摘要
目的 探讨AR如何通过调控微小RNA(miRNA,miR)-9-5p和N-myc下游调节基因1(NDRG1)通路,从而影响胰腺导管腺癌(PDAC)细胞的侵袭.方法 对PDAC组织和相应非肿瘤组织样本的分析.我们通过在PDAC细胞系PANC-1、BxPC-3细胞中过表达或敲低AR,用Transwell方法观察AR对PANC-1、BxPC-3及侵袭的影响.通过生信分析寻找能够调控靶基因NDRG1表达的miRNA,并用定量聚合酶链反应(qPCR)和蛋白质印迹法(Western blot)验证该miRNA与NDRG1的调控关系,观察其对PANC-1、BxPC-3细胞侵袭的影响.通过转染miR-9-5p和NDRG1等基因来评估对PANC-1、BxPC-3细胞侵袭能力的影响,采用t检验比较各组间差异.结果 相对于载体对照细胞(PANC-1、BxPC-3-Ctrl),过表达 AR促进了 PDAC 细胞(PANC-1、BxPC-3-oeAR)的 PDAC 细胞侵袭(0.65±0.07 比 2.11±0.52、0.74±0.05 比 2.28±0.65),差异有统计学意义(t=21.160、10.120,P＜0.05).相对对照细胞(PANC-1、BxPC-3-Scr),敲除AR抑制了 PDAC细胞的侵袭(PANC-1-shAR、BxPC-3-shAR)(0.75±0.12 比 0.34±0.02、0.84±0.13 比 0.44±0.03)差异有统计学意义(t=45.117、13.155,P＜0.01).miR-9-5p模拟物能显著抑制AR过表达PDAC细胞PANC-1、BxPC-3 的侵袭能力(0.67±0.06 比 2.25±0.22、0.88±0.07 比 2.07±0.32,t=15.124、8.156,P＜0.05),而miR-9-5p抑制剂则能增强对照组细胞的侵袭能力(1.64±0.23比0.75±0.09、1.48±0.26比0.84±0.15,t=11.143、9.175,P＜0.05).PDAC组织里,miR-9-5p的产量明显低于正常胰腺组织(0.23±0.02比1.58±0.57,t=8.122,P＜0.05).利用双荧光素酶基因报告实验,我们更深入地肯定了 miR-9-5p直接影响NDRG1的3'非翻译区(3'UTR),以此压低其表达.miR-9-5p的过表达显著降低了 NDRG1 蛋白水平(0.76±0.04 比 0.42±0.22,t=7.143、11.106,P＜0.05),而miR-9-5p 的抑制剂则提高了 NDRG1 蛋白表达(0.46±0.02 比 1.22±0.26,t=6.121、15.106,P＜0.05).oeAR 显著增加了 NDRG1 的表达(0.37±0.02 比 1.56±0.45,t=8.197、8.996,P＜0.05),而 AR 的抑制则减少了NDRG1 的表达(0.66±0.05 比 0.22±0.04,t=11.751、6.196,P＜0.05).过表达 NDRG1 显著抑制PDAC 细胞的侵袭能力(1.13±0.12 比 0.42±0.06,t=5.175、7.776,P＜0.05).NDRG1 敲除 PDAC 细胞侵袭能力显著升高(0.56±0.06比1.36±0.27,t=16.151、17.135,P＜0.01).结论 雄激素受体通过miR-9-5p/NDRG1通路抑制PDAC细胞侵袭的机制,为PDAC的治疗提供治疗策略.
Abstract
Objective Androgen receptor(AR)is widely expressed in pancreatic ductal adenocar-cinoma(PDAC)cells and may be associated with tumor invasion and metastasis.This study aimed to ex-plore how AR affects PDAC cell invasion by regulating the miR-9-5p and N-myc downstream-regulated gene 1(NDRG1)pathway.Methods We analyzed PDAC tissue and corresponding non-tumor tissue samples.AR was overexpressed or knocked down in PDAC cell lines PANC-1 and BxPC-3,and the effects on inva-sion were observed using the Transwell method.A miRNA that could regulate the expression of the target gene NDRG1 was identified through bioinformatic analysis.The regulatory relationship between this miRNA and NDRG1 was verified by quantitative polymerase chain reaction(qPCR)and Western blotting,and its effect on the invasion of PANC-1 and BxPC-3 cells was observed.Finally,transfection of miR-9-5p and NDRG1 genes was performed to evaluate their effect on the invasion ability of PANC-1 and BxPC-3 cells,and a t-test was used to compare the differences among all groups.Results Overexpression of AR promo-ted PDAC cell invasion in PDAC cells(PANC-1,BxPC-3-oeAR)compared with vector control cells(PANC-1,BxPC-3-Ctrl)(0.65±0.07 vs.2.11±0.52,0.74±0.05 vs.2.28±0.65,t=21.160,10.120,P＜0.05).AR knockout inhibited the invasion of PDAC cells(PANC-1-SHAR,BxPC-3-shAR)compared with control cells(PANC-1,BxPC-3-Scr)(0.75±0.12 vs.0.34±0.02,0.84±0.13 vs.0.44±0.03,t=45.117,13.155,P＜0.01).miR-9-5p mimics significantly inhibited the invasion ability of AR-overexpressed PDAC cells(PANC-1,BxPC-3)(0.67±0.06 vs.2.25±0.22,0.88±0.07 vs.2.07±0.32,t=15.124,8.156,P＜0.05).miR-9-5p inhibitors enhanced the invasion ability of control group cells(PANC-1,BxPC-3)(1.64±0.23 vs.0.75±0.09,1.48±0.26 vs.0.84±0.15,t=11.143,9.175,P＜0.05).miR-9-5p production in PDAC tissues was significantly lower than in normal pancreatic tissues(0.23±0.02 vs.1.58±0.57,t=8.122,P＜0.05).Dual-luciferase gene reporter as-says confirmed that miR-9-5p directly targets the 3'untranslated region(3'UTR)of NDRG1,thereby sup-pressing its expression.Overexpression of miR-9-5p significantly decreased the level of NDRG1 protein(0.76±0.04 vs.0.42±0.22,t=7.143,11.106,P＜0.05),while miR-9-5p inhibitors increased NDRG1 protein expression(0.46±0.02 vs.1.22±0.26,t=6.121,15.106,P＜0.05).Overexpres-sion of AR significantly increased NDRG1 expression(0.37±0.02 vs.1.56±0.45,t=8.197,8.996,P＜0.05),while AR inhibition decreased NDRG1 expression(0.66±0.05 vs.0.22±0.04,t=11.751,6.196,P＜0.05).Overexpression of NDRG1 significantly inhibited PDAC cell invasion(1.13±0.12 vs.0.42±0.06,t=5.175,7.776,P＜0.05),while NDRG1 knockout significantly increased PDAC cell in-vasion(0.56±0.06 vs.1.36±0.27,t=16.151,17.135,P＜0.01).Conclusion This study reveals that androgen receptors inhibit PDAC cell invasion through the miR-9-5p/NDRG1 pathway,providing new insights and potential therapeutic strategies for PDAC treatment.
基金项目
广东省钟南山医学基金会科研基金(ZNSXS-20220072)
NETL
NSTL
万方数据