抑制酪氨酸磷酸酶2活性介导DNA损伤修复影响宫颈癌肿瘤放射敏感性的机制研究

Mechanism of DNA damage repair mediated by inhibition of tyrosine phosphatase 2 activity affecting radiosensitivity of cervical cancer tumors

目的 探究抑制酪氨酸磷酸酶2(SHP2)活性介导DNA损伤修复对人宫颈癌细胞放射敏感性的影响及机制。方法 使用不同浓度的SHP2特异性抑制剂PHPS1(5、10、15、20μmol/L)处理人宫颈癌细胞株SiHa,Western blot测定SHP2磷酸化水平变化,MTT法检测细胞增殖抑制率,不同剂量放射线(2、4、6、8 Gy)处理SiHa细胞,MTT法检测细胞增殖抑制率。实验分为对照组、PHPS1组、6 Gy组、PHPS1+6 Gy组,经过20μmol/L PHPS1或(和)6 Gy处理后,MTT法检测4组细胞增殖抑制率,EdU染色检测4组细胞增殖水平,流式细胞术测定4组细胞凋亡率,细胞免疫荧光染色实验观察4组细胞内组蛋白H2AX磷酸化(γ-H2AX)焦点形成情况,Western blot测定4组细胞内DNA损伤修复相关途径分子DNA依赖的蛋白激酶催化亚基(DNA-PKcs)、X线修复交叉互补蛋白4(XRCC4)、重组蛋白A51(RAD51)、乳腺癌易感蛋白1(BRCA1)、p53结合蛋白1(53BP1)蛋白表达。结果 与未经PHPS处理的细胞比较,10、15、20μmol/LPHPS1处理的细胞内p-SHP2蛋白相对表达量下调(P<0.05),5、10、15、20μmol/LPHPS1处理的细胞增殖抑制率升高(P<0.05);与未经放射线处理的细胞比较,2、4、6、8Gy放射线处理的细胞增殖抑制率也升高(P<0.05)。与6 Gy组比较,PHPS1+6 Gy组细胞增殖抑制率升高(P<0.05),EdU阳性率减少(P<0.05),细胞凋亡率增加(P<0.05),细胞内γ-H2AX焦点增加(P<0.05),细胞内DNA-PKcs、XRCC4、RAD51、BRCA1、53BP1蛋白相对表达量下调(P<0.05)。结论 抑制SHP2活性能够增加宫颈癌细胞放射敏感性,从而进一步促进细胞凋亡,该作用可能与其抑制DNA损伤修复过程有关。

Objective To explore the effect of DNA damage repair mediated by src homology-2 domain-containing phosphatase 2(SHP2)activity on the radiosensitivity of human cervical cancer cells and its mechanism.Methods Human cervical cancer cell line SiHa was treated with different concentrations of SHP2 specific inhibitor PHPS1(5,10,15,20μmol/L),the phosphorylation levels of SHP2 were measured by Western blot,the inhibition rate of cell proliferation was detected by MTT assay.SiHa cells were treated with different doses of radiation(2,4,6,8 Gy),and the inhibitory rate of cell proliferation was detected by MTT assay.The experiment was divided into control group,PHPS1 group,6 Gy group and PHPS1+6 Gy group.After treated with 20μmol/L PHPS1 or 6 Gy,MTT assay was used to detect the inhibition rate of cell proliferation in 4 groups,EdU staining was used to detect cell proliferation in the 4 groups,flow cytometry was used to determine cell apoptosis rate in the 4 groups,cell immunofluorescence staining was used to observe the formation of histone H2AX phosphorylation(gamma-H2Ax)focus in the 4 groups,Western blot was used to determine the protein expression of DNA-dependent protein kinase lytic subunit(DNA-PKcs),X-ray repair cross complementing protein4(XRCC4),recombination protein A51(RAD51),breast cancer suscepti-bility protein 1(BRCA1),p53-binding protein 1(53BP1)in the 4 groups.Results Compared to cells that were not treated with PHPS,the relative expression level of p-SHP2 protein in the cells treated with 10,15 and 20μmol/L PHPS1 was down-regulated(P<0.05).After 5,10,15,20μmol/L PHPS1 treatment,the cell proliferation inhibition rate was increased(P<0.05).Compared to cells that were not treated with Gy,the proliferation inhibition rate of cells treated with 2,4,6 and 8 Gy was also increased(P<0.05).Compared with the 6 Gy group,the inhibition rate of cell proliferation in PHPS1+6 Gy group was increased(P<0.05),the positive rate of EdU was decreased(P<0.05),the apoptosis rate was increased(P<0.05),and the intracellular-H2AX focus was increased(P<0.05),the relative protein expressions of DNA-PKcs,XRCC4,RAD51,BRCA1 and 53BP1 proteins were down-regulated(P<0.05).Conclusion Inhibition of SHP2 activity can increase the radiosensitivity of cervical cancer cells and further promote apoptosis,which may be related to the inhibitionofDNAdamagerepairprocess.