西格列汀调节Hippo/Yes相关蛋白信号通路对非小细胞肺癌A549/CDDP细胞增殖、迁移、凋亡与化疗敏感性的影响实验研究

Effects of sitagliptin on proliferation,migration,apoptosisand chemosensitivity of NSCLC A549/CDDP cells by regulating Hippo/YAP signaling pathway

目的:探究西格列汀(SIT)调节Hippo/Yes相关蛋白(YAP)信号通路对非小细胞肺癌(NSCLC)A549/CDDP细胞增殖、迁移、凋亡和化疗敏感性的影响。方法:常规培养顺铂耐药的NSCLC细胞株A549/CDDP,随机分为对照(Control)组(常规培养)、CDDP组(给予10μmol/L CDDP)、SIT组(给予10μmol/L SIT)、CDDP+SIT组(给予10μmol/L CDDP+10μmol/L SIT)和CDDP+SIT+YAP激活剂Jasplakinolide(CDDP+SIT+JASP)组(给予10μmol/L CDDP+10μmol/L SIT+50 nmol/L JASP)。CCK-8法检测细胞增殖能力,划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力,流式细胞术检测各组细胞凋亡率,实时荧光定量PCR(RT-qPCR)检测细胞YAP mRNA表达水平,Western blot检测Hippo/YAP信号通路相关蛋白与B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达水平。结果:与Control组比较,CDDP组和SIT组OD_(450)值(48、72 h)、细胞侵袭数目、细胞迁移率、YAP mRNA水平及YAP、Bcl-2蛋白表达下降,细胞凋亡率、磷酸化YAP(p-YAP)和Bax蛋白表达及哺乳动物STE20样激酶1/2(MST1/2)、大肿瘤抑制激酶1/2(LATS1/2)的磷酸化水平增高(均P<0.05)。与CDDP组比较,CDDP+SIT组上述指标趋势增强(均P<0.05)。与CDDP+SIT组比较,CDDP+SIT+JASP组减弱了SIT对A549/CDDP细胞化疗敏感性的提高。结论:SIT能够促进A549/CDDP细胞凋亡,抑制细胞迁移、侵袭和增殖,提高A549/CDDP细胞化疗敏感性,其机制可能与激活Hippo/YAP信号通路有关。

Objective:To investigate the effects of sitagliptin(SIT)on the proliferation,migration,apoptosis and chemosensitivity of non-small cell lung cancer(NSCLC)A549/CDDP cells by regulating the Hippo/Yes-associated protein(YAP)signaling pathway.Methods:The cisplatin resistant NSCLC cell line A549/CDDP was routinely cultured and randomly grouped into Control group(conventional culture),CDDP group(treated with 10μmol/L CDDP),SIT group(treated with 10μmol/L SIT)and CDDP+SIT group(treated with 10μmol/L CDDP+10μmol/L SIT),CDDP+SIT+YAP activator Jasplakinolide(CDDP+SIT+JASP)group(treated with 10μmol/L CDDP+10μmol/L SIT+50 nmol/L JASP).CCK-8 method was applied to detect cell proliferation.Scratch experiment was applied to detect the ability of cellsmigration.Transwell experiment was applied to detect the invasiveness of cells.The rate of apoptosis was detected by flow cytometry.The expression level of YAP mRNA was detected by RT-qPCR.Western blot was applied to detect the expression levels of cell Hippo/YAP signaling pathway related proteins and apoptosis related proteins Bcl-2 and Bax.Results:Compared with the Control group,the OD 450 value(48 and 72 hours),cell migration rate,cell invasion number,YAP mRNA expression,YAP and Bcl-2 protein expressions of A549/CDDP cells in the CDDP group and SIT group were decreased,the apoptosis rate,expressions of p-YAP,Bax,p-MST1/2 and p-LATS1/2 protein were increased(all P<0.05).Compared with the CDDP group,the above indexes in the CDDP+SIT group showed an enhanced trend(all P<0.05).Compared with CDDP+SIT group,CDDP+SIT+JASP group attenuated the enhancement of chemosensitivity of A549/CDDP cells induced by SIT.Conclusion:SIT can promote apoptosis of A549/CDDP cells,inhibit cell migration,invasion and proliferation,and increase chemotherapy sensitivity of A549/CDDP cells,the mechanism may be related to the activation of Hippo/YAP signaling pathway.