The Study On The Expression,Function And Mechanism Of Long Non-coding RNA LALR1 In Hepatocellular Carcinoma

Primary liver cancer is the sixth most common and fourth approximately fatal cancer in the world,and the fifth most common fatal disease in China.Hepatocellular carcinoma(HCC)accounts for 75%-85% of primary liver cancer,and more than 50% of global HCC occurs in China.HCC patients have high mortality rates due to the lack of symptoms of early HCC,the lack of diagnostic biomarkers and other detection methods.most cases are found to have advanced and patients with limited treatment options.Patients with advanced HCC have a poor prognosis and a short survival time.Therefore,it is urgent to understand the molecular mechanisms of HCC development and put forward new ideas for the treatment of HCC.It has been reported that lncRNA-LALR1 could promote liver regeneration,but its role in HCC was unclear.We found that lncRNA-LALR1 was highly expressed in HCC,suggesting that lncRNA-LALR1 might promote the progression of HCC.However,the biological role and related molecular mechanisms of lncRNA-LALR1 in HCC are not clear.Our study will discuss the effects of lncRNA-LALR1 on the biological functions of HCC cells such as proliferation and invasion from three parts,and explain the related molecular mechanism.Part 1 The expression of lncRNA-LALR1 in HCC tissues and its relationship with the patients' clinicopathological parametersObjective: To detect the expression of lncRNA-LALR1 in clinical tissue samples and explore the relationship between lncRNA-LALR1 and clinical pathological parameters.Methods: 46 pairs fresh HCC tissues and their adjacent cancerous tissues were collected in the Second Affiliated Hospital of Chongqing Medical University.The expression of lncRNA-LALR1 in 46 pairs of clinical tissue samples was detected by qRT-PCR.Paired t test was used to analyze the relative expression level of lncRNA-LALR1 in HCC cancer and adjacent tissues.Chi-square test was used to analyze the relationship between its expression and clinical pathological parameters.Results:(1)The expression of lncRNA-LALR1 in HCC cancer tissues was significantly higher than that in corresponding adjacent cancer tissues in 46 cases of HCC tissues and corresponding adjacent tissues;(2)The expression of lncRNA-LALR1 was related to the advanced TNM stage of patients,tumor differentiation and distant metastasis.Conclusions: lncRNA-LALR1 is highly expressed in HCC tissues,and positively correlated with the patient's TNM stage,tumor differentiation,and distant metastasis,suggesting that lncRNA-LALR1 may promote HCC progression.Part 2 Study on the effects of lncRNA-LALR1 on the growth and invasion of HCC cellsObjective: To investigate the biological effects of lncRNA-LALR1 on the proliferation,clone formation and invasion of HCC cells.Methods: qRT-PCR and FISH assays were used to detect the expression of lncRNA-LALR1 in six HCC cell lines including Huh7,HepG2,Sk-Hep1,SMMC-7721,PLC/PRF/5 and MHCC-97 H.Lentiviruses carrying small hairpin RNA(shRNA)sequence of human lncRNA-LALR1 were used to infect HCC cells.qRT-PCR and FISH were used to validate the efficiency of lncRNA-LALR1-knockdown cell lines.CCK-8,colony formation,flow cytometry,and transwell invasion assays were used to detect the proliferation,growth,apoptosis,cell cycle and invasion of lncRNA-LALR1-knockdown cells.the effect of lncRNA-LALR1 on the growth of HCC cells in vivo was measured using subcutaneous tumor transplantation in nude mice.Results:(1)The expression of lncRNA-LALR1 was higher in SMMC-7721 and HepG2 cells than other HCC cell lines.(2)LncRNA-LALR1 was located in the nucleus and cytoplasm,especially in nucleolus.(3)The proliferation,growth and invasion ability of HCC cells were significantly reduced in vitro after knockdown of lncRNA-LALR1.(4)The growth of subcutaneous tumors of nude mice was inhibited by knockdown of lncRNALALR1.Conclusions: LncRNA-LALR1 is located in the cytoplasm and nucleus,especially in nucleolus.Knockdown of lncRNA-LALR1 significantly reduces the proliferation,growth and invasion of HCC cells,indicating that lncRNA-LALR1 can promote the malignant phenotype of HCC.Part 3 Study on the mechanism of lncRNA-LALR1 promoting HCC cell growth and invasion by up-regulating SNORD72Objective: To investigate the molecular mechanism of lncRNALALR1 in promoting the growth and invasion of HCC cells.Methods: Transcriptome sequencing was used to analyze the expression profile after lncRNA-LALR1 silencing.qRT-PCR was used to verify gene expression changes after lncRNA-LALR1 knockdown.GO enrichment analysis was used to analyze cell biological processes,cell location and cell molecular functions involved in lncRNA-LALR1.KEGG signal pathway was used to analyze the signaling pathway changes after lncRNA-LALR1 silencing.qRT-PCR,Western Blot,and FISH assays were used to detect the effects of lncRNA-LALR1 on SNORD72 and ID2 expression.RNA-RNA pulldown was used to detect the binding of lncRNA-LALR1,SNORD72 and ID2 mRNA.Actinomycin D assay was used to detect the effect of lncRNA-LALR1 on the stability of SNORD72 and ID2 mRNA.CCK-8,colony formation,transwell invasion assay were used to determine SNORD72 on HCC cell proliferation,growth and invasion.The effect of SNORD72 on lncRNA-LALR1 was tested by SNORD72 rescue experiments.Results:(1)After lncRNA-LALR1 silencing,the expression of 540 genes was up-regulated and the expression of 145 genes was down-regulated.The expression of SNORD72 and ID2 were most obviously changed by qRTPCR.(2)GO analysis showed that lncRNA-LALR1 was significantly related to nuclear components and processes.(3)KEGG analysis showed that lncRNA-LALR1 was related to p53,NF-?B,and MAPK signal pathways.(4)qRT-PCR,Western Blot and FISH assay showed that lncRNA-LALR1 silencing significantly down-regulated SNORD72 and ID2.(5)RNA-RNA pulldown assay showed that lncRNA-LALR1 bound to both SNORD72 and ID2 mRNA.(6)Actinomycin D assay showed that lncRNA-LALR1 knockdown could reduce ID2 mRNA stability,but have no significant effect on the stability of SNORD72.(7)SNORD72 was highly expressed in HCC and significantly promoted HCC cell proliferation,colony formation and cell invasion ability.(8)SNORD72 up-regulated ID2 expression and overexpression of SNORD72 significantly increased the stability of ID2 mRNA.(9)Overexpression of SNORD72 in lncRNA-LALR1 knockdown cells rescued the cell proliferation,colony formation,and invasion caused by knockdown of lncRNA-LALR1.Conclusions: LncRNA-LALR1 promotes the growth and invasion of HCC cells by up-regulating SNORD72 to stabilize ID2 mRNA.