| Objective:Hepatocellular carcinoma(HCC)is one of the most common malignancies worldwide and the second leading cause of cancer-related death.Despite recent progress in the development of therapeutic strategies,including surgical resection,liver transplantation,radiofrequency ablation,interventional therapy and drug targeted therapy,the overall prognosis of most HCC patients remains poor because they are typically diagnosed at advanced stages of the disease with large recurrence rate and metastasis rate.So it is of great significance to explore the pathogenesis of HCC and find new therapeutic targets and prognostic biomarkers.Circular RNA is a type of closed endogenous RNA that is covalently bound.Recent studies have shown thatcircular RNA plays an important role in the development of many human cancers.We screened aberrantly expressed circular RNAs by high-throughput sequencing.We found that circ-BIRC6 was overexpressed in HCC tissues compared to adjacent normal tissues by qRT-PCR.The aim of this study was to investigate the role of circ-BIRC6 in the development and progression of HCC and related molecular mechanisms.Methods:1.High-throughput sequencing technology was used to screen for aberrantly expressed circular RNA in HCC tissues and adjacent normal tissues.We detected the expression level of circ-BIRC6 in HCC tissues and adjacent normal tissues by qRT-PCR.We also testified the circ-BIRC6 expression in tumor tissues from different TNM stages and presence or absence of vascular invasion.In vitro experiment,we detected the expression of circ-BIRC6 in HCC cell lines(HepG2,Bel-7402,SMMC-7721,Huh7 cells)and normal liver cell line L02.We analyzed the relationship between circ-B1RC6 expression in tumor tissues and clinical pathological factors(age,gender,serum alpha-fetoprotein,tumor size.-TNM stages,presence or absence of vascular invasion).We assessed whether circ-BIRC6 is an independent risk factor for the prognosis of patients with HCC by univariate and multivariate Cox regression analysis.We analyzed the correlation between circ-BIRC6 expression and postoperative survival time of HCC patients by Kaplan-Meier survival curves.2.HCC cell lines HepG2 and Bel-7402 were transfected with small interfering RNA(si-circ-B1RC6)and overexpressing plasmid(oe-circ-BIRC6).respectivelhy.The transfection efficiency was detected by qRT-PCR.We detected the effects of circ-BIRC6 knockdown and overexpression on proliferation,apoptosis and invasion and miration of HCC by CCK-8 assays,flow cytometry analysis.and transwell experiments,respectively.3.We found the miR-3918 was a potential target of circ-BIRC6 by bioinformatics analysis.Fluorescence in situ hybridization(FISH)was used to detect the localization of circ-BIRC6 and miR-3918 in cells.We detected the interactive relationship between circ-BIRC6 and miR-3918 by dual luciferase reporter assay and RNA-binding proteins immunoprecipitation experiments.By qRT-PCR.we detected the expression of miR-3918 in HCC tissues and normal tissues.We also analyzed the linear correlation between circ-BIRC6 expression and miR-3918 expression in tumor tissues.4.HepG2 and Bel7402 cells were transfected with miR-3918 inhibitor and miR-3918 mimics,respectively.The transfection efficiency was detected by qRT-PCR.We detected the proliferation,invasion,migration and apoptosis in HepG2 and Bel7402 cells transfected with miR-3918 inhibitor and miR-3918 mimics by CCK-8 assays,transwell experiments and flow cytometry analysis.5.Bv bioinformatics analysis,we found that the Bcl2 is a potential downstream target gene of miR-3918.We testified the interactive relationship between miR-3918 and Bcl2 by dual luciferase reporter assay.By qRT-PCR and western blotting experiments,we detected the expression level of Bcl2 mRNA and protein in HepG2 and Bel7402 cells after knockdown.overexpression of circ-BIRC6 and inhibition,overexpression of miR-3918,respectively.We also analyzed the linear correlation between Bcl2 mRNA and miR-3918,Bcl2 mRNA and circ-BIRC6 in HCC tissues.6.We further verify the function of circ-BIRC6 in HCC by rescue experiments.HepG2 and Bel7402 cells were transfected with si-NC,si-circ-BIRC6,the mixture of si-circ-BIRC6 and miR-3918 inhibitor,respectively.By CCK-8,qRT-PCR,flow cytometry,transwell and western blotting experiments,we detected the variation of proliferation,apoptosis,invasion and migration in HepG2 and Bel7402 cells.7.In vivo experiments:Subcutaneous tumor formation in nude mice further verified the effect of circular RNA circ-BIRC6 deletion and overexpression on hepatocellular carcinoma progressionResults:1.The expression of circ-BIRC6 in tumor tissues is higher than that in normal tissues;the expression level of circ-BIRC6 in HCC tissues is related to the TNM stages and presence or absence of vascular invasion.Circ-BIRC6 expression is obviously higher in HCC cell lines(HepG2,Bel7402,SMMC7721,HuH7)than normal cell L02.The results of correlation between circ-BIRC6 expression and clinical pathological factors showed that the expression of circ-BIRC6 was correlated with TNM stages(P=0.0143),with or without vascular invasion(P=0.0151).Univariate and multivariate Cox regression analysis indicated that the expression level of circ-BIRC6 was an independent risk factor in the prognosis of patients with HCC.Survival analysis showed that patients with high expression of circ-BIRC6 had a poor prognosis compared with patients with low expression of circ-BIRC6(The median value of circ-BIRC6 expression is used as the demarcation point).2.Functional experiments showed that circ-BIRC6 knockdown significantly inhibited the proliferation,promoted the apoptosis and reduced the migration and invasion in HepG2 and Bel7402 cells compared with the normal control(NC)group.Hoverer,over-expression of circ-BIRC6 increased the proliferation,decreased the apoptosis and promoted the invasion and migration.3.FISH experiments showed that circ-BIRC6 and miR-3918 were primarily co-localized in the cytoplasm of HepG2 cells.The dual luciferase report assays revealed that miR-3918 mimics suppressed luciferase activity in 293T cells transfected with the wide type(WT)circ-BIRC6 sequence,while mutation of the predicted circ-BIRC6 binding site abrogated this effect.RIP assays verified that circ-BIRC6 is combined with miR-3918.qRT-PCR results showed that the expression of miR-3918 was increased in HepG2 and Bel7402 cell after circ-BIRC6 knockdown.and the expression of miR-3918 in HCC tissues is significantly lower than that of normal tissues.Linear correlation analysis also indicated that the expression of miR-3918 and circ-BIRC6 was negatively correlated in HCC tissues.4.Overexpression of miR-3918 reduced the proliferation.invasion,migration and promoted the apoptosis in HepG2 and Be17402 cells compared with the normal control(NC)group.However,inhibition of miR-3918 increased the proliferation,invasion,migration and inhibited the apoptosis in HepG2 and Be17402 cells compared with the normal control(NC)group.5.miR-3918 overexpression reduced the luciferase activity of the WT Bcl2 3'UTR in 293T cells but had no effect when the predicted binding site was mutated by the dual lucifeise reporter assay.miR-3918 overexpression and circ-BIRC6 silencing reduced Bcl2 mRNA and protein expression levels in HepG2 and Bel-7402 cells.as determined by qRT-PCR and western blotting,respectively.Conversely.miR-3918 inhibition and circ-BIRC6 overexpression had the opposite effects.In addition,Bcl2 mRNA expression was negatively correlated with that of miR-3918 and positively correlated with that of circ-BIRC6 in HCC tissues6.miR-3918 inhibition increased cell proliferation in circ-BIRC6-depleted HepG2 and Bel7402 cells.miR-3918 inhibition decreased the expression of miR-3918:this was accompanied by an upregulation on mRNA and protein level of Bcl2.Moreover.miR-3918 inhibition abolished the effects of circ-BIRC6 knockdown on cell migration,invasion,and apoptosis.7.The results of tumor formation in nude mice showed that the volume and weight of tumors were significantly decreased in the nude mice that inoculated by Bel7402 cells transfected with shRNA-circ-BIRC6 compared with the control group Circ-BIRC6 expression was downregulated in sh-Circ-BIRC6-derived tumor tissues while miR-3918 was upregulated.Furthermore,circ-BIRC6 deletion decreased the protein expression of Bcl2.However,the stable overexpression of circ-BIRC6 produced the opposite effect on HCC progression.Conclusion:1.The expression of circ-BIRC6 is higher in HCC tissues than that in normal tissues;the expression level of circ-BIRC6 is significantly associated with TNM stages and vascular invasion.The high expression of circ-BIRC6 has an adverse effect on the prognosis of the patients with HCC.The circ-BIRC6 expression is an independent risk factor of prognosis of patients with HCC.2.The expression of circ-BIRC6 can promote the proliferation,invasion and migration and inhibit apoptosis in HCC;3.In terms of mechanism,circ-BIRC6 functions as a competing endogenous RNA that regulates Bcl2 expression by sponging miR-3918.Therefore,circ-BIRC6 may serve as a prognostic biomarker and therapeutic target of HCC.Significance:This study investigated the aberrant expression of circ-BIRC6 in HCC:tissues and analyzed the relationship between the expression of circ-BIRC6 and prognosis of patients with HCC,clinicopathological factors.We further studied the effect of circ-BIRC6 on the proliferation,invasion,migration and apoptosis in HCC and its molecular mechanism,which provided the experimental and theoretical basis for the prognosis and targeted treatment for the patients with HCC. |
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