circ_0000263通过miR-338-3p/TERT抑制端粒酶蛋白活性影响HeLa细胞生物学功能提高放射敏感性  

Circ_0000263 improves radiosensitivity of Hela cells by inhibiting the activity of telomerase protein through miR-338-3p/TERT

作　　者：王冲[1] 霍研锟 李梦亚 李潺[1] 申晓辉 王树娟[1] 刘延方[1] 姜中兴[1] Wang Chong;Huo Yankun;Li Mengya;Li Chan;Shen Xiaohui;Wang Shujuan;Liu Yanfang;Jiang Zhongxing(Department of Hematology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院血液科,郑州450052

出　　处：《中华肿瘤杂志》2024年第7期676-685,共10页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目(U1804191);河南省重点研发与推广专项(科技攻关)项目(232102310308);河南省卫生健康科技创新领军人才项目(YXKC2021011)。

摘　　要：目的探讨circ_0000263对HeLa细胞活性、凋亡、端粒酶活性以及放射敏感性的影响及其分子机制。方法采用实时荧光定量聚合酶链反应检测circ_0000263和miR-338-3p mRNA表达水平,细胞克隆形成实验检测细胞存活情况,细胞计数试剂盒8检测细胞增殖,流式细胞术检测细胞凋亡,Western blot法检测蛋白增殖细胞核抗原(PCNA)、Cleaved-casp3、端粒酶逆转录酶(TERT)的表达,双荧光素酶实验检测circ_0000263和miR-338-3p、miR-338-3p和TERT的靶向关系,端粒重复序列扩增酶联免疫吸附试验检测端粒酶活性。结果Hela细胞中circ_0000263呈高表达,miR-338-3p呈低表达,TERT呈高表达,射线辐射的HeLa细胞中circ_0000263也呈高表达(均P<0.05)。敲减circ_0000263可抑制HeLa细胞克隆形成和细胞增殖能力,增强HeLa细胞的辐射敏感性和细胞凋亡。敲减circ_0000263可降低HeLa细胞中PCNA蛋白表达水平,增强HeLa细胞中Cleaved-casp3蛋白表达水平(P<0.05)。si-circ_0000263(si-circ)组细胞凋亡率为(13.19±1.12)%,高于si-NC组[(6.80±0.62)%,P<0.05];si-circ+4 Gy组细胞凋亡率为(24.82±1.57)%,高于si-NC+4 Gy组[(17.00±0.96)%,P<0.05]。circ_0000263靶向调控miR-338-3p,miR-338-3p靶向调控TERT。miR-338-3p在Hela细胞中呈低表达,敲减circ_0000263可提高HeLa细胞中miR-338-3p表达水平。circ_0000263通过miR-338-3p调控TERT表达,抑制端粒酶活性。miR-338-3p/TERT均能恢复抑制circ_0000263对Hela细胞放射敏感性的影响。si-circ+anti-NC组细胞凋亡率为(27.37±0.89)%,高于si-circ+anti-miR-338-3p组[(18.22±1.18)%,P<0.05];si-circ+vector组细胞凋亡率为(27.55±0.48)%,高于si-circ+TERT组[(20.10±0.68)%,P<0.05]。经4 Gy射线照射72 h后,si-circ+anti-NC组细胞存活分数(0.41±0.02)低于si-circ+anti-miR-338-3p组(0.66±0.03,P<0.05);si-circ+vector组细胞存活分数(0.42±0.05)低于si-circ+TERT组(0.70±0.03,P<0.05)。结论抑制circ_0000263的表达通过调控miR-338-3p/TERT抑制Hela细胞增殖,促进细胞凋亡,�Objective To explore the effect and molecular mechanism of circ_0000263 on HeLa cell activity,apoptosis,telomerase activity,and radiosensitivity.Methods The Hela cells were divided into si-NC,si-circ,vector,circ_0000263,anti-NC,anti-miR-338-3p,miR-NC,miR-338-3p,si-circ+anti-NC,si-circ+anti-miR-338-3p,si-circ+vector,si-circ+TERT,sh-NC,sh-circ groups.Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the expressions of circ_0000263 and miR-338-3p.Cell clone formation array was used to detect cell survival;cell counting kit-8(CCK-8)to detect cell proliferation;flow cytometry to detect apoptosis;western blot method to detect the expressions of proliferating cell nuclear antigen(PCNA),Cleaved-casp3,telomerase reverse transcriptase(TERT)proteins;double luciferase assay to detect the targeting relationships of circ_0000263 and miR-338-3p,miR-338-3p and TERT;telomere repeat amplification enzyme linked immunosorbent assay(TRAR-ELISA)to detect telomerase activity.Results Circ_0000263 was highly expressed in Hela cells,miR-338-3p was low expressed,and TERT was highly expressed;circ_0000263 was also highly expressed in Hela cells treated with radiation(P<0.05).Knockdown of circ_0000263 inhibited the clone formation and cell proliferation ability of HeLa cells,and enhanced the radiosensitivity and apoptosis of HeLa cells.In contrast,knockdown of circ_0000263 decreased PCNA protein expression level and enhanced Cleaved-casp3 protein expression level in HeLa cells(P<0.05).The apoptosis rate in the si-circ group was(13.19±1.12)%,which was higher than(6.80±0.62)%of si-NC group(P<0.05).The apoptosis rate in the si-circ+4 Gy group was(24.82±1.57)%,which was higher than(17.00±0.96)%of si-NC+4 Gy group(P<0.05).Circ_0000263 targeted regulated miR-338-3p,and miR-338-3p targeted regulated TERT.MiR-338-3p was lowly expressed in HeLa cells,and knockdown of circ_0000263 elevated miR-338-3p expression level in HeLa cells.Circ_0000263 regulated TERT expression and inhibited telomerase activi

关 键 词：妇科肿瘤 HELA细胞 circ_0000263 miR-338-3p 端粒酶逆转录酶 放射敏感性 细胞凋亡 

分 类 号：R737.3[医药卫生—肿瘤]