| Background:Hepatocellular carcinoma(HCC)is one of the most common cancers in the world and the third leading cause of cancer-related deaths worldwide.In recent years,its morbidity and mortality have been increasing,and it is at the forefront of malignant tumors in the world.Especially in C hina,due to the large number of people infected with hepatitis B virus,HCC patients account for more than half of the total number of HCC in the world.It is well known that surgical resection is the treatment of choice for HCC.However,in view of the concealment of HCC,high degree of malignancy and rapid disease progression,most patients have reached local later period or distant metastasis at the first visit,so surgical treatment is only used in less than 30% of patients.There is an urgent need to find new gene therapy targets and treatments.More and more studies have shown that lncRNA can participate in tumor development and drug resistance,and it is related to the prognosis of cancer patients,and it is expected to become a new diagnostic marker and therapeutic target.By the bioinformatics analysis,this experimental group revealed that lncRNA mi R143 HG was significantly down-regulated in HCC tissues.Method:1.Collect 50 cases of HCC patients with surgically resected cancer tissues and their corresponding adjacent tissues.Real-time quantitative PCR(q RT-PCR)was used to detect the gene expression level of lnc RN A mi R143 HG.These patients were divided into two groups based on the median lncRNA mi R143 HG expression.The correlation between mi R143 HG expression and prognosis,and the correlations between mi R143 HG expression and the patients’ clinicopathological characteristics— such as tumor size,number of tumors,TNM stage,and lymph node metastasis—were evaluated based on the two groups.2.The expression of lncRNA mi R143 HG was detected by q RT-PCR in normal liver cell line(LO2)and hepatoma cell line(Hep G2,Hu H7,smmc7721,Bel7402,Hep3B),and two hepatocellular carcinoma cell lines with the lowest expression were selected for subsequent experiments.3.Overexpression and knockdown of lncRNA mi R143 HG expression in cell lines by transfection.The effects of lncRNA mi R143 HG on the proliferation,apoptosis and invasion and metastasis of hepatocellular carcinoma cells and the expression of related protein were observed by CCK8 assay,Ed U,plate cloning,flow cytometry,Transwell chamber and Western blot.4.Using bioinformatics software to predict target genes,using luciferase reporter experiments,q PCR and Western blot techniques to verify the relationship between lncRNA mi R143 HG and mi R155,Wnt,MAPK.Result:1.By q PCR verification,it was found that lnc RN A mi R143 HG expression was markedly downregulated in HCC tissues and cells and its expression was associated with the presence or absence of portal vein tumor thrombus(PVTT),hepatitis B virus(HBV)infection,relapse and metastasis,and Barcelona C linic Liver Cancer(BCLC)stage.Additionally,mi R143 HG expression predicted a good prognosis and acted as an independent prognostic factor in HCC for overall survival(OS).Compared with the LO2 hepatocyte cell line,lncRNA mi R143 HG showed low expression in HCC cell lines,among which the expression level was the lowest in smmc and Hu H7 cell lines,so the two HCC cell lines were used in subsequent experiments.After overexpression of lncRNA mi R143 HG,the expression of lncRNA mi R143 HG in smmc and Hu H7 cells was significantly up-regulated;the results of CCK8 and Ed U showed that compared with the negative control group,the cell proliferation ability of the experimental group was significantly reduced;G2/M phase arrest was observed after up-regulation of lncRNA mi R143 HG.Flow cytometry showed that the apoptosis rate of HCC cells up-regulated lncRNA mi R143 HG expression was significantly higher than that of the negative control group.The results of transwell chamber showed the invasion and migration ability of the experimental group was significantly lower than the negative control group.Western blot showed that the expression levels of caspase3,cleave-caspase3,Bax and E-cadherin in HCC cell lines were up-regulated lncRNA mi R143 HG,and the expression levels of bcl-xl,CDK1,C yclin B1,MMP2 and MMP9 were decreased in HCC cell lines down-regulated lncRNA mi R143 HG,the opposite result was observed.Consistently,the depletion of mi R143 HG resulted in the opposite phenomenon of the aforementioned resultsBy bioinformatics analysis,luciferase reporter and q PC R experiments,it was found that lncRNA mi R143 HG inhibits mi R-155 expression,while mi R-155 directly targets APC which is a negative regulator of the Wnt/β-catenin pathway,so we can speculate that lncRNA mi R143 HG can acts on the Wnt pathway.In our study,mi R143 HG is further found to reduce the expression of β-catenin and block the nuclear accumulation of β-catenin,ultimately inhibit the activation of Wnt pathway.It inhibits the expression of Wnt downstream target gene ZEB1,and then E-cadherin expression is increased and cell motility is inhibited.5.Western blot confirmed that mi R143 HG exerts its anti-proliferative effect by affecting the MAPK signaling pathway and then inducing G2/M phase arrest in HCC cel.Conclusion:1.Lnc RN A mi R143 HG is significantly downregulated in HCC tissues and cell lines,and its expression is related to the status of PVTT,BCLC,HBV infection,recurrence and metastasis.In addition,lncRNA mi R143 HG expression was well correlated with good prognosis and served as an independent prognostic factor for OS.2.LncRNA mi R143 HG expression is associated with proliferation,invasion,metastasis and apoptosis of HCC cells,and ma y play a role as a tumor suppressor gene in HCC.3.This study demonstrates that lnc RN A mi R143 HG plays a key role in the development and progression of HCC by inhibiting MAPK and Wnt signaling pathways. |
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