摘要
目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因沉默对人黑色素瘤细胞替莫唑胺(TMZ)敏感性的影响。方法将前期构建的靶向MGMT基因特异性短发夹型RNA(shRNA)干扰载体转染人黑色素瘤A375细胞。根据转染质粒的不同及是否加药分组如下:对照组、TMZ组、阴性对照(NC)组、NC-TMZ组、MGMT干扰(MGMTi)组和MGMTi-TMZ(MGMT干扰联合TMZ)组。通过实时荧光定量PCR(qPCR)、Western blotting和免疫荧光验证质粒载体的干扰效率,MTT法和流式细胞术检测TMZ处理24、48和72 h后A375细胞的增殖和凋亡情况。Western blotting和免疫荧光检测ATR、ATM和γ-H2AX水平以评价细胞DNA的损伤程度。结果转染MGMT shRNA干扰载体后,MGMTi组MGMT在mRNA和蛋白水平与对照组相比降低(P<0.05)。MTT、流式细胞仪、Western blotting和免疫荧光检测结果显示,MGMTi-TMZ组的细胞抑制率、凋亡率及ATR、ATM和γ-H2AX水平较其他组明显升高(P<0.05)。结论沉默MGMT基因的表达能够增强TMZ对A375细胞增殖的抑制作用并促进细胞凋亡,同时增强DNA损伤作用。通过沉默MGMT基因可以提高人黑色素瘤A375细胞对TMZ的敏感性。
Objective To explore the influence of O6-methylguanine-DNA methyltransferase(MGMT)gene silencing on the sensitivity of human melanoma cells to temozolomide(TMZ).Methods The previously constructed MGMT gene specific short hairpin RNA(shRNA)interference vector was transfected into human melanoma A375 cells.According to the different transfection plasmids and whether or not to add drugs,A375 cells were divided into Control group,TMZ group,negative control(NC)group,NC-TMZ group,MGMT interference(MGMTi)group and MGMTi-TMZ(MGMT interference combined with TMZ)group.Real-time quantitative PCR(qPCR),Western blotting and immunofluorescence were used to verify the interference efficiency of plasmid vectors.MTT method and flow cytometry were used to detect the proliferation and apoptosis of A375 cells after 24,48 and 72 h of TMZ treatment.Western blotting and immunofluorescence were used to detect the levels of ATR,ATM andγ-H2AX to evaluate the DNA damage induced by TMZ.Results After transfection with MGMT shRNA interference vector,mRNA and protein levels of MGMT in MGMTi group were lower than those in Control group(P<0.05).Results of MTT,flow cytometry,Western blotting and immunofluorescence showed that the cell inhibition rate,apoptotic rate and levels of ATR,ATM andγ-H2AX in MGMTi-TMZ group were significantly higher than those in other groups(P<0.05).Conclusion Silencing of MGMT gene can enhance the inhibitory effect of TMZ on proliferation,promote apoptosis,and enhance DNA damage of A375 cells.MGMT gene silencing can improve the sensitivity of human melanoma A375 cells to TMZ.
作者
任冰洁
李强
赵晶
陶敏
梁容瑞
胡颖熹
REN Bingjie;LI Qiang;ZHAO Jing;TAO Min;LIANG Rongrui;HU Yingxi(Department of Oncology, the First Affiliated Hospital of Suzhou University, Suzhou 215006, China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2020年第12期1057-1063,共7页
Chinese Clinical Oncology
基金
国家自然科学基金资助项目(81402477,81602802)
江苏省自然科学基金资助项目(BK20140295)
江苏政府留学奖学金(JS-2018-179)。
