瞬时受体电势通道6对胃癌细胞增殖的作用及机制研究

Essential role of TRPC6 in the proliferation of gastric cancer and its mechanism

目的探讨瞬时受体电势通道6（TRPC6）基因对胃癌细胞增殖的作用及其分子机制。方法采用Westernblot法和免疫组化方法检测30例胃癌及癌旁组织中TRPC6蛋白的表达水平。以腺病毒载体介导的TRPC6显性负突变体抑制胃癌AGS细胞内源性TRPC6通道后绘制细胞生长曲线，检测单细胞克隆形成能力和裸鼠移植瘤的皮下成瘤能力；流式细胞仪检测细胞周期；Westernblot法检测Cdc2磷酸化水平。结果TRPC6蛋白在胃癌及癌旁组织中的相对表达量分别为（21．60±8．32）％和（7．14±2．24）％，差异有统计学意义（P〈0．05）。转染24、48、72、96h后，与Ad—GFP转染组相比，Ad-DNC6转染组的细胞增殖率分别下降（36．90±1．13）％、（44．06±2．17）％、（52．12±2．76）％和（50．89±1．97）％。Ad—DNC6转染组和Ad．GFP转染组胃癌AGS细胞的克隆形成率分别为（14．70±3．00）％和（43．80±7．00）％。在转染0、24、36、48h后，Ad—DNC6转染组和Ad-GFP转染组的G2／M期细胞比例分别为（20．34±1．98）％、（24．31±2．37）％、（27．70±2．36）％、（35．10±3．07）％和（18．40±2．01）％、（18．30±1．72）％、（17．50±1．74）％、（16．80±1．71）％。抑制TRPC6通道能明显抑制裸鼠移植瘤的生长（P〈0．05）。结论TRPC6通道通过调节细胞周期G2期的进程来影响胃癌细胞的增殖。

Objective To investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer. Methods The expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 （DNC6）. The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided. Results The TRPC6 protein in the tumor tissues and para-tumor tissues was （21.60 ± 8.32） % versus （7.14 ±2.24） %. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were （36.90 ± 1.13） %, （44. 06 ± 2. 17 ） %, （52. 12 ± 2.76） % and （50. 89 ± 1.97） %, respectively. The clone formation rates of control group and DNC6 group were （ 14.70 ± 3.00） % versus （43.80 ± 7.00） %. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the GJM phase arrest was （20. 34 ± 1.98）%, （24.31 ±2.37）%, （27.70 ±2.36）%, （35.10 ±3.0）% in the DNC6 group and （18.40 ± 2.01 ） %, （ 18.0% ± 1.72） %, （ 17.50 ± 1.74） %, （ 16.80 ± 1.71 ） % in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors （ P 〈 0.05 ）. Conclusion In the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G2/M phase transition.