| Objective: In this study,the expression of Cytoplasmic activation/proliferation-associated protein-1(Caprin-1)in clinical samples of Hepatocellular carcinoma(HCC)was analyzed by immunohistochemical(IHC)method.The Cancer Genome Atlas(TCGA)database was used to analyze the relationship between Caprin-1 expression and prognosis in patients with HCC.On this basis,(ultrasound-targeted microbubble destruction,UTMD)-mediated Caprin-1 knockout plasmid(Caprin-1-KO)was transfected into human hepatocellular carcinoma HepG2 cells by ultrasonic targeting microbubbles to observe the effects of target plasmid transfection on the proliferation,cycle,invasion and migration of human hepatocellular carcinoma HepG2 cells,and to explore the role of Caprin-1 in the pathogenesis of HCC and its relationship with the clinicopathological characteristics and prognosis of HCC,thus providing basis for prognostic evaluation and ultrasound-mediated gene targeted therapy of HCC.Methods: 1.The difference in the expression of Caprin-1 between HCC tissues and normal liver tissues was detected by IHC.2.To analyze the relationship between Caprin-1 gene expression and clinicopathological features of patients based on the clinical data of 154 patients with HCC in TCGA database,and to evaluate the relationship between Caprin-1gene expression and prognosis of patients with HCC.3.The Caprin-1 knockout plasmids Caprin-1-KO-1 and Caprin-1-KO-2 were constructed and the recombinant plasmids were confirmed by sequencing. 4.In order to explore the optimal microbubble concentration for subsequent experiments,Cell Counting Kit-8(CCK8)method was used to detect the changes of cell activity of human hepatocellular carcinoma HepG2 cells treated with different concentrations of SF6 microbubbles.5.On the basis of the above-mentioned most suitable microbubble concentration,more parameters were explored by UTMD mediated Caprin-1 knockout plasmid transfection into human hepatocellular carcinoma HepG2 cells,including irradiation time and mechanical index(MI).The optimal ultrasonic transfection conditions for subsequent cell experiments were then selected.6.After transfecting the target plasmid into human hepatocellular carcinoma HepG2 cells by optimal ultrasound transfection conditions,the protein expression of Caprin-1 gene in transfected human hepatocellular carcinoma HepG2 cells was detected by Western Blot method.The best quality granules for subsequent cell function experiments were then selected.7.After transfecting the optimal plasmid into human hepatocellular carcinoma HepG2 cells to inhibit the expression of Caprin-1.CCK8 assay was used to detect cell proliferation,flow cytometry was used to detect cell cycle,and Transwell method was used to detect cell invasion.Woundhealing assay was used to detect cell migration.Results: 1.Immunohistochemical analysis showed that the expression level of Caprin-1 in HCC tissues was significantly higher than that in normal liver tissues(P < 0 05).2.When TMA clinical data were used to analyze the correlation between Caprin-1and clinical characteristics of patients,the results showed that the high expression of Caprin-1 protein was associated with HCC patients with advanced clinical stage and enhanced tumor invasion.The difference was statistically significant(P< 0.05).Further analysis of TCGA database showed that the high expression of Caprin-1 protein was related to age and the degree of tumor invasion(P < 0.05).Kaplan-Meier analysis showed that the overall survival rate and disease-free survival rate of HCC patients with high expression of Caprin-1 were significantly shorter than those with low expression of Caprin-1(P<0.05).But in patients without metastasis,the metastasis-free rate and disease-free survival rate in patients with high expression of Caprin-1 were significantly lower than those with low expression of Caprin-1,and the difference was statistically significant(P<0.05).Univariate analysis and multivariate survival comparison of Cox proportional hazard regression model showed that high expression of Caprin-1 could be used as a significant independent prognostic factor for HCC(P <0.05).3.The primers of Caprin-1 knockout plasmid Caprin-1-KO-1 and Caprin-1-KO-2were designed and inserted into the restriction endonuclease Bsmb1 site of PX330-puro plasmid vector.The plasmid was extracted by electrophoresis and sequencing.The identification plasmids Caprin-1-KO-1 and Caprin-1-KO-2 were constructed successfully.4.The microbubble concentration experiment showed that When the concentration was greater than or equal to 5%,the cell survival rate was more than 80%.As the microbubble concentration increased,the cell viability began to decrease.When the concentration rose to 10%,the cytotoxic reaction began to appear.when the concentration was 20%,the cell survival rate was less than<70%,and the difference between the groups was statistically significant(P<0.05).In this experiment,according to the principle of not affecting cell activity and maximizing the transfection rate,5% microbubble concentration was applied to subsequent experimental studies.5.Optimization of the parameters of UTMD-mediated Caprin-1 knockout plasmid transfection into human hepatocellular carcinoma HepG2 cells:(1)the transfection efficiency of microbubble + plasmid + ultrasound irradiation group was significantly higher than that of blank cell control group,simple plasmid group and microbubble + plasmid group(P < 0.05).(2)When the concentration of microbubbles was 5%,the transfection rate increased at first and then decreased with the increase of MI value,and the difference was statistically significant(P<0.05).When the microbubble concentration was 5% and the irradiation time was 20 s,the gene transfection rate(24.15±1.75)% was the highest when MI was 0.37,which was significantly higher than that in other groups(P<0.05).6.Western Blot method was used to detect the protein expression of Caprin-1 gene after transfection of target plasmid.The results showed that compared with negative control(NC)group and Caprin-1-KO-1 group,the expression of Caprin-1 in Caprin-1-KO-2 group decreased significantly after transfection into cells(P<0.05).7.Caprin-1-KO-2(renamed as Caprin-1-KO)was transfected into human hepatoma HepG2 cells mediated by UTMD method.After inhibiting the expression of Caprin-1,compared with the control group,the proliferation,invasion and migration of HepG2 cells were significantly inhibited(P<0.05).The number of cells in GO/G1 phase in the experimental group(64.56 ± 1.20)% was significantly higher than that in the control group(43.11±2.14)%,and the difference was statistically significant(P<0.05),suggesting that the cells cycle were blocked.Conclusion: 1)The high expression of Caprin-1 in HCC and the high expression of Caprin-1 were associated with the poor prognosis of HCC,such as late clinical stage and enhanced tumor invasion,which can be used as an independent prognostic factor for HCC suggesting that Caprin-1 may be a new molecular target for gene therapy in patients with HCC.2)The survival rate of human hepatocellular carcinoma HepG2 cells was inversely proportional to the concentration of microbubbles.UTMD method could enhance gene transfection.When the concentration of microbubbles was 5%,MI 0.37,irradiation time was 20 s,the gene transfection rate was the highest.3)Caprin-1-KO was transfected into human hepatoma HepG2 cells by UTMD method.After inhibiting the expression of Caprin-1,the proliferation,invasion and migration of HepG2 cells were inhibited and the cells cycle were blocked.It was expected to provide a basis for further exploring the potential role and clinical significance of Caprin-1 in the pathogenesis of HCC. |
PDF Full Text Request