| Primary hepatocellular carcinoma(PHC)is one of the most common malignant tumors worldwide.In China,the incidence of hepatocellular carcinoma is second only to that of lung cancer.At present,the treatment of hepatocellular carcinoma is still mainly surgical treatment,and the 5-year survival rate is low.The main bottleneck restricting the therapeutic effect of hepatocellular carcinoma surgery is the low rate of early diagnosis and high rate of recurrence after operation,but there is still a lack of effective adjuvant therapy besides surgery.Therefore,in-depth study of the molecular mechanism of the occurrence and development of hepatocellular carcinoma,development of new diagnostic markers and therapeutic targets has become a hot and important issue in current cancer research.Long-non-coding RNA is a non-coding RNA with a length of more than 200 nucleotides.For a long time,most studies on genes have focused on protein-coding genes.People follow the central rule of DNA-RNA-protein.Non-coding genes are considered as "noise" or "dark matter" in genomes.In recent years,a large number of experiments have proved that long-chain non-coding RNA transcribed by "dark matter" in some genomes is an epigenetic legacy.Transfer level,transcription level,translation level and protein modification process can play an important regulatory role.Antisense LncRNAs also play an important role in the occurrence and development of tumors.Cell adhesion molecule 1 antisense strand RNA 1(LncRNA CADM1-AS1),an antisense LncRNA,was first found to be low expression in renal clear cell carcinoma.Low expression of CADM1-AS1 is associated with tumor size,TNM stage,vascular invasion and poor prognosis,and can affect the proliferation of cancer cells and promote tumor metastasis.However,there is no evidence that CADM1-AS1 can affect the development of hepatocellular carcinoma.Objective:To detect the expression of cell adhesion molecule 1 antisense RNA 1(LncRNA CADM1-AS1)in hepatocellular carcinoma(HCC)tissues and cell lines and its relationship with clinicopathological features.Secondly,the relationship between lncRNA CADM1-AS1 and proliferation,migration and invasion of hepatocellular carcinoma cells was studied.Finally,the possible molecular mechanism of the role of lncRNA CADM1-AS1 in hepatocellular carcinoma was analyzed to provide a new theoretical basis for elucidating the mechanism of the occurrence and development of hepatocellular carcinoma.Methods:1.Differential expression of LncRNA CADM1-AS1 in hepatocellular carcinoma1.1 The expression level of CADM1-AS1 in cancer tissues and adjacent normal tissues of 90 patients with gastric cancer was detected by ISH.The personal information and detailed clinical data of patients were collected.The correlation between the expression level of CADM1-AS1 and pathological factors such as sex,age,tumor size,lymph node metastasis and TNM stage was analyzed.The correlation between CADM1-AS1 and clinicopathological characteristics of patients was examined by χ2 test.K-M survival curve was used to analyze the effect of CADM1-AS1 expression on survival.Multivariate COX regression analysis was used to determine independent predictors of gastric cancer.1.2.The expression of CADM1-AS1 in three human gastric cancer cell lines(HepG2,BEL-7402,Huh-7)and human normal liver cell line LO2 was analyzed by qRT-PCR.2.The effects of LncRNA CADM1-AS1 on the proliferation,migration,invasion and cell cycle of hepatocellular carcinoma cell lines HepG2 and BEL-7402 and the related molecular mechanisms.2.1 Cells were transfected with specific small interfering RNA(siRNA)chemical fragments,down-regulated the expression of relatively high expression of lncRNA CADM1-AS1 in hepatocellular carcinoma cell lines,transfected with specific lentivirus chemical fragments,up-regulated the expression of relatively low expression of lncRNA CADM1-AS1 in hepatocellular carcinoma cell lines,and observed the biological behavior of lncRNA CADM1-AS1 on hepatocellular carcinoma cells.Influence2.2 The effect of lncRNA CADM1-AS1 on cell proliferation was detected by CCK-8,EDU and cloning assay.2.3 The effects of lncRNA CADM1-AS1 on cell invasion and migration were detected by Transwell chamber and scratch test.2.4 The effect of lncRNA CADM1-AS1 on cell cycle was detected by flow cytometry.2.5 The effects of lncRNA CADM1-AS1 on PTEN/AKT/GSK-3 beta signaling pathway and expression of cyclin(CDK2,CDK4,CDK6,CyclinD,CyclinE,P15,P21,P27)were detected by Western blot.2.6.Subcutaneous tumorigenesis in nude mice was used to detect the tumorigenicity of hepatocellular carcinoma cells in vivo.Results:1.Differential expression of LncRNA CADM1-AS1 in hepatocellular carcinoma1.1 ISH results showed that the expression level of LncRNA CADM1-AS1 was closely related to hepatocellular carcinoma.There was an obvious downregulation of LncRNA CADM1-AS1 in hepatocellular carcinoma.Further analysis of clinical data(age,sex,tumor size)and the relationship between TMN grading and LncRNA CADM1-AS1 expression showed that LncRNA CADM1-AS1 was negatively correlated with tumor grade and TNM stage.QRT-PCR results also showed that the expression level of LncRNA CADM1-AS1 in hepatocellular carcinoma cell lines was significantly lower than that in normal hepatocellular cell lines.2.Experimental study of LncRNA CADM1-AS1 on proliferation,migration and invasion of HepG2 and BEL-7402 hepatocellular carcinoma cells2.1 LV CADM1-AS1 and LV-control lentiviruses were used to infect HepG2 and BEL-7402 cells,which significantly increased the expression of ADM1-AS1 in HepG2 or BEL-7402 cells.After transfection of HepG2 and BEL-7402 cells with specific si-CADM1-AS1-1 and si-CADM1-AS1-2,the expression of ADM1-AS1 in HepG2 or BEL-7402 cells was significantly inhibited.2.2 CCK8 and EDU results showed that the proliferation ability of HepG2 and BEL-7402 hepatocellular carcinoma cells decreased significantly after overexpression of LncRNA CADM1-AS1.After low expression of LncRNA CADM1-AS1,the proliferation of HepG2 and BEL-7402 hepatocellular carcinoma cells increasedsignificantly.The results of plate cloning experiment showed that the in vitro cloning ability of hepatocellular carcinoma cells was significantly weakened after overexpression of LncRNA CADM1-AS1,while the in vitro cloning ability of hepatocellular carcinoma cells was significantly enhanced after low expression of LncRNA-CADM1-AS1.2.3 Flow cytometry showed that over-expression of LncRNA CADM1-AS1 resulted in significant G0/G1 arrest in hepatocellular carcinoma cells.After low expression of LncRNA CADM1-AS1,the G0/G1 phase of hepatocellular carcinoma cells decreased significantly.2.4 Transwell test and scratch test showed that after overexpression of LncRNA CADM1-AS1,the ability of migration and invasion of hepatocellular carcinoma cells decreased significantly,while the ability of migration and invasion of hepatocellular carcinoma cells increased significantly after low expression of LncRNA CADM1-AS1.2.5 Western blot results showed that p-AKT(S473,T308)and p-GSK-3 beta(S9)in the AKT/GSK-3 beta pathway were significantly lower in the over-expressed CADM1-AS1 group and significantly higher in the low-expressed CADM1-AS1 group than in the control group.Compared with the control group,CDK2,CDK4,CDK6,cyclinD and cyclinE in the overexpression CADM1-AS1 group were significantly lower,while those in the low expression CADM1-AS1 group were significantly higher.P15,P21 and P27 were significantly increased in the overexpression CADM1-AS1 group and significantly decreased in the low expression CADM1-AS1 group.2.6.The results of subcutaneous tumorigenesis experiment in nude mice showed that after increasing the expression of CADM1-AS1 in hepatocellular carcinoma cells,the rate of subcutaneous tumorigenesis in nude mice was significantly slowed down,and the final volume and tumors were significantly lower than those in the control group,indicating that CADM1-AS1 could significantly inhibit the proliferation of HCC cells in vivo.Conclusion:1.Compared with the adjacent tissues,the expression of CADM1-AS1 in HCC islower,which is related to the grade and prognosis of HCC.CADM1-AS1 can be used as a prognostic marker for HCC patients.2.CADM1-AS1 inhibits the proliferation,migration,invasion and cell cycle of HCC cells by regulating AKT/GSK-3 beta pathway.3.CADM1-AS1 could significantly inhibit the proliferation of HCC cells in nude mice. |
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