CBX7对维持肺癌干细胞表型及化疗敏感性的影响

Effects of CBX7 on phenotype maintenance and chemotherapy sensitivity of lung cancer stem cells

目的:探讨色素框同源蛋白7(CBX7)对肺癌干细胞(LCSCs)表型和化疗敏感性的调节作用及部分潜在机制。方法:培养肺癌A549、NCL-H460和H1299细胞株,分选CD44^(+)细胞进行后续实验。转染后,将CD44^(+)LCSCs分为空白组、阴性组(转染空载体)、shCBX7(沉默CBX7表达)。细胞计数试剂盒-8(CCK-8)测定和集落形成实验分析细胞增殖能力和对化疗药物的敏感性,使用染色质免疫沉淀(ChIP)和双荧光素酶报告基因测定法验证CBX7和CD44之间的关系,Western blot检测蛋白含量。结果:成功的从A549、H460和H1299细胞中富集了CD44^(+)LCSCs,CD44^(+)细胞百分比为97.1%~99.5%。与CD44^(-)细胞相比,CD44^(+)细胞中CBX7、CD44、Sox2、Nanog蛋白表达量更高(P<0.001),且CD44^(+)H460细胞CBX7蛋白表达量最高(P<0.05),可用作后续沉默CBX7表达实验。shCBX7组沉默CBX7表达后,CD44^(+)H460细胞的增殖率、集落形成能力、肿瘤球形成数量及细胞内CD44、Sox2、Nanog蛋白表达量明显低于空白组和阴性组(P<0.05)。ChIP-qPCR实验结果显示CBX7可与CD44基因启动子结合。CBX7过表达也显著增加了CD44启动子的荧光素酶活性(P<0.05)。此外,与空白组DDP(0.98±0.04)μmol/L和DOX(17.5±0.97)μmol/L相比,阴性组CD44^(+)H460细胞对DDP(0.87±0.03)μmol/L和DOX(16.3±1.04)μmol/L的IC_(50)基本一致(P>0.05);与阴性组相比,shCBX7组CD44^(+)H460细胞对DDP和DOX的IC 50分别降低为(0.08±0.01)μmol/L和(0.43±0.03)μmol/L(P<0.05)。结论:CBX7可维持LCSCs的干性特征并提高化疗敏感性,在肺癌临床治疗中具有潜在的研究价值。

Objective:To investigate the regulatory effects and potential mechanisms of chromobox protein homolog 7(CBX7)on the phenotype and chemotherapy sensitivity of lung cancer stem cells(LCSCs).Methods:Lung cancer cell lines A549,NCL-H460 and H1299 were cultured,and CD44^(+)cells were sorted for follow-up experiments.After transfection,CD44^(+)LCSCs were divided into blank group,negative group(transfected with empty vector)and shCBX7 group(silenced CBX7 expression).Cell count Kit 8(CCK-8)assay and colony formation assay were used to analyze cell proliferation capacity and sensitivity to chemotherapy drugs.The relationship between CBX7 and CD44 was verified using chromatin immunoprecipitation(ChIP)and dual luciferase reporter gene assays.The protein content was detected by Western blot.Results:CD44^(+)LCSCs were successfully enriched from A549,H460 and H1299 cells,and the percentage of CD44^(+)cells was 97.1%~99.5%.Compared with CD44^(-)cells,the expression of CBX7,CD44,Sox2 and Nanog proteins was higher in CD44^(+)cells(P<0.001),CBX7 protein expression was the highest in CD44^(+)H460 cells(P<0.05).Therefore,H460 cells were selected for CBX7 silting experiment.After silencing of CBX7 expression in the shCBX7 group,compared with blank group and negative group,the proliferation rate,colony formation ability,the number of tumor balls formed and the protein expressions of CD44,Sox2 and Nanog of CD44^(+)H460 cells were significantly decreased(P<0.05).The results of ChIP-qPCR showed that CBX7 could bind to CD44 gene promoter.The overexpression of CBX7 also significantly increased the luciferase activity of CD44 promoter(P<0.05).In addition,compared with blank groupDDP:(0.98±0.04)μmol/L and DOX(17.5±0.97)μmol/L,the IC 50 of DDP(0.87±0.03)μmol/L and DOX(16.3±1.04)μmol/L in CD44^(+)H460 cells of the negative group were basically the same(P>0.05).Compared with the negative group,the IC_(50) of DDP was decreased to(0.08±0.01)μmol/L and DOX was decreased to(0.43±0.03)μmol/L in CD44^(+)H460 cells of the shCBX7 group(P<0.05).Conclusion:CBX7 can maintain the stemness characteristics of LCSCs and improve the sensitivity of chemotherapy,which has potential research value in the clinical treatment of lung cancer.