Study On The Effects Of Cell Proliferation And Cell Cycle Of MiR-181c-shRNA Plasmid Transfected Into Human Hepatoma HepG2 Cells By Microbubble Ultrasound Contrast Agents

Objective Based on the preliminary analysis of the relationship between the expression of miR-181 c and the prognosis of patients with hepatocellular carcinoma(HCC),the cancer genome atlas(TCGA)database was used in this study.To evaluate the feasibility of ultrasound-targeted microbubble destruction(UTMD)mediated miR-181 c interference plasmid(miR-181c-shRNA)transfection into human hepatoma HepG2 cells and to screen the optimal ultrasonic transfection conditions.To observe the effects of transfection of target plasmid on the proliferation and cycle of HepG2 cells.It provides a basis for further exploring the potential role and clinical significance of miR-181 c in the pathogenesis of HCC.Methods1.The data set of HCC patients in TCGA was downloaded to analyze the relationship between miR-181 c gene expression and clinicopathological features of HCC patients,as well as the effect on the survival time and prognosis of HCC patients.2.The interfering plasmid miR-181c-shRNA was constructed to silence the expression of miR-181 c.3.Cell Counting Kit-8(CCK8)assay was used to detect the effect of different concentrations of sulfur hexafluoride(SF6)microbubbles on the activity of human hepatoma HepG2 cells.4.Human hepatoma HepG2 cells were transfected with miR-181c-shRNA mediated by UTMD method.the optimum transfection conditions,including microbubble concentration,irradiation time and mechanical index(MI),were screened.5.Human hepatoma HepG2 cells were transfected with miR-181c-shRNA under the optimum conditions.the expression of miR-181 c after transfection was detected by real-time fluorescence quantitative PCR(qPCR).6.CCK8 assay was used to detect the proliferation of cells after inhibiting the expression of miR-181 c,and flow cytometry was used to detect the cell cycle after inhibiting the expression of miR-181 c.Results1.The results of TCGA database analysis showed that the abnormal expression of miR-181 c was related to the clinical stage and tumor invasion of HCC patients,and the difference was statistically significant(P<0.05).The disease-free survival time of patients with high expression of miR-181 c was significantly lower than that of patients with low expression of miR-181 c.The difference was statistically significant(P<0.05),but there was no significant difference in the total survival time(P>0.05).Univariate and multivariate analysis of Cox proportional hazard model suggested that miR-181 c expression was an independent prognostic factor in HCC(P <0.05).2.The primer sequence of miR-181 c silencing expression vector shRNA was designed.the miRNA silencing expression vector was digested by restriction endonuclease Eco R1-HF and Bam H1-HF and ligated with shRNA,for transformation and extraction.the plasmid miR-181c-shRNA was identified by electrophoresis and sequencing.3.The survival rate of human hepatoma HepG2 cells decreased with the increase of microbubble concentration.when the microbubble concentration was less than or equal to 5%,the cell survival rate was above 80%,and when the concentration reached 20%,The cell survival rate decreased to 62% and the cytotoxicity increased significantly.the survival rate of each group was significantly different(P < 0.05).4.The results of ultrasonic irradiation parameters were as follows:(1)The transfection efficiency of blank cell control group,simple plasmid group and microbubble + plasmid group was less than 0.4%,which was significantly lower than that of microbubble + plasmid group + ultrasound irradiation group;The difference was statistically significant(P<0.05).(2)The results of microbubble+plasmid group+ultrasound irradiation group indicated that when the concentration of microbubbles was 5% and 10%,the transfection rate of each group increased first and then decreased with the increase of MI,and the difference was statistically significant(P<0.05);when the microbubble concentration reached 20%,the transfection rate of each group had no significant change with time or MI,and both were less than 4%,the difference was not statistically significant(P>0.05).When the microbubble concentration was 5%,the irradiation time was 20 s,and the MI was 0.37,the gene transfection rate was the highest,up to(23.33±1.47)%.5.After qPCR identification,the relative expression of miR-181 c in HepG2 cells in the experimental group(0.11 ± 0.06)was significantly lower than that in the control group(1.02 ± 0.26)(P<0.05).It is suggested that the expression of miR-181 c in HepG2 cells was inhibited after transfection of the target plasmid.6.Mi R-181c-shRNA was transfected into human hepatoma HepG2 cells by UTMD method.after silencing the expression of miR-181 c,the proliferation of human hepatoma HepG2 cells was inhibited,the difference was statistically significant(P<0.05),and the greater the difference with the prolongation of culture time.The percentage of GO/G1 phase cells in the experimental group(65.83 ±1.31)%was significantly higher than that in the control group(61.19±1.15)%(P< 0.05),suggesting that the cell cycle was blocked.Conclusion1.The abnormal expression of miR-181 c is associated with the poor prognosis of HCC and can be used as an independent prognostic factor of HCC,indicating that miR-181 c may become a new target of HCC gene therapy.2.The survival rate of human hepatoma HepG2 cells was inversely proportional to the concentration of microbubbles.UTMD method could promote gene transfection.when the concentration of microbubbles was 5%,the irradiation frequency was 2.0 MHz,MI was 0.37,and the irradiation time was 20 s,the gene transfection rate was the highest.3.Mi R-181c-shRNA was transfected into human hepatoma HepG2 cells by UTMD method.after silencing the expression of miR-181 c,the proliferation of human hepatoma HepG2 cells was inhibited and the cell cycle was blocked.It provides a basis for further exploring the potential role and clinical significance of miR-181 c in the pathogenesis of HCC.