miR-141-3p、KLF9异常表达对前列腺癌细胞株药物敏感性和雄激素受体表达的影响及靶向关系  

Effects of abnormal expression of miR-141-3p and KLF9 on drug sensitivity and androgen receptor expression of prostate cancer cells and the targeting relationship

作　　者：刘彼得 靳宏勇[1,2] 王书恒 李循[1,2] 李九智[1,2] LIU Bide;JIN Hongyong;WANG Shuheng;LI Xun;LI Jiuzhi(Department of Urology,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China;不详)

机构地区：[1]新疆维吾尔自治区人民医院泌尿中心,乌鲁木齐830001 [2]新疆维吾尔自治区人民医院泌尿外科研究室

出　　处：《山东医药》2024年第15期1-8,共8页Shandong Medical Journal

基　　金：新疆维吾尔自治区科学技术厅自治区自然科学基金项目(2020D01C121);新疆维吾尔自治区人民医院院内科研项目(20190110)。

摘　　要：目的观察miR-141-3p、Krüppel样因子9(KLF9)对前列腺癌细胞株药物敏感性、雄激素受体(AR)表达的影响,验证miR-141-3p、KLF9之间的靶向关系,以探讨miR-141-3p、KLF9对PCa药物敏感性的调控作用及机制。方法选择雄激素敏感且表达AR的LNCaP细胞株。将细胞分为miR-141-3p低表达组、miR-141-3p正常组、KLF9过表达组、KLF9正常组,采用脂质体转染法分别转染miR-141-3p-inhibitor、inhibitor阴性对照物(inhibitor-NC)、KLF9过表达质粒、空白质粒。上述各组细胞加入不同浓度(0、10、25、50、75、100µmol/L)比卡鲁胺或(0.5、1.0、2.5、5.0 nmol/L)多西他赛后培养48 h,检测各组细胞OD值,计算细胞增殖率(反映药物敏感性)。采用qRT-PCR和Western blotting法检测各组细胞中AR及AR-V7。采用TargetScan数据库预测miR-141-3p与KLF9是否存在结合位点,然后采用荧光素酶报告基因实验验证miR-141-3p与KLF9的靶向调控关系[将LNCaP细胞株分别分为A、B、C、D组,A组先后转染野生型KLF9荧光素酶报告基因质粒(KLF9-WT)、inhibitor-NC,B组先后转染KLF9-WT、miR-141-3p-inhibitor,C组先后转染突变型KLF9荧光素酶报告基因质粒(KLF9-Mut)、inhibitor-NC,D组先后转染KLF9-Mut、miR-141-3p-inhibitor,转染24 h后检测各组荧光素酶活性]。通过细胞功能回复实验进一步验证miR-141-3p通过靶向调控KLF9抑制前列腺癌细胞药物敏感性[将LNCaP细胞株分别分为a、b、c、d组,a组先后转染si-KLF9阴性对照(si-Ctrl)、inhibitor-NC,b组先后转染si-Ctrl、miR-141-3p-inhibitor,c组先后转染si-KLF9、inhibitor-NC,d组先后转染si-KLF9、miR-141-3p-inhibitor,转染24 h后分别使用75µmol/L比卡鲁胺或2.5 nmol/L多西他赛处理细胞48 h,使用CCK-8法检测细胞OD值,并计算细胞增殖率]。结果LNCaP细胞培养48 h,在无药物加入时,miR-141-3p低表达组细胞增殖率低于miR-141-3p正常组(P均<0.05),KLF9过表达组细胞增殖率低于KLF9正常组(P均<0.05);随着药物浓度升�Objective To observe the effects of microRNA-141-3p(miR-141-3p)and Krüppel-like factor 9(KLF9)on the drug sensitivity and androgen receptor(AR)expression of prostate cancer(PCa)cells,and to verify the targeting relationship between miR-141-3p and KLF9 in order to explore the regulatory effects and mechanism of miR-141-3p and KLF9 on the drug sensitivity of PCa cells.Methods Androgen-sensitive LNCaP cell line with AR expression was selected for this study.The cells were divided into low miR-141-3p expression group,normal miR-141-3p group,KLF9 overexpression group,and normal KLF9 expression group,respectively,which were transfected with the miR-141-3p-inhibitor,inhibitor-negative control(NC)group,KLF9 overexpression plasmid,and empty plasmid,respectively,using liposome transfection method.The cells were treated with different concentrations of bicalutamide(0,10,25,50,75,and 100µmol/L)or docetaxel(0,0.2,0.5,1.0,2.5,and 5.0 nmol/L).After 48 h of culture,cell OD value was detected by CCK-8,and cell proliferation rate was calculated to evaluate drug sensitivity.The expression of AR and AR-V7 of LNCaP cells was analyzed by qRT-PCR and Western blotting.TargetScan database was used to predict binding sites between miR-141-3p and KLF9.Then,the luciferase reporter gene experiment was used to verify the targeted regulatory relationship between miR-141-3p and KLF9.LNCaP cells were divided into groups A,B,C and D.Cells in the group A were transfected with wild-type KLF9 luciferase reporter gene plasmid(KLF9-WT)and inhibitor-NC;cells in the group B were transfected with KLF9-WT and miR-141-3p-inhibitor;cells in the group C were transfected with mutant KLF9 luciferase reporter gene plasmid(KLF9-Mut)and inhibitor-NC;cells in the group D were transfected with KLF9-Mut and miR-141-3pinhibitor.The relative luciferase activity of cells in each group was detected by dual luciferase reporter gene assay at 24 h after transfection.The inhibitory effect of miR-141-3p targetedly regulating KLF9 on the drug sensitivity of LNCaP cell was f

关 键 词：微小RNA-141-3p Krüppel样因子 药物敏感性 比卡鲁胺 多西他赛能 雄激素受体 雄激素受体剪接变异体7 前列腺癌 

分 类 号：R737.25[医药卫生—肿瘤]