IQGAP1高表达抑制食管鳞癌细胞对顺铂化疗的敏感性

IQGAP1 overexpression inhibits the chemosensitivity of esophageal squamous cell carcinoma cells to cisplatin

本文旨在研究含IQ模序的GTP酶激活蛋白1(IQ motif containing GTPase-activating protein 1,IQGAP1)过表达或基因干扰是否影响食管鳞癌细胞对顺铂化疗的敏感性。质粒转染构建IQGAP1高表达和基因干扰的稳定细胞系,并采用Western blot进行鉴定。然后,采用不同浓度(2.5μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L)的顺铂处理细胞,通过3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑噻唑蓝[3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide,MTT]法检测IQGAP1高表达、IQGAP1基因干扰和相应对照细胞的活力,通过4′,6-二脒基-2-苯基吲哚(4′,6-Diamidino-2-phenylindole,DAPI)染色和流式细胞技术检测细胞凋亡率,Western blot检测凋亡相关蛋白的表达。结果显示,IQGAP1高表达和基因干扰的稳定细胞系成功建立。在不同浓度的顺铂处理后,MTT结果表明,IQGAP1高表达细胞活力明显高于对照细胞(P<0.05);而IQGAP1基因干扰细胞活力明显低于对照细胞(P<0.05)。DAPI染色和流式细胞检测结果显示,IQGAP1高表达细胞凋亡率明显低于对照细胞(P<0.05),IQGAP1基因干扰细胞凋亡率明显高于对照细胞(P<0.05)。Western blot结果表明,IQGAP1高表达细胞的含半胱氨酸的天冬氨酸蛋白水解酶-3酶原(pro-cysteinyl aspartate specific proteinase-3,Procaspase-3)和多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)原带表达水平均高于对照细胞,裂解PARP(cleaved-PARP)的表达水平低于对照细胞,IQGAP1基因干扰细胞的Procaspase-3和PARP原带表达水平均低于对照细胞。研究结果表明,IQGAP1过表达影响凋亡抑制食管鳞癌细胞对顺铂的敏感性,提示IQGAP1过表达是导致顺铂在食管鳞癌细胞中产生耐药的重要机制。干扰IQGAP1表达后能提高食管鳞癌细胞对顺铂化疗的敏感性,提示IQGAP1可能是提高食管鳞癌化疗敏感性的重要治疗靶点。

In order to investigate whether overexpression or interference of IQ motif containing GTPaseactivating protein 1(IQGAP1)affects cisplatin chemosensitivity in esophageal squamous cell carcinoma,stable cell lines with IQGAP1 overexpression and IQGAP1 interference were constructed by plasmid transfection and detected by Western blot.Then,the cells were treated with different concentrations of cisplatin(2.5μmol/L,5μmol/L,10μmol/L,20μmol/L,40μmol/L).The cell viability of IQGAP1 overexpression,IQGAP1 interference and control cells was detected by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The apoptosis rates were detected by 4′,6-Diamidino-2-phenylindole(DAPI)staining and flow cytometry.And the expression levels of apoptosis-related protein were detected by Western blot.Stable cell lines with IQGAP1 overexpression and IQGAP1 interference were successfully established.After treatment with cisplatin at different concentrations,the result of MTT assay showed that the viability of IQGAP1 overexpression cells was significantly higher than that of control cells,and the viability of IQGAP1interference cells was significantly lower than that of control cells(P<0.05).DAPI staining and flow cytometry assay showed that the apoptosis rate of IQGAP1 overexpression cells was significantly lower than that of control cells,and the apoptosis rate of IQGAP1 interference cells was significantly higher than that of control cells(P<0.05).The Western blot results showed that the expression levels of pro-cysteinyl aspartate specific proteinase-3(Procaspase-3)and poly ADP-ribose polymerase(PARP)in IQGAP1 overexpression cells were higher than those of control cells,and the expression levels of cleaved-PARP were lower than those of control cells.The expression levels of Procaspase-3 and PARP in IQGAP1 interference cells were lower than those of control cells.In summary,IQGAP1 can affect the sensitivity of tumor cells to cisplatin by inhibiting cell apoptosis,which suggests that the IQGAP1 overexpression is a molecular mechanism of cisplatin resistance in esophageal squamous cell carcinoma cells.Moreover,knockdown of IQGAP1 expression can improve the sensitivity of tumor cells to cisplatin chemotherapy,suggesting that IQGAP1 may be an important therapeutic target to improve the chemosensitivity of esophageal squamous cell carcinoma.