摘要
目的探讨长链非编码RNA(long non-coding RNA,LncRNA)核仁小RNA宿主基因6(SNHG6)对前列腺肿瘤干细胞增殖、凋亡及化疗敏感性的影响及其机制。方法采用流式分选技术从前列腺肿瘤细胞PC-3中分选出CD44+CD24-肿瘤干细胞和非CD44+CD24-细胞,采用实时荧光定量PCR检测两种细胞中SNHG6和微小RNA(miR)-26a表达,5-溴脱氧尿嘧啶核苷(BrdU)法检测细胞增殖,流式细胞仪检测细胞凋亡,四甲基偶氮唑盐(MTT)法检测细胞对顺铂的化疗敏感性,免疫印迹法(Western blot)检测细胞中细胞核增殖相关抗原(Ki67)、B淋巴细胞瘤-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达;双荧光素酶报告基因实验检测SNHG6、miR-26a和zeste基因增强子同源物2(EZH2)的靶向关系,Western blot检测miR-26a模拟物对EZH2蛋白表达的影响。结果与非CD44+CD24-细胞比较,SNHG6在CD44+CD24-细胞中的表达水平明显升高(P<0.05);与NC-siRNA组[(1.00±0.06)%、(96.85±6.48)%、(0.72±0.06)%、(0.43±0.03)%、(5.32±0.15)%、(0.35±0.03)]比较,SNHG6-siRNA组细胞中SNHG6表达水平(0.25±0.03)、细胞增殖活力(75.36±5.1)%和细胞中Ki67(0.38±0.03)、Bcl-2蛋白(0.21±0.02)表达水平均明显降低,而miR-26a表达水平、细胞凋亡率(13.83±2.36)%和Bax蛋白(0.48±0.03)表达水平均明显升高,且细胞对顺铂化疗敏感性明显增强(P<0.05);miR-26a是SNHG6的靶基因,而EZH2是miR-26a的靶基因,miR-26a模拟物可使EZH2蛋白表达水平降低。结论SNHG6在前列腺肿瘤干细胞中表达上调,干扰其表达可抑制肿瘤干细胞增殖,促进细胞凋亡,并增强肿瘤干细胞对顺铂的敏感性,其作用机制可能与靶向调控miR-26a/EZH2轴有关。
Objective To investigate the effects and its mechanism of long non-coding RNA(LncRNA)small nucleolar RNA host gene 6(SNHG6)on proliferation,apoptosis and chemosensitivity of prostate cancer stem cells.Methods CD44+CD24-tumor stem cells and non-CD44+CD24-cells were selected from prostate cancer cell PC-3 by flow separation technology,and the expression level of SNHG6 and microRNA(miR)-26a were detected by real-time fluorescence quantitative PCR.Cell proliferation was detected by 5-bromodeoxyuridine(Br-dU),the apoptosis was detected by flow cytometry,the chemosensitivity of cells to cisplatin was detected by methyl thiazolyl tetrazolium(MTT),and Western blot was used to detect the protein expressions of proliferating cell nuclear antigen(Ki-67),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax);moreover,the target relationships of SNHG6,miR-26a and zeste enhancer of zeste homolog 2(EZH2)were detected by double luciferase reporter gene assay,and Western blot was used to detect the effect of miR-26a analog on EZH2 protein expression.Results Compared with non-CD44+CD24-cells,the expression level of SNHG6 in CD44+CD24-cells was significantly higher(P<0.05);compared with NC-siRNA group[(1.00±0.06)%,(96.85±6.48)%,(0.72±0.06)%,(0.43±0.03)%,(5.32±0.15)%,(0.35±0.03)%],SNHG6 expression level(0.25±0.03),cell proliferation activity[(75.36±5.1)%],Ki67(0.38±0.03)and Bcl-2 protein(0.21±0.02)expression levels in SNHG6-siRNA group were significantly lower,while miR-26a expression level,apoptosis rate[(13.83±2.36)%]and Bax protein(0.48±0.03)expression level were significantly higher,and the sensitivity of the cells to cisplatin was significantly higher(P<0.05);in addition,miR-26a was the target gene of SNHG6,EZH2 was the target gene of miR-26a,and miR-26a analog could reduce the expression level of EZH2 protein.Conclusions SNHG6 is up-regulated in prostate cancer stem cells.Interfering SNHG6 expression can inhibit the proliferation of cancer stem cells,promote apoptosis,and enhance the sensitivity of cancer stem
作者
郑建波
于芳
史建华
王冬梅
于鹏
Zheng Jianbo;Yu Fang;Shi Jianhua;Wang Dongmei;Yu Peng(Reproductive Medicine Centre,Yantai Yantaishan Hospital,Yantai 264001,China;Cardiac Catheterization Laboratory,Yantai Yantaishan Hospital,Yantai 264001,China;Department of Urology Surgery,Hospital of the Joint Service Support Force of the PLA,Yantai 264001,China)
出处
《中华内分泌外科杂志》
CAS
2021年第5期536-541,共6页
Chinese Journal of Endocrine Surgery
基金
烟台市科技计划项目(2017YD018)。
