磷脂酰肌醇3激酶调节亚基基因对HepG2细胞增殖的影响

Roles of PIK3R1 gene in development of hepatocellular carcinoma

目的：初步探讨磷脂酰肌醇3激酶调节亚基（PIK3R1）在肝癌发生发展中的生物学作用及其机制.方法：首先通过蛋白质印迹法和免疫组织化学法检测p85α（PIK3R1所编码的蛋白）在肝癌组织标本中的表达情况;接着利用脂质体转染法将PIK3R1的小分子干扰RNA（siRNA）和含有PIK3R1 cDNA的重组质粒转染入HepG2细胞中,并利用实时定量PCR检测其干扰效率和过表达效率;采用MTT法和集落形成实验检测PIK3R1对细胞增殖的影响,应用流式细胞术分析PIK3R1对细胞周期的影响;最后蛋白质印迹法检测PI3K/AKT信号通路下游效应分子的表达变化.结果：p85α在肝癌组织中的表达高于其癌旁组织,差异有统计学意义（P ＜0.05）;PIK3R1被干扰后,HepG2细胞增殖速度减慢,集落形成率减少,差异有统计学意义（均P ＜0.05）;PIK3R1过表达后,HepG2细胞增殖速度加快,集落形成率增多,差异有统计学意义（均P＜0.05）;PIK3R1提高了HepG2细胞中S期细胞的比例（P＜0.01）;且PIK3R1可提高PI3 K/A KT通路下游效应分子p-AKT的水平,并降低p53的水平.结论：PIK3R1在肝癌组织中表达上调,PIK3R1可能通过激活PI3K/AKT信号通路促进HepG2细胞的增殖.

Objective： To investigate the roles of phosphatidylinositol 3-kinase regulatory subunit alpha （PIK3R1） gene in the development of hepatocellular carcinoma （HCC）. Methods： Surgical specimens of liver cancer and corresponding pericancerous liver tissue were collected from 20 patients with hepatocellular carcinoma. Expression of p85α, encoded by PIK3R1, in HCC tissue specimens was detected by Western blotting and immunohistochemistry. HCC HepG2 cells were transfected with PIK3R1 siRNA or PIK3R1-cDNA. The expression of PIK3R1 in transfected HepG2 cells or control cells were detected by real-time PCR. Cell proliferation was evaluated by MTF, colony formation assays and flow cytometry respectively. The expression of PI3K/AKT pathway-related proteins were detected by Western blotting. Results： The expression of p85α in liver tissue was higher than that in pericancerous tissues （ 1.27 ±0.58 vs 0.99±0.47, t = - 3.25, P 〈0.05）. The expression of PIK3R1 was decreased by 0. 19 ±0. 03 fold in PIK3RlsiRNA-transfected HepG2 cells （t =46.77, P 〈0.05）, and increased by 32.36± 3.33 fold in PIK3R1 cDNA-transfected cells （t= - 16.31, P 〈0. 05）. MTT result showed that PIK3R1 siRNA inhibited growth of HepG2 cells （0. 611±0. 072 vs 0. 807 ±0. 059, t =3.65, P 〈0. 05）, while PIK3R1 cDNA increased the cell growth （0. 937 ± 0. 060 vs 0. 693 ±0. 065, t = - 4.78, P 〈 0. 05）. PIK3RI siRNA transfected cells presented lower colony-forming efficiency than control group （3.8% ±0.84% vs 15.0%±2.3%, t =7.92, P 〈0.05）, while PIK3R1 eDNA transfected cells had higher colony-forming efficiency than control group （23.6% ±3.4% vs 12.0% ± 1.5%, t = -5.40, P 〈0.05）. PIK3R1 siRNA reduced the ratio of S phase cells （ 13.9% ±0. 015% vs 32.9% ±0. 07%, t =45.97, P 〈 0. 01 ） , while PIK3R1 cDNA increased S phase cells （ 56. 33%± 0. 024% vs 31.94%±0.042%, t = -8.73, P〈0.01）. PIK3R1 increased the level ofp-AKT and decreased p53 level. Conclusion： p85α is highly expressed in HCC, and PIK3R1 gene may promote proliferation of HepG2 cells by activating PI3K/AKT pathway.