Study On The Role And Mechanism Of Long Non-coding RNA LINC02163 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide,ranking sixth in incidence and third in mortality.In China,the death rate of HCC is secondary only to lung cancer.The number of people dying of HCC every year accounts for more than 50% of the overall HCC mortality in the worl d.Although great progress has been made in the treatment of HCC in the areas of surgery,chemoradiotherapy and molecular targeted therapy,the poor prognosis of HCC patients has not been fundamentally changed due to the characteristics of high invasiveness,high metastasis and high recurrence.Up to now,the exact molecular mechanism of HCC development is not fully understood.Therefore,it is critical for HCC research to investigate the molecular mechanism of HCC invasion and metastasis and to find effective therapeutic targets as well as biomarkers for early diagnosis and prognosis prediction.Recent studies have found that long non-coding RNA(lnc RNA)is abnormally expressed in a variety of tumors,including HCC,and plays an important role as oncogene or tumor suppressor gene in the occurrence and development of HCC.In this study,we focused on the study of a new lncRNA LINC02163 with up-regulated expression in HCC.We analyzed its expression in HCC tissues and HCC cells and its clinical significance in HCC,and studied its biological function and possible mechanism of action.The specific contents are as follows:Part Ⅰ The molecular characteristics of LINC02163 and the expression and clinical significance of LINC02163 in hepatocellular carcinoma.ObjectivesTo lay a theoretical foundation for further cell function experiments,we first analyzed the molecular characteristics of LINC02163.Then we investigated the expression of LINC02163 in HCC tissues and its correlation with the clinicopathological pa rameters.We also studied its prognostic value in HCC.MethodsThe gene characteristics and coding ability of LINC02163 were analyzed by NCBI gene database and UCSC gene browser,and further confirmed by Coding Potential Assessment Tool(CPAT)and Open Reading Frame(ORF)Finder.The expression level of LINC02163 in HCC tissues and its paired adjacent tissues was detected by quantitative real-time PCR(qRT-PCR).The relationship between LINC02163 expression and the clinicopathological parameters of HCC patients was analyzed.Kaplan-Meier analysis was used to analyze the relationship between LINC02163 and overall survival(OS)of HCC patients based on the clinical information of HCC patients in TCGA database.Results1.LINC02163 is an intergenic lncRNA,located in the region of human chromosome 5q21.2.The total length of LINC02163 is 316 bp.LINC02163 contains 3 exons but its protein-coding potential is very low,which is consistent with the molecular characteristics of lncRNAs.2.The qRT-PCR results of 60 pairs of HCC tissues and its paired adjacent tissues showed that the expression level of LINC02163 in HCC tissues was significantly higher than that in paired adjacent tissues(P<0.001).3.The expression of LINC02163 in HCC was positively correlated with T NM stage and microvascular invasion(MVI)(P<0.01).4.Kaplan-Meier survival analysis of TCGA database information showed that high LINC02163 expression in HCC tissues was significantly correlated with poor overall survival rate(P < 0.05).ConclusionsLINC02163 is an intergenic lncRNA located in the 5q21.2 region of the human chromosome.The expression level of LINC02163 was significantly increased in HCC tissues.Moreover,the high expression of LINC02163 was significantly associated with TNM stage,MVI and poor prognosis.Part Ⅱ The effect of LINC02163 on the biological behavior of HCC cellsObjectivesThe effect of LINC02163 on the biological behavior of HCC cells was observed in vitro and in vivo.MethodsQRT-PCR was used to detect the expression level of LINC02163 in 5 HCC cell lines(Hu H-7、HCCLM3、MHCC-97H、SNU-182、SMMC-7721)and human normal liver cell line(L02).LINC02163 si RNA and pcDNA3.1-LINC02163 overexpression plasmid were designed and constructed,and LINC02163 interference cell model and LINC0 2163 overexpression cell model were constructed by transfection with HCC cells.The effects of LINC02163 interference and overexpression on the proliferation of liver cancer Cell lines were examined by Cell Counting kit-8(CCK-8)experiment.The effects of LINC02163 interference and overexpression on the migration and invasion ability of HCC cell lines were examined by Transwell migration and invasion assay.The effect of LINC02163 on the growth and metastasis of HCC cells in vivo was examined by constructing subcutaneous tumorigenicity model and liver orthotopic transplantation model of nude mice.Results1.Compared with normal liver cell line(L02),the expression level of LINC02163 was up-regulated in 5 HCC cell lines,among which the Hu H-7 and HCCLM3 cell lines showed higher expression level,whereas the SMMC-7721 cell line showed the lowest expression level.2.In vitro cell functional experiments showed that interference with LINC02163 expression significantly inhibited proliferation,migration and invasion of Hu H-7 and HCCLM3 cell lines.On the contrary,overexpression of LINC02163 significantly promoted the proliferation,migration and invasion of SMMC-7721 cell line.3.The subcutaneous tumorigenicity model showed that LINC02163 promoted the growth of HCC cells in nude mice.The liver orthotopic transplantation model showed that LINC02163 promoted the metastasis of HCC cells in nude mice.ConclusionsLINC02163 plays the role of oncogenic gene in HCC and can promote the occurrence and development of HCC.Part Ⅲ The molecular mechanism of LINC02163 in regulating the migration and invasion of HCC cellsObjectivesTo explore the molecular mechanism of LINC02163 in regulating the migration and invasion of HCC cells.MethodsThe subcellular localization of LINC02163 in HCC cells was observed by RNA fluorescence in situ hybridization(FISH)and plasmonuclear separation.Bioinformatics software star Base and TargetScan 7.2 were used to predict the miRNA that LINC02163 might bind and the downstream target gene of that miRNA.QRT-PCR was used to detect the expression level of LINC02163,mi R-338-3p,and LAMC1.Western blot was used to detect the protein level of LAMC1 and Epithelial Mesenehymal Transition(EMT)-related markers(E-cadherin,Vimentin).Results1.FISH and plasmonuclear separation experiments showed that LINC02163 was mainly distributed in cytoplasm in HCC cells.2.Bioinformatics software star Base and TargetScan 7.2 predicted that LINC02163 might specifically bind to mi R-338-3p as competing endogenous RNA(ce RNA),and LAMC1 might be a downstream target gene of miR-338-3p,and that there were targeting binding sites among LINC02163,miR-338-3p,and LAMC1-3 ’UTR.3.We analyzed the LINC02163 and LAMC1 m RNA expression in clinical HCC tissue samples and observed a significant positive correlation.Interference with LINC02163 expression in HuH-7 cells inhibited LAMC1 expression and epithelial mesenchymal transformation.On the contrary,overexpression of LINC02163 in SMMC-7721 cells promoted LAMC1 expression and epithelial mesenchymal transformation.4.Interfering with LINC02163 expression in HuH-7 cells increased mi R-338-3p expression.5.Overexpression of miR-338-3p in HuH-7 cells inhibited LAMC1 expression and epithelial mesenchymal transition.ConclusionsLINC02163 may act as a molecular sponge for mi R-338-3p to down-regulate the expression of mi R-338-3p.In this way,LINC02163 played a role in promoting the migration and invasion of HCC cells by inhibiting mi R-338-3p.In addition,LINC02163 could also up-regulate LAMC1,which is a oncogenic gene targeted by miR-338-3p.