RNA干扰肝癌MHCC97-H细胞N-Ras基因对放射敏感性的影响

Effect of N-Ras by RNA interference on radiosensitivity of hepatoma carcinoma cell MHCC97-H line

目的探讨RNA干扰（RNAi）N—Ras基因增强人肝癌MHCC-97细胞的放射敏感性。方法通过构建干扰N-Ras基因siRNA，采用免疫组织化学法、实时荧光定量RT—PCR、Westernblot、MTT法观察RNAi前后肝癌MHCC97-H细胞RNA水平、蛋白水平、生长情况等变化，照射后用细胞克隆计数实验检测放射敏感性变化。结果MHCC97-H细胞株RNAi后mRNA表达抑制率为96．9％±0．159％，RNAi前后差异有统计学意义（t=40．377，P〈0．05），蛋白表达抑制率为89．8％±0．012％，RNAi前后差异有统计学意义（t=31．595，P〈0．05），免疫组织化学显示90％表达被抑制，细胞生长抑制率为21．9％，RNAi前后差异均有统计学意义（F=4．63，P〈0．05）。MHCC97-H细胞RNAi后增敏比（SER）=1．15。结论RNAi肝癌MHCC97一H细胞N—Ras基因可以达到放射增敏的目的。

Objective To investigate the radiosensitivity of silencing N-Ras by RNA interference in hepatoma carcinoma cell MHCC-97. Methods N-Ras RNA interference （RNAi） vector was constructed by using pcDNA 6.2-GW/EmGFP-mir plamid. The RNAi effect was detected by RT-PCR, Western bolt,immunohistochemisty and MTT method. Survival curve for each cell line were obtained by measuring the clone forming abilities of irradiated cell populations. Results After silencing the N-Ras by RNAi,The expression level of N-Ras mRNA,N-Ras protein, immunohistochemisty were decreased 96.9% ±0.159%（t=40.377,P〈0.05）,89.8% ±0.012%（t=31.595,P〈0.05）, 90%, respectively, and The survival of hepatoma carcinoma cell MHCC-97 line were inhibited 21.9% （ F = 4.63, P 〈 0. 05 ）. Which have significant difference in statistics. The SER of hepatoma carcinoma cell MHCC-97 line after interference was 1.15. Conclusions RNAi targeting silence N-Ras may increase the radiosensitivity of hepatoma carcinoma cell MHCC-97 line.