| Background:Hepatocellular carcinoma(HCC)is a kind of primary liver cancer with high mortality.It is the second leading cause of death in the world.It is harmful to people's life and health,and brings heavy economic and mental burden to patients and their families.The early symptoms of most patients with primary liver cancer are not obvious.At the time of diagnosis,most patients have been in the middle and late stage of cancer with varying degrees of cirrhosis.Although the diagnosis and treatment of liver cancer has made great progress,the therapeutic effect and prognosis of liver cancer are still not optimistic.In a word,the high recurrence rate of liver cancer after operation or interventional treatment seriously affects the survival and prognosis of patients.It has been found that the deletion of many oncogenes,mutation of tumor suppressor genes,regulation of transcription modification and epigenetic changes lead to malignant transformation of hepatocytes,which leads to tumorigenesis.Therefore,it is of great significance to find new or further reveal the function of existing oncogenes or tumor suppressor genes for elucidating the mechanism of carcinogenesis and progression and developing potential therapeutic targets.In recent years,with the development of high-throughput sequencing technology,more and more noncoding RNA has been found and plays an important regulatory role,so the research field of noncoding RNA is still a very broad mysterious area,which has great research value.It has been reported that long-chain noncoding RNA(lncRNA)derived from noncoding region plays a very complex regulatory role in the process of tumorigenesis and development.It can participate in the process of tumorigenesis and development through the regulation of chromatin remodeling,transcriptional regulation and post transcriptional processing,but the specific role and function are still unclear.Up to now,it has been reported that many lncRNAs may have the characteristics of competitive endogenous RNA(ceRNA).Such lncRNA,as a competitive regulator of ceRNA and miRNA,miRNA,as an important regulator of post transcriptional regulation,can be adsorbed by lncRNA through"molecular sponge",thus regulating the protein expression level of downstream coding genes,and then participating in regulating many biological behaviors of tumors.LINC00460 is a transcriptional product of human chromosome 13,with a length of 935bp.At present,little is known about its function.Recent studies have shown that LINC00460 is associated with a variety of tumors.LINC00460 can be used as a cancer gene to combine miRNA through competitive binding,and up regulate the expression of downstream mRNA to promote the proliferation,invasion and migration of tumor cells.Up to now,it is still unclear whether LINC00460 participates in the process of hepatocarcinogenesis and development through the mechanism of ceRNA.We will mainly try to study the relationship between LINC00460 and hepatocellular carcinoma,and explore its role in hepatocarcinogenesis and development and related molecular mechanism.The purpose of this project is to clarify the role of LINC00460 in the progression of liver cancer,and to provide a new theoretical basis and experimental basis for the early diagnosis,treatment,efficacy evaluation and prognosis of liver cancer.Objective:1.To investigate the expression of LINC00460 in hepatocellular carcinoma and its adjacent tissues and its correlation with clinicopathological features and prognosis of patients with hepatocellular carcinoma.2.To investigate the effect of LINC00460 expression on the proliferation,migration and invasion of hepatocellular carcinoma cells.3.To explore the regulatory mechanism of LINC00460 in promoting the proliferation,migration and invasion of hepatocellular carcinoma.Method:1.The expression level of LINC00460 in 50 pairs of liver cancer tissues and adjacent tissues in the People's Hospital of Yichun City and the Third Affiliated Hospital of Nanchang University was detected by real-time fluorescent quantitative PCR(qRT-PCR),and the correlation between the expression level and the clinicopathological characteristics of patients with liver cancer was analyzed.At the same time,the basic expression of LINC00460 in hepatocellular carcinoma cell lines(HepG2,Huh7,SMMC7721,BEL-7402,HCCLM3 and SK-Hep-1)and normal human hepatoepithelial cell line LO2 was detected by qRT-PCR.Kaplan Meier and log rank methods were used to analyze the relationship between the expression level of LINC00460 and the overall survival(OS)and disease free survival(DFS)of patients with liver cancer.2.siRNA interfering with LINC00460 was transfected into HepG2 and Huh7 cells,pcDNA3.1-LINC00460 overexpression plasmid of LINC00460 was transfected into HepG2 and Huh7 cells,and the transfection efficiency of LINC00460 was verified by qRT-PCR 48 hours after transfection.The effects of LINC00460 on the proliferation,plate clone formation assay,migration and invasion of hepatoma cells were detected by CCK-8,plate clone formation assay and transwell experiments.3.qRT-PCR and FISH were used to detect the subcellular localization of LINC00460 in hepatoma cells;Miranda database was used to predict miR-342-3p as the downstream miRNA molecule of LINC00460 and double luciferase experiment was used to verify the targeted binding of LINC00460 and miR-342-3p;luciferase experiment was used to verify the targeted binding of LINC00460 and miR-342-3p;Pearson correlation analysis was used to analyze the correlation between LINC00460 and miR-342-3p;Rescue experiments verify that LINC000460 affects the biological function of hepatoma cells through miR-342-3p/AGR2 axis.In vivo,the effect of down-regulation of LINC00460 expression on tumor formation and expression of AGR2,miR-342-3p in nude mice was verified.4.SPSS21.0 statistical software and graph pad prism 7.0 software were used for statistical analysis and mapping;student's t test was used to compare the expression level of LINC00460 in hepatocellular carcinoma and adjacent tissues.?2 test was used for correlation between LINC00460 expression and clinical pathological parameters.Kaplan Meier method was used to draw the survival curve,log rank method was used to test the relationship between the level of LINC00460 in patients with liver cancer and the prognosis.The differences between the two or more groups of data were statistically analyzed by independent sample t-test or one-way ANOVA.All measurement data were expressed as meanąstandard deviation(x?s),P<0.05,indicating that the difference was statistically significant.Result:1.The results of qRT-PCR showed that the expression level of LINC00460 in hepatocellular carcinoma tissue was significantly higher than that in adjacent tissues,the difference was statistically significant(**P<0.01);the expression level of LINC00460 in HepG2,Huh7,SMMC7721,BEL-7402,HCCLM3 and SK-Hep-1was significantly higher than that in LO2,the difference was statistically significant(**P<0.01).The expression of LINC00460 was closely related to tumor size,tumor differentiation,lymph node metastasis and TNM stage(P<0.01),but not to patients'age and gender(P>0.05).2.Compared with the patients with low expression of LINC00460,the patients with high expression of LINC00460 had shorter overall survival(OS)(P=0.0419)and disease free survival(DFS)(P=0.031).3.Compared with the negative control group(NC),the number of HepG2 and Huh7 cells was significantly inhibited by the interference of LINC00460 expression,the difference was statistically significant(**P<0.01);in addition,the number of HepG2 and Huh7 cells was significantly increased after the up-regulation of LINC00460 expression,the difference was statistically significant(**P<0.01).4.The results of CCK-8 showed that after interfering with the expression of LINC00460 in HepG2 and Huh7 cells,the proliferation ability of hepatoma cells was significantly lower than that of the control group(*P<0.01);at the same time,after up regulating the overexpression of LINC00460 in HepG2 and Huh7 cells,the proliferation ability of hepatoma cells was significantly enhanced compared with the control group(**P<0.01).5.The results of transwell experiment showed that:compared with the negative control group(si-NC),the number of migration and invasion cells of HepG2 and Huh7 cells was significantly inhibited after the interference of LINC00460expression(**P<0.01),while the number of migration and invasion cells of hepatocellular carcinoma cells increased significantly after the up-regulation of LINC00460 expression in HepG2 and Huh7 cells(**P<0.01).6.The results of qRT-PCR and FISH showed that LINC00460 was mainly located in the cytoplasm of HepG2 and Huh7 cells,but its expression in the nucleus was low(*P<0.05).Miranda database predicted that miR-342-3p was a LINC00460 targeted miRNA molecule,and the overexpression of miR-342-3p significantly inhibited the luciferase activity of wild type(WT)LINC00460(**P<0.01).RNA pull down test showed that the expression of miR-342-3p was more abundant in biotin labeled LINC00460 probe(**P<0.01).7.The expression of miR-342-3p in HCC was significantly lower than that in adjacent tissues by qRT-PCR(***P<0.01);Pearson correlation analysis showed that the expression of LINC00460 in HCC was negatively correlated with miR-342-3p(R~2=0.8288,P<0.001).8.qRT-PCR showed that the over expression of miR-342-3p significantly inhibited the expression of AGR2 mRNA and protein in HepG2 and Huh7 cells,while after co-transfection of LINC00460,the expression of AGR2 mRNA and protein increased,and the inhibition of miR-342-3p on AGR2 decreased.qRT-PCR was used to detect the expression level of AGR2 in 50 pairs of clinical specimens of liver cancer and its adjacent tissues.The results showed that AGR2 was significantly up-regulated in liver cancer tissues(***P<0.01);Pearson correlation analysis showed that AGR2 and mimics were positively correlated in HCC samples(R~2=0.9187,P<0.001).9.CCK8 test showed that over expression of miR-342-3p or AGR2 gene knockout saved the proliferation effect of over expression of LINC00460 in HepG2and Huh7 cells(**P<0.01).In addition,after co-transfection with miR-342-3p or si-AGR2,the over expression of LINC00460 also inhibited the migration and invasion of HCC cells(**P<0.01).10.The results of in vivo experiments showed that compared with the control group,the tumor size and weight of nude mice were significantly reduced after LINC00460 interference(**P<0.01);after LINC00460 interference,the expression of AGR2 in HepG2 xenografts was decreased,but the expression of miR-342-3p was increased(**P<0.01).In addition,Western blot was used to detect the protein expression of AGR2 in the tumor tissues of nude mice.The results showed that the expression of AGR2 in the si-LINC00460 group was down regulated compared with that in the si-NC control group(**P<0.01).Conclusion:1.The expression level of LINC00460 in hepatocellular carcinoma tissue and cell line was significantly higher than that in adjacent tissues and normal liver epithelial cells,and the prognosis of patients with high expression of LINC00460was poor.LINC00460 could be a potential marker of poor prognosis of hepatocellular carcinoma;2.LINC00460 plays a role similar to"oncogene"in hepatocellular carcinoma,promoting the proliferation,migration and metastasis of hepatocellular carcinoma cells;3.miR-342-3p,as a downstream miRNA molecule of LINC00460,can target each other and reverse the effect of LINC00460 on the proliferation of liver cancer.4.LINC00460 can inhibit the expression of miR-342-3p by competitive binding with AGR2,so as to promote the expression level of AGR2,and then promote the proliferation,migration and invasion of hepatocellular carcinoma cells,which is a potential target for molecular targeted therapy of hepatocellular carcinoma. |
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