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

目的　通过RNA干扰（RNA interference，RNAi）技术沉默喉癌He p - 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。

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

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 1.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/Neo-siRNA1, pRNAT-U6.1/Neo-siRNA2 and pRNAT-U6.1/Neo-siRNA3 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 β-actin in siRNA-1group 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-cadherin's mRAN can enhance the proliferation of Hep-2 cells.