双特异性磷酸酶2对胃癌细胞增殖凋亡的影响及机制研究

The effect of DUSP2 on proliferation and apoptosis of gastric cancer cells and its mechanism

目的探讨双特异性磷酸酶2(DUSP2)基因对胃癌细胞增殖和凋亡的影响及其可能的机制。方法首先利用在线分析工具KM plotter分析876例胃癌患者DUSP2表达高低对其总体生存率的影响,并在多种胃癌细胞株(MKN-45、SGC-7901、HGC-27、N-87)中检测DUSP2的表达。进一步构建DUSP2过表达慢病毒载体,包装病毒感染MKN-45细胞,筛选出DUSP2稳定过表达的胃癌细胞株,以空载慢病毒转染并筛选后的胃癌细胞为对照。采用MTS细胞增殖实验检测DUSP2上调表达对胃癌细胞增殖能力的影响;Annexin V-FITC/PI双染流式检测细胞凋亡变化;蛋白质印迹法(Western blot)检测DUSP2、细胞外调节蛋白激酶(ERK)、p-ERK(Thr202/Tyr204)、P38、p-P38等蛋白表达水平。结果高表达DUSP2的胃癌患者较低表达者有明显的生存优势,并且DUSP2在多株胃癌细胞中呈低表达。Western blot结果显示DUSP2稳定过表达的胃癌细胞(实验组)中DUSP2表达较对照组明显上调,DUSP2稳定过表达的胃癌细胞株构建成功。MTS实验结果表明,实验组细胞活力较对照组明显下降。实验组细胞凋亡率明显高于对照组。Western blot结果表明,实验组细胞中p-ERK(Thr202/Tyr204)及p-P38表达较对照组显著下调。结论上调DUSP2的表达可显著抑制胃癌细胞的增殖,并促进其凋亡,其机制与DUSP2抑制了ERK、P38的磷酸化水平有关。

Objective To investigate the effect of dual-specificity phosphatase-2(DUSP2)on the cell proliferation and apoptosis in the gastric cancer and its mechanisms.Methods Firstly,the effects of different expressions of DUSP2 on the overall survival of 876 gastric cancer patients were analyzed by online analysis tool KM plotter,and the expressions of DUSP2 in various gastric cancer cell lines(MKN-45,SGC-7901,HGC-27 and N-87)were verified.Secondly,DUSP2 overexpressed lentiviral vector was constructed,and MKN-45 was transfected by packaged virus.DUSP2-overexpression gastric cancer cell line was gained by drug screening.Meanwhile,gastric cancer cells infected with empty vector virus were used as control.Then the effect of DUSP2 upregulation on the proliferation ability of gastric cancer cells was evaluated by MTS cell proliferation assay,and the apoptosis was determined by Annexin V-FITC/PI double staining.The protein expressions of DUSP2,ERK,p-ERK(Thr202/Tyr204),P38 and p-P38 were tested by the Western blot analysis.Results Gastric cancer patients with high DUSP2 expression showed a significant survival advantage compared with those with low DUSP2 expression,and DUSP2 levels were decreased in several gastric cancer cell lines.The Western blot analysis revealed that the expression of DUSP2 markedly increased in overexpressed DUSP2 group(experimental group)compared with that of control group.The MTS experiment showed that the cell viability was significantly decreased in experimental group than that of the control group.Correspondingly,the cell apoptosis test showed that the cell apoptosis rate was obviously higher in the experimental group than that of the control group.The results of Western blot assay indicated that p-ERK(Thr202/Tyr204)and p-38 were significantly down-regulated in the experimental group compared with those of control group.Conclusion The over-expression of DUSP2 can efficiently inhibit cell proliferation and promote its apoptosis in gastric cancer cells,and the mechanism is related to DUSP2 inhibiting the phosphorylation levels of ERK and P38.