P-Rex2a干扰PTEN-long对胃癌细胞增殖和凋亡的影响

P-Rex2a interferes with the effect of PTEN-long on the proliferation and apoptosis of gastric cancer cells

目的 探讨P-Rex2a干扰PTEN-long对胃癌细胞增殖和凋亡的影响。方法 回顾性选取2018年1月至12月在上海市第八人民医院行胃癌根治术患者的胃癌组织和癌旁组织(距离癌组织5 cm)病理标本30例;收集胃癌细胞系BGC-823、AGS、MGC-803和SGC-7901,采用qRT-PCR检测胃癌和癌旁组织和细胞中PTEN-long mRNA的表达;将SGC-7901细胞处于对数生长期时进行实验,实验分为对照组(SGC-7901细胞未转染慢病毒)、NC组(SGC-7901细胞转染空白质粒)、PTEN-long-RFP组(将PTEN-Long-RFP转染到的SGC-7901细胞)、si-NC组(转染siP-Rex2a NC)、siP-Rex2a组(siRNA干扰技术对Rex2a进行沉默)和siP-Rex2a+PTEN-long组(将PTEN-long-RFP和siP-Rex2a共同转染到的SGC-7901细胞)。采用CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,免疫共沉淀(Co-IP)检测P-Rex2a与PTEN-long相互作用,蛋白质印迹法检测SGC-7901细胞中PI3K活性相关蛋白;构建P-Rex2a干扰表达载体,检测siP-Rex2a对细胞增殖和凋亡的影响。结果 胃癌组织中PTEN-long mRNA水平显著低于癌旁组织,差异有统计学意义(P<0.05);胃癌细胞系BGC-823、AGS、MGC-803、SGC-7901中mRNA明显低于胃上皮细胞GES-1,差异均有统计学意义(P<0.05)。与对照组和NC组相比,PTEN-Long-RFP组中的胃癌细胞SGC-7901增殖明显降低,凋亡能力显著升高,差异均有统计学意义(P<0.05)。P-Rex2a与PTEN-long可以相互作用,P-Rex2a过表达可以增强P-Rex2a与PTEN-long的结合;当P-Rex2a蛋白水平随着时间增加时,PTEN-long的蛋白水平逐渐降低。培养后48~96 h,与对照组和si-NC组相比,siP-Rex2a组中的胃癌细胞SGC-7901增殖明显降低,差异均有统计学意义(P<0.05);与对照组和si-NC组相比,siP-Rex2a组中的胃癌细胞SGC-7901凋亡能力显著升高,差异均有统计学意义(P<0.05);与对照组和si-NC组相比,siP-Rex2a组中的P-Akt、P-PI3K、Bcl-2显著降低,Caspase3、PARP、Bax、Bad明显升高,差异均有统计学意义(P<0.05)。培养后48~96 h,与对照组和si-NC组相比,siP-Rex2a组中的细胞增殖率明显降低;与siP-Rex2a组相比,siP-Rex2a+PTEN-long组中培养后48~96 h的细胞增殖率显著降低,差异均有统计学意义(P<0.05)。与对照组和si-NC组相比,siP-Rex2a组中的细胞凋亡率明显升高;与siP-Rex2a组相比,siP-Rex2a+PTEN-long组中的细胞凋亡率显著升高,差异均有统计学意义(P<0.05)。结论 PTEN-long参与胃癌的发生和发展,干扰P-Rex2a可以促进PTEN-long表达从而抑制胃癌细胞SGC-7901增殖,促进其凋亡,有望成为胃癌潜在治疗的新靶标。

Objective To investigate the effect of P-Rex2a interfering with PTEN-long on the proliferation and apoptosis of gastric cancer cells. Methods Thirty pathological specimens of gastric cancer tissue and adjacent tissue(5 cm away from the cancer tissue) from patients with gastric cancer who underwent radical resection of gastric cancer in Shanghai Eighth People’s Hospital from January to December 2018 were retrospectively selected, and the gastric cancer cell line BGC-823, AGS, MGC-803 and SGC-7901 was collected. The expression of PTEN-long mRNA in gastric cancer and adjacent tissues and cells was detected by qRT-PCR;the SGC-7901 cells were in the logarithmic growth phase for the experiment, and the experiment was divided into the control group(SGC-7901 cells were not transfected with lentivirus), NC group(SGC-7901 cells were transfected with blank plasmid), PTEN-long-RFP group(SGC-7901 cells transfected with PTEN-Long-RFP), si-NC group(NC transfected with siP-Rex2a), siP-Rex2a group(Rex2a was silenced by siRNA interference technology) and siP-Rex2a+PTEN-long group(SGC-7901 cells co-transfected with PTEN-long-RFP and siP-Rex2a). Cell proliferation was detected by CCK-8 assay, cell apoptosis was detected by flow cytometry, interaction between P-Rex2a and PTEN-long was detected by co-immunoprecipitation(Co-IP), and PI3K activity-related proteins in SGC-7901 cells were detected by Western blotting. The P-Rex2a interference expression vector was constructed to detect the effect of siP-Rex2a on cell proliferation and apoptosis. Results The level of PTEN-long mRNA in gastric cancer tissues was significantly lower than that in adjacent tissues, the difference was statistically significant(P<0.05). The levels of PTEN-long mRNA in gastric cancer cell lines BGC-823, AGS, MGC-803 and SGC-7901 were significantly lower than those in gastric epithelial cells GES-1, the differences were statistically significant(P<0.05). Compared with the control group and NC group, the proliferation of gastric cancer cell SGC-7901 in PTEN-Long-RFP group was significantly decreased, the apoptotic ability of gastric cancer cells SGC-7901 in the PTEN-Long-RFP group was significantly increased, the differences were statistically significant(P<0.05). P-Rex2a can interact with PTEN-long, and overexpression of P-Rex2a can enhance the binding of P-Rex2a to PTEN-long;when the protein level of P-Rex2a increases with time, the protein level of PTEN-long gradually decreases. After 48-96 h culture, compared with the control group and the si-NC group, the proliferation of gastric cancer cells SGC-7901 in the siP-Rex2a group was significantly decreased;compared with the control group and the si-NC group, the apoptotic ability of SGC-7901 the gastric cancer cells in the siP-Rex2a group was significantly increased, the differences were statistically significant(P<0.05). P-Akt, P-PI3K and Bcl-2 in the siP-Rex2a group were significantly decreased compared with the control group and the si-NC group, Caspase3, PARP, Bax, Bad in the SiP-Rex2a group were significantly increased, the differences were statistically significant(P<0.05). After 48-96 h culture, the cell proliferation rate in the siP-Rex2a group was significantly lower than that in the control and si-NC groups, the cell proliferation rate in the siP-Rex2a+PTEN-long group was significantly lower than that in the siP-Rex2a group, the differences were statistically significant(P<0.05);the cell apoptosis rate in the siP-Rex2a group was significantly higher than that in the control group and si-NC group, the apoptosis rate in siP-Rex2a+PTEN-long group was significantly higher than that in siP-Rex2a group, the differences were statistically significant(P<0.05). Conclusion PTEN long is involved in the occurrence and development of gastric cancer, thereby inhibiting the proliferation of gastric cancer cell SGC-7901 and promoting its apoptosis, which is expected to become a new target for potential treatment of gastric cancer.