA was tested by qRT-PCR.IRX5 protein level was tested by western blot.Results:Part one: Explore the expression and biological function of CRNDE in HCC1.CRNDE was localized in the nucleus of HCC cells.2.CRNDE were elevated in HCC tissues and HCC cell lines.3.Overexpression of CRNDE enhanced the function of HCC cells.Interference of CRNDE decreased its function.4.CRNDE overexpression promoted the growth of HCC cells in nude mice by subcutaneous colonies tumor assay.Interference of CRNDE decreased its growth.5.CRNDE overexpression promoted metastasis of HCC cells in liver and lung of nude mice by orthotopic tumor models and HE staining.Interference of CRNDE decreased its metastasis.Part two: Investigate the relationship between CRNDE and its adjacent gene IRX5 and its biological function.1.CRNDE overexpression increased the expression of IRX5 mRNA and protein.Interference of CRNDE decreased its expression.2.In the liver and lung tissues of nude mice metastasis model which was induced by CRNDE gene,IRX5 protein expression was increased.3.IRX5 mRNA was elevated in HCC tissues and HCC cell lines.It was positively correlated with CRNDE.4.IRX5 protein was elevated in HCC tissues and HCC cell lines.5.Ectopic IRX5 expression enhanced the function of HCC cells.Interference of CRNDE supressed its function.Part three: Investigate the molecular mechanism of CRNDE regulating IRX51.Eight miRNAs were predicted by biological software.MiR-136-5p was especially downregulated(P<0.01),other miRNAs had no difference.2.CRNDE overexpression decreased the expression of MiR-136-5p.Interference of CRNDE upregulated its expression.3.MiR-136-5p mimic inhibited CRNDE expression.Interference of mi R-136-5p elevated the expression of CRNDE.4.MiR-136-5p was downregulated in HCC tissues and HCC cell lines.5.MiR-136-5p mimic repressed the function of HCC cells.MiR-136-5p inhibitor promoted its function.6.MiR-136-5p suppressed the function of CRNDE.7.MiR-136-5p inhibited the luciferase activity of PGL3-CRNDE,but there was no effect on PGL3-CRNDE-Mut.8.AGO2 antibody could enrichment AGO2 protein by RIP assay.But there was no AGO2 protein in IgG group.The enrichment of CRNDE was more in NC group than Ig G antibody group.HCC cells were transfected with miR-136-5p inhibitor,the enrichment of CRNDE was obviously decreased.9.MiR-136-5p mimic inhibited the expression of IRX5 mRNA and protein.MiR-136-5p inhibitor elevated the expression of IRX5 mRNA and protein.10.MiR-136-5p inhibited the luciferase activity of PGL3-IRX5-3'UTR,but there was no effect on PGL3-IRX5-3'UTR-Mut.11.MiR-136-5p mimic inhibited the IRX5 mRNA and protein level which was induced by CRNDE.Conclusion:In the study,CRNDE and its nearby gene IRX5 was elevated in HCC.CRNDE and IRX5 enhanced the function of HCC cells.MiR-136-5p was downregulated in HCC.MiR-136-5p suppressed the function of HCC.CRNDE and miR-136-5p was reciprocal repression.MiR-136-5p was directly combined to CRNDE.CRNDE was interacted with miR-136-5p in the same RISC complex through AGO2 protein.MiR-136-5p was directly targeted to IRX5.MiR-136-5p was involved in the CRNDE regulated in IRX5.CRNDE may as the endogenous RNA of miR-136-5p to regulate IRX5 and enhance the function of HCC.