长链非编码RNA H19通过靶向miR-18b调控Wnt/β-catenin信号通路对宫颈癌细胞顺铂耐药性的影响  

Effects of long non-coding RNA H19 on cisplatin resistance in cervical cancer cells by targeting miR-18b and regulating Wnt/β-catenin signaling pathway

作　　者：王乾印 祁青玲[1] 王爱敏 高文君 巩雪 王烈宏[1] WANG Qianyin;QI Qingling;WANG Aimin;GAO Wenjun;GONG Xue;WANG Liehong(Department of Gynecology,Qinghai Red Cross Hospital,Xining,Qinghai 810000,China)

机构地区：[1]青海红十字医院妇科,青海西宁810000

出　　处：《中国优生与遗传杂志》2022年第4期570-575,共6页Chinese Journal of Birth Health & Heredity

基　　金：青海省(应用)基础研究计划项目(2021-ZJ764)。

摘　　要：目的 探究长链非编码RNA(lncRNA)H19对宫颈癌顺铂耐药性的影响及分子机制。方法 体外培养人宫颈癌细胞株HeLa,采用顺铂梯度暴露法构建宫颈癌顺铂耐药细胞HeLa/DDP;qRT-PCR检测正常宫颈细胞ECT1/E6E7、HeLa细胞和HeLa/DDP细胞中H19、miR-18b的相对表达水平;以HeLa/DDP细胞为研究对象,根据转染载体不同将细胞分为pcDNA-NC组、pcDNA-H19组、si-H19组、si-H19+inhibitor-NC组、si-H19+miR-18binhibitor组;qRT-PCR检测细胞转染后H19和miR-18b表达水平;MTT法检测细胞存活率和顺铂半数抑制浓度(顺铂IC50值);流式细胞术检测细胞凋亡;双荧光素酶报告实验验证H19与miR-18b的靶向关系;Western blot检测细胞中凋亡相关蛋白cleavedcaspase-3、Bax、Bcl-2及Wnt/β-catenin信号通路相关蛋白表达水平。结果 与HeLa细胞比较,HeLa/DDP细胞中H19表达水平显著升高(P<0.05),miR-18b表达水平显著降低(P<0.05);干扰H19的表达后,HeLa/DDP细胞存活率和顺铂IC50、Bcl-2蛋白表达显著降低(P<0.05),细胞凋亡率、cleavedcaspase-3、Bax蛋白表达显著升高(P<0.05);双荧光素酶报告实验证实H19可负向调控miR-18b的表达;抑制miR-18b表达可明显逆转干扰H19对HeLa/DDP细胞增殖及凋亡的影响,从而降低HeLa/DDP细胞对顺铂的耐药性。结论 干扰H19可能通过负向调控miR-18b表达,抑制Wnt/β-catenin信号通路活性,降低宫颈癌细胞对顺铂的耐药性。Objective To explore the effect and molecular mechanism of long non-coding RNA(lncRNA) H19 on drug resistance of cervical cancer. Methods Human cervical cancer cell line HeLa was cultured in vitro, and cisplatin resistant cervical cancer cells HeLa/DDP were constructed by cisplatin gradient exposure method;qRT-PCR was used to detect the relative expression levels of H19 and miR-18b in normal cervical cells ECT1/E6E7, HeLa cells and HeLa/DDP cells;HeLa/DDP cells were studied, According to different transfection vectors, the cells were divided into pcDNA NC group,pcDNA-H19 group, si-NC group, si-H19 group, si-H19+inhibitor-NC group, si-H19+miR-18b inhibitor group;The expression levels of H19 and mir-18b were detected by qRT-PCR;MTT assay was used to detect cell survival rate and cisplatin half inhibitory concentration(IC50);flow cytometry was used to detect cell apoptosis;double Luciferase Report experiment was used to verify the targeting relationship between H19 and miR-18b. Western blot was used to detect the expression of apoptosis related proteins cleaved caspase-3, Bax, Bcl-2 and Wnt/β-catenin signaling pathway related proteins. Results Compared with HeLa cells, the expression level of H19 in HeLa/DDP cells increased significantly(P<0.05), and the expression level of miR-18b decreased significantly(P<0.05). Interfere with the expression of H19, HeLa/DDP cell survival rate and cisplatin IC50, Bcl-2 protein expression significantly reduced(P<0.05), cell apoptosis rate, cleave-caspase 3, Bax protein expression was significantly increased(P<0.05). Dual luciferase reporting assay confirmed that H19 could negatively regulate the expression of miR-18b. Inhibition of miR-18b expression could significantly reverse the effects of interfering H19 on the proliferation and apoptosis of HeLa/DDP cells, thus reducing HeLa/DDP cells to cisplatin resistance. Conclusion Interference of H19 may inhibit the activity of Wnt/β-catenin signaling pathway and reduce cisplatin resistance of cervical cancer cells by negatively regula

关 键 词：长链非编码RNA H19 miR-18b 宫颈癌 顺铂 耐药性 

分 类 号：R737.33[医药卫生—肿瘤]