| Objective: Hepatocellular carcinoma(HCC)is still one of the common tumors that threaten human health.The preliminary data of this study showed that LINC01268 was significantly overexpressed in liver cancer cell lines,and the specific mechanism of action was unclear.With the implementation of various techniques from molecular cell biology,clinical samples and animal models,1).the expression of LINC01268 in HCC tissues and cells will be detected,2).the effects of LINC01268 on the biological effects of HCC and MAPK pathway through MAP3K7 will be analyzed,3).the effects of LINC01268 on the regulation of HCC cell transcriptome and its role in the pathogenesis of HCC in mouse models will be investigated.Methods : 1.Bioinformatics analysis: The gene expression data and clinical information data downloaded from TCGA database were used to analyze the differential expression of LINC01268 in HCC tissues and adjacent tissues,and to perform its clinical correlation analysis,prognosis analysis,functional enrichment analysis,etc.The target gene MAP3K7 of LINC01268 screened out by GEPIA database was used to analyze the differential expression of MAP3K7 in HCC tissue and adjacent tissue,and to perform clinical correlation analysis,prognosis analysis,and functional enrichment analysis,etc.The correlation between LINC01268 and MAP3K7,and the active pathway were analyzed.2.Experiments on cells and human tissue samples: The main research objects are normal hepatocyte LO2,liver cancer cells(Huh7,Hep G2,Hep3B),HCC tissue and adjacent tissue.The differential expression of LINC01268 between normal hepatocytes and hepatoma cells was verified by real-time PCR.The differential expression and localization of LINC01268 were verified by cell in situ hybridization and tissue in situ hybridization.Differential expression of target gene MAP3K7 was verified by real-time PCR in normal hepatocytes and hepatoma cells.The HCC tissues and adjacent tissues of 32 patients were selected to detect the differential expressions of LINC01268 and MAP3K7 by real-time PCR,and the clinical correlations were analyzed respectively.LINC01268 was knocked down or overexpressed in liver cancer cells,and the expression changes of MAP3K7,ERK,p-ERK were verified by real-time PCR and western blot.Meanwhile,the changes of cell proliferation,apoptosis,cell cycle and other cell functions were detected.Stable overexpression of LINC01268 in liver cancer cells and knockdown of the target gene MAP3K7 were performed to detect changes in cell proliferation and apoptosis.3.Experiments in vivo : Tumors were formed subcutaneously in nude mice.The tumors size and weight were measured.The expressions of MAP3K7,ERK and p-ERK were detected by immunohistochemistry,western blot and real-time PCR.Result:1.374 HCC samples and 50 paracancerous samples were downloaded from the TCGA database,including clinical data and survival data of patients.Bioinformatics analysis found that the expression of LINC01268 in HCC tissues was significantly higher than that in paracancerous tissues.Combined with the clinical survival data of liver cancer patients,time-dependent survival analysis of LINC01268 expression was done by R language.The results showed that for liver cancer,the survival rate of the LINC01268 high expression group was lower than that of the LINC01268 low expression group along with the prolongation of time.Clinical correlation analysis showed that the differential expression of LINC01268 in stage II and stage IV of liver cancer was statistically significant.Based on R language GO database,the analysis of molecular function,cellular components and biological processes on differential expression of LINC01268 in HCC was conducted.Using GSEA software and KEGG pathway database,the active promotion pathways when LINC01268 was highly expressed were screened.2.The expression of MAP3K7 in HCC tissues was significantly higher than that in adjacent tissues by bioinformatics analysis.Based on R language GO database,the analysis of molecular function,cellular components and biological processes on differential expression of MAP3K7 in HCC was conducted.Combined with the clinical survival data of HCC patients,time-dependent survival analysis of MAP3K7 expression was done by R language.With the prolongation of time,the survival rate of the MAP3K7 high expression group was lower than that of the MAP3K7 low expression group for HCC.Clinical correlation analysis showed that the differential expression of MAP3K7 in HCC G1 and G3,G2 and G3 was statistically significant.Using GSEA software and KEGG pathway database,the active promotion pathways,including the MAPK signaling pathway,were screened out when MAP3K7 was highly expressed.3.The expression of LINC01268 in hepatoma cells was significantly higher than that in normal hepatocytes,which was verified by real-time PCR.4.Using cell in situ hybridization technology and tissue in situ hybridization technology,the experiment verified that LINC01268 is mainly located in the cytoplasm.5.The expression of MAP3K7 in hepatoma cells was significantly higher than that in normal hepatocytes by real-time PCR.6.The HCC tissues and adjacent tissues of 32 HCC patients were used to detect the differential expression of LINC01268 and MAP3K7 by real-time PCR.The results showed that the differential expressions of LINC01268 and MAP3K7 in liver cancer tissue and adjacent cancer tissues were closely related to the degree of pathological differentiation,and the expression was higher in moderately/poorly differentiated hepatocellular carcinoma tissues.7.LINC01268 was silenced in hepatoma cells,and the expression of MAP3K7 and p-ERK was lower than that in the control group when detected by real-time PCR and western blot.8.After silencing LINC01268 in hepatoma cells,the results showed that the cell proliferation ability decreased and the apoptosis increased.9.LINC01268 was overexpressed in hepatoma cells,and the expression of MAP3K7 and p-ERK increased when detected by real-time PCR and western blot.10.After overexpressing LINC01268 in hepatoma cells,the results showed that the cell proliferation ability was increased and the apoptosis was decreased.11.Compared with the control group,overexpression of LINC01268 and knockdown of MAP3K7 in hepatoma cells showed no significant difference in cell proliferation and apoptosis.12.The experiments in vivo showed that the tumor volume and weight of the LINC01268 overexpression group were significantly higher than those of the control group,and the tumor volume and weight of the LINC01268 silenced group were significantly lower than those of the control group.The results of immunohistochemistry showed that the expression of MAP3K7/p-ERK protein in the LINC01268 overexpression group was increased,and the expression of MAP3K7/p-ERK protein in the LINC01268 knockdown group was decreased.The results of western blot showed that the expressions of MAP3K7 and p-ERK in the LINC01268 overexpression group were increased,and the expressions of MAP3K7 and p-ERK in the LINC01268 knockdown group were decreased.Conclusion: 1.LINC01268 is highly expressed in HCC cells.2.The high expression of LINC01268 is positively correlated with the pathological moderately/poorly differentiation and poor prognosis of HCC patients.3.The high expression of LINC01268 regulates the target gene MAP3K7 positively,and activates the MAPK signaling pathway.It also promotes the proliferation of hepatoma cells and inhibits the apoptosis of hepatoma cells. |
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