HMGB1/STAT3/Nanog信号通路在宫颈癌顺铂化疗耐药中的作用

The Role of HMGB1/STAT3/Nanog Signal Pathway in Cisplatin Chemoresistance during the treatment of Cervical Cancer

目的:研究HMGB1/STAT3/Nanog信号通路是否介导了宫颈癌顺铂化疗敏感性的调控。方法:体外培养宫颈癌耐药细胞株(Siha/DDP),SiRNA HMGB1和过表达STAT3后分别利用克隆形成法检测细胞增殖水平和Western blot检测细胞DNA损伤相关蛋白p H2AX,p53BP1,通路相关蛋白p STAT3和干细胞相关标志物Nanog蛋白表达的变化;并通过Annexin V/PI双染法检测细胞凋亡、PI单染法检测细胞周期和流式细胞技术检测干细胞标记物(OCT4、Sox2和Nanog)的表达。结果:SiHa/DDP细胞顺铂耐药指数为8.58,提示SiHa/DDP细胞显著耐药。与空载体组相比,HMGB1低表达组和HMGB1低表达+过表达STAT3组中集落形成计数明显减低(P <0. 05);p53BP1、p H2AX升高(P<0.05),p STAT3和Nanog蛋白明显降低(分别为P<0.05和P<0.001);细胞凋亡率增加和G1期分布增高(P<0.05),干细胞标记物(OCT4、Sox2和Nanog)表达降低(P<0.05)。实验说明HMGB1低表达可致细胞生长缓慢、抑制DNA损伤修复、肿瘤细胞增殖变慢和干细胞标记物(OCT4、Sox2和Nanog)比例减少。结论:HMGB1/STAT3/Nanog信号通路可能在顺铂对宫颈癌的化疗敏感性中起到重要的调控作用,SiRNA HMGB1表达可能有助于增强顺铂对宫颈癌化疗的敏感性。

Objective To examine whether HMGB1/STAT3/Nanog pathway mediates the regulation of chemosensitivity of cisplatin in cervical cancer.Methods Cell proliferation,DNA damage-related proteins pH2AX and p53BP1,pathway-related protein pSTAT3and stem cell marker Nanog were detected by clone formation assay and Western blot respectively after culturing drug-resistant cervical cancer cell lines (Siha/DDP)under different conditions,SiRNA HMGB1 and STAT3 overexpression.Annexin V/PI double staining was used to detect apoptosis,PI single staining was used to detect cell cycle and flow cytometry was used to detect the expression of stem cell markers (OCT4,Sox2and Nanog).Results The cisplatin resistance index of SiHa/DDP ceils was 8.58,suggesting that SiHa/DDP cells were significantly resistant to cisplatin.Compared with the empty vector group,the number of colony formation decreased significantly in HMGB1 low-expression group and HMGB1 low-expression+over-expression STAT3 group (P<0.05).p53BP1 and pH2AX increased (P<0.05).But pSTAT3 and Nanog protein decreased significantly (P<0.05 and P<0.001 respectively).The apoptotic rate and G1 phase distribution increased (P<0.05),and the expression of stem cell markers (OCT4,Sox2 and Nanog)decreased (P<0.05). The results showed that the low expression of HMGB1 could slow down the growth of cells,inhibit the repair of DNA damage;slow the proliferation of tumor ceils and reduce the proportion of stem cell markers (OCT4,Sox2and Nanog).Conclusion HMGB1/STAT3/Nanog signaling pathway may play an important regulatory role in the cervical cancer cell sensitivity to cisplatin chemotherapy.Down-regulation of HMGB1 may contribute to the enhancement of cervical cancer cell sensitivity to cisplatin chemotherapy.