RNA干扰技术沉默上皮型钙黏蛋白表达对体外培养的喉癌Hep-2细胞增殖的影响

Influence of RNAi on the silencing expression of E-cadherin and the proliferation ability of Hep-2 trained in vitro

目的通过RNA干扰(RNA interference,RNAi)技术沉默喉癌HeP-2细胞上皮型钙黏蛋白(E-cadherin)的表达,探讨E-cadherin对Hep-2细胞增殖能力的影响。方法设计、合成特异性小干扰RNA(siRNA)和无基因沉默功能的阴性siRNA转染Hep-2细胞,获得下调E-cadherin基因表达的体外培养的Hep-2细胞;荧光定量PCR检测E-cadherin基因的沉默效果;体外细胞增殖实验(MTT)检测Hep-2细胞体外增殖能力的变化。结果①重组质粒和脂质体质量体积按1:1转染时,转染效率最高组siRNA-3达65%。②荧光定量PCR:重组质粒pRNAT-U6.1/Neo-siRNA1、pRNAT-U6.1/Neo-siRNA2、pRNAT-U6.1/Neo-siRNA3使E-cadherin mRNA的表达下调。以空白对照组为基准(设为1),E-cadherin相对于β-actin的改变倍数siRNA-1组=0.00092,siRNA-2组=0.00143,siRNA-3组=0.00045,阴性对照组=3.44898,差异有统计学意义。③MTT:经特异性siRNA处理的Hep-2细胞生长速度较对照组增快,差异有统计学意义。结论有效抑制Hep-2细胞E-cadherin mRNA的表达使Hep-2细胞的增殖能力增强。

OBJECTIVE To explore the effect of E-cadherin on the proliferation ability of Hep-2 by method of RNA interference technology to silence the expression of E-cadherin.METHODS The specific siRNA sequences and non-silencing siRNA were designed and synthesized.Hep-2 cells were transfected and then the down expression of E-cadherin gene in vitro cultured Hep-2 cells were got.The silencing effect of E-cadherin gene was explored by Fluorescence Quantitative Polymerase Chain Reaction and the proliferation of the transfected Hep-2 cells were detected in vitro by MTT assay.RESULTS l.When transfected with the ratio of recombinant plasmid and the quality of liposome volume at 1：1,the transfection efficiency at the siRNA-3 group was the highest and can be up to 65%.2.The results of Fluorescence Quantitative Polymerase Chain Reaction：recombinant plasmid pRNAT-U6.1/NeosiRNAl,pRNAT-U6.1/Neo-siRNA2 and pRNAT-U6.1/NeosiRNA3 can down regulate the expression of E-cadherin mRAN.Set blank control group as a baseline（set to 1）,the changes of expression of E-cadherin relative to P-actin in siRNA-lgroup was 0.00092,siRNA-2 group was 0.00143,siRNA-3 group was 0.00045 and the negative control group was 3.44898.The difference was statistically significant（P〈0.05）.3.MTT：The growth rate of Hep-2 cells treated by specific siRNA was faster than that of the control group,and the difference was statistically significant（P〈0.05）.CONCLUSION Effectively inhibition the expression of E-cadheriris mRAN can enhance the proliferation of Hep-2 cells.