The Function And Machanism Of LncRNA-BC200 In Hepatocellular Carcinoma Progression

BackgroundHepatocellular carcinoma(HCC)is a malignant tumor with strong invasiveness,high metastasis rate and poor prognosis.More than 90%of primary liver cancer(PLC)is hepatocellular carcinoma.According to the latest statistics,the global incidence of HCC is ranked 6th in cancer,and the mortality rate is ranked 4th.In China,the incidence of HCC is the fourth among malignant tumors,and the mortality rate is the second;With the continuous improvement of the treatment methods and techniques of HCC,the recurrence and metastasis rate and the five-year survival rate have improved,but the mechanism of metastasis and recurrence of HCC has not been fully elucidated.Many studies have shown that long noncoding RNA(lncRNA)is associated with the development and recurrence of HCC.According to the literature,lncRNA-BC200 plays an important role in a variety of tumors,but the role of lncRNA-BC200 in the expression of liver cancer and its role in the development of liver cancer is poorly studied.ObjectiveTo detect the expression level of lncRNA-BC200 in hepatocarcinoma tissues and adjacent tissues,and to analysis the relationship between clinicopathological parameters and the expression level of lncRNA-BC200;To clarify the effect of lncRNA-BC200 on the biological function of hepatocarcinoma cells,explore the possible regulatory mechanisms of lncRNA-BC200,and provide a theoretical basis for further searching for new molecular markers and targeted therapies for early diagnosis and early treatment of HCC.Methods1.Microarray technology was used to screen lncrnas with two or more times of differential expression in liver cancer tissues and adjacent tissues.The IncRNA microarray technique was used to screen the expression profile of IncRNA in 5 cases of HCC tissues and 5 cases of adjacent tissues.The independent sample t-test was used to screen out the differential IncRNA,and 9 differentially expressed IncRNAs were selected and detected by qPCR,and comparison of qPCR results with gene chip results.2.The expression of lncRNA-BC200 in hepatocellular carcinoma and its relationship with clinicopathological parameters of liver cancer patients.The expression of lncRNA-BC200 in 45 pairs of HCC tissues and adjacent tissues was detected by qPCR.Paired t-test was used to analyze the differential expression of lncRNA-BC200 in HCC tissues and adjacent tissues.The clinical and pathological parameters of 45 patients with liver cancer were collected.The relationship between the expression level of lncRNA-BC200 and clinical pathological parameters of HCC patients was analyzed by chi-square test.3.To study the function and mechanism of lncRNA-BC200 in hepatoma cells.Firstly,this study used CRISPR Cas9 gene editing technology to construct plasmids,transfected into HepG2 cells,screened stably transfected cells with puromycin,extracted total cellular RNA,and detected the expression level of lncRNA-BC200 by qPCR.Verify transfection efficiency.Further,at the cellular level,CCK8 assay,flow cytometry,transwell assay,etc.were used to detect the effects of BC200 on cell proliferation,apoptosis,cell cycle and cell migration,The effect of lncRNA-BC200 on related proteins was detected by Western blot.Secondly,HepG2 cells were infected with lentivirus,and stable transfected cells were screened by puromycin,and total RNA was extracted.The expression level of lncRNA-BC200 was detected by qPCR,and the infection efficiency was verified.Further,at the cellular level,CCK8 assay,flow cytometry,transwell assay,etc.were used to detect the effects of lncRNA-BC200 on cell proliferation,apoptosis,cell cycle and cell migration,and the effect of lncRNA-BC200 on related proteins was detected by Western blot.Furthermore,a blank group of HepG2 cells,HepG2 cells knocked out of lncRNA-BC200,and HepG2 cells overexpressing lncRNA-BC200 were injected subcutaneously into the right thigh of the nude mice.After one week of injection,the body weight of nude mice was weighed every other week.After 8 weeks,the nude mice were sacrificed and the tumors were weighed.Finally,using RNA pull-down technology combined with protein profiling techniques,proteins that may bind to lncRNA-BC200 were obtained.Results1.1ncRNA is differentially expressed in HCC tissues and adjacent tissues.The results of the microarray showed that compared with the adjacent tissues,34 lncRNA expression was up-regulated and 11 lncRNA expression was down-regulated.Eight lncRNAs were selected from the different lncRNAs and detected by qPCR.The results showed that the qPCR results were consistent with the results of the microarray,indicating that the microarray results were reliable.2.lncRNA-BC200 is highly expressed in liver cancer tissues and is associated with liver metastasis.The qPCR results of 45 pairs of HCC tissues and adjacent tissues showed that the expression level of lncRNA-BC200 in HCC tissues was significantly higher than that in adjacent tissues,and the expression difference was statistically significant(p<0.05).Chi-square test analysis showed that the expression of lncRNA-BC200 was closely related to metastasis(p=0.041)and serum ALT concentration(p=0.048),and no correlation with age,gender,pathological grade,cirrhosis,preoperative serum AFP concentration,and hepatitis B virus,hepatitis B quantitative DNA,portal vein tumor thrombus,tumor size,BCLC stage,serum AST concentration,T lymphocytes,and B lymphocytes.3.The expression level of lncRNA-BC200 can affect the migration of hepatoma cells,tumor growth,c-myc protein expression and binding to hnRNP A2/B1 protein.Stable transfected HepG2 cells were constructed and cell function experiments were performed at the cellular level.The results of this experiment showed that knockdown of lncRNA-BC200 inhibited the migration of HepG2 cells compared with the blank group and the control group,but the cell cycle and cell Death,cell proliferation has no effect.Western blot results showed that the expression of c-myc protein and bax protein decreased and the expression of bcl-xl protein increased in the knockout lncRNA-BC200 group compared with the blank group and the control group.Construction of stably infected HepG2 cells,and cell function experiments were performed at the cellular level,the results of this experiment showed that overexpression of lncRNA-BC200 can promote the migration of HepG2 cells compared with the blank group and the control group,but the cell cycle and cell Death,cell proliferation has no effect.The results of Western blot showed that the expression of c-myc protein and bax protein increased and the expression of bcl-xl protein decreased in the overexpressed lncRNA-BC200 group compared with the blank group and the control group.Subcutaneous tumor formation in nude mice showed that the average tumor volume of the overexpressed group was the largest(1.43±0.29 cm3),and the mean tumor volume of the knockout group was the smallest(0.49±0.36 cm3).The difference was statistically significant(p<0.05).The average tumor weight of the overexpression group was the heaviest(2.0±0.16g),and the mean tumor weight of the knockout group was the lightest(0.70±0.61 g),the difference was statistically significant(p<0.05).The weight growth rate of the overexpressing group was lower than that of the knockout group,and the difference was statistically significant(p<0.05).RNA pull-down experiments and protein profiling showed that 77 proteins may bind to lncRNA-BC200,and the identified protein has hnRNP A2/B1 protein.Conclusions1.lncRNA-BC200 is highly expressed in HCC tissues.2,lncRNA-BC200 is associated with cancer cell metastasis in HCC patients,which may be used as a molecular marker for the diagnosis of HCC.3,lncRNA-BC200 may affect the migration ability of HCC cells and tumor proliferation by binding hnRNP A2/B1.