P300/CBP相关因子在BCa细胞DNA损伤修复与肿瘤干细胞干性维持中的作用  

The role of p300/cbp related factors in the repair of DNA damage in BCa cells and the maintenance of tumor stem cells

作　　者：李挺云 周治彦 周邦奋 王声兴[1] 梁培育[1] LI Tingyun;ZHOU Zhiyan;ZHOU Bangfen;WANG Shengxing;LIANG Yingyu(Department of Urology,The First Affiliated Hospital of Hainan Medical College,Haikou 570102,China)

机构地区：[1]海南医学院第一附属医院泌尿外科,海南海口570102

出　　处：《西部医学》2023年第9期1264-1269,共6页Medical Journal of West China

基　　金：海南省卫生健康行业科研项目(21A200117)。

摘　　要：目的探究P300/CBP相关因子(PCAF)在人膀胱癌(BCa)细胞DNA损伤修复与肿瘤干细胞干性维持中的作用。方法利用Ad-PCAF RNAi转染人膀胱癌细胞系5637,荧光显微镜和Western blot检测转染效果,选择顺铂诱导膀胱癌细胞DNA损伤,MTS法检测损伤细胞的增殖活性,免疫荧光染色检测损伤细胞内PCAF与γ-H2AX的募集情况,细胞彗星实验分析顺铂诱导细胞DNA损伤的程度;从膀胱癌细胞中分选CD44^(+)标记的肿瘤干细胞,Western blot检测Bca干细胞标志物67LR、OCT4及YAP1蛋白表达水平变化,细胞成球实验检测细胞干性变化。结果Ad-PCAF RNAi转染5637细胞后,明显抑制了细胞中PCAF表达(P<0.05);在下调PCAF表达后,20、40、60、80、100 nmol/L的顺铂处理下5637细胞活性下降,增强了5637细胞对顺铂的敏感性,细胞内γ-H2AX焦点形成明显增加,细胞拖尾较长,拖尾细胞数目和尾距均增加(P<0.05);经分选得到CD44^(+)比例为93%且67LR、OCT4及YAP1蛋白呈阳性表达的膀胱癌干细胞,通过下调PCAF表达,肿瘤干细胞的球体体积变小,成球个数也减少(P<0.05)。结论通过下调PCAF的表达能够抑制膀胱癌细胞DNA损伤修复,调控肿瘤干细胞的干性维持。Objective To explore the role of P300/CBP-associated factor(PCAF)in the repair of human bladder carcinoma(bladder carcinoma)cell DNA damage and the maintenance of tumor stem cell stemness.Methods Human bladder cancer cell line 5637 was transfected with Ad-PCAF RNAi.The transfection effect was detected by fluorescence microscopy and Western blot.Cisplatin was selected to induce DNA damage in bladder cancer cells.The proliferative activity of the injured cells was detected by MTS.The recruitment of PCAF andγ-H2AX in injured cells was detected by immunofluorescence staining.The degree of DNA damage induced by cisplatin was analyzed by comet assay.CD44^(+)labeled tumor stem cells were sorted from bladder cancer cells.The expression levels of Bca stem cell markers 67LR,OCT4 and YAP1 were detected by Western blot.Cell pelletizing assay was used to detect the changes of cell stem.Results An Ad-PCAF RNAi transfected into 5637 cells significantly inhibited the expression of PCAF(P<0.05).After the PCAF expression was down-regulated,the activity of 5637 cells was decreased under 20,40,60,80,100 nmol/L cisplatin treatment,and the sensitivity of 5637 cells to cisplatin was enhanced,the formation ofγ-H2AX focal point was significantly increased,the cell tail was longer,and the number and distance of trailing cells were increased(P<0.05).The bladder cancer stem cells with 93%CD44^(+)and positive expression of 67LR,OCT4 and YAP1 were obtained.By down-regulating the expression of PCAF,the spheroids of tumor stem cells became smaller and the number of spheroids formed was also decreased(P<0.05).Conclusion Down-regulating the expression of PCAF can inhibit DNA damage and repair of bladder cancer cells and regulate the maintenance of stemness of cancer stem cells.

关 键 词：膀胱癌 P300/CBP相关因子 DNA损伤 肿瘤干细胞 干性维持 

分 类 号：R737.14[医药卫生—肿瘤]