舒尼替尼耐药肾癌细胞中circTMBIM6表达变化及其与miR-19a-3p/PPARα轴的调控关系  

Expression of circTMBIM6 and its relationship with miR-19a-3p/PPARαaxis in sunitinib-resistant renal carcinoma cells

作　　者：艾尼瓦尔·艾木都拉[1] 张骥驰 张瑞丽[1] 阿不来提·买买提明[2] Ainiwaer·Aimudula;ZHANG Jichi;ZHANG Ruili;Abulaiti·Maimaitimimg(Cancer Center,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;不详)

机构地区：[1]新疆医科大学第一附属医院肿瘤中心,乌鲁木齐830054 [2]新疆医科大学第一附属医院泌尿中心

出　　处：《山东医药》2022年第26期6-11,共6页Shandong Medical Journal

基　　金：国家自然科学基金项目(81760452)。

摘　　要：目的观察舒尼替尼耐药的肾癌细胞中circTMBIM6表达变化,并探讨其与miR-19a-3p/PPARα轴的关系。方法取舒尼替尼耐药的肾癌细胞A498、Caci-1、780-O,采用qRT-PCR法检测细胞中TMBIM6表达。采用Star⁃base、Targetscan数据库,分别预测miR-19a-3p与circTMBIM6 mRNA的3'非翻译区(3'UTR)和PPARαmRNA的3'UTR是否存在互补结合。构建包含结合位点的野生型和突变型circTMBIM6 mRNA 3'UTR双荧光报告质粒。将circTMBIM6-WT、circTMBIM6-MUT与miR-19a-3p mimics或miR-NC阴性对照共转染至786-O细胞。同时将PPARα-WT,PPARα-MUT与miR-19a-3p mimics或miR-NC阴性对照共转染至786-O细胞,构建野生型和突变型的PPARαmRNA 3'UTR双荧光报告质粒,检测miR-19a-3p对circTMBIM63'UTR和PPARαmRNA 3'UTR荧光素酶报告基因活性的影响。si-circTMBIM6和si-NC+miR-NC阴性对照共转染至A498、Caci-1、780-O细胞,将3种肾癌细胞分为抑表达组、对照组,分别转染si-circTMBIM6和si-NC+miR-NC,采用qPCR法检测三种细胞中miR-19a-3p表达。将三种细胞分为miR-19a-3p过表达组、miR-19a-3p抑表达组和对照组,分别转染miR-19a-3p mimics、miR-19a-3p inhibitor及si-NC+miR-NC阴性对照,采用qRT-PCR法和Westernblotting法检测PPARαmRNA和蛋白表达。将三种肾癌细胞分成阴性对照组、miR-19a-3p抑制组、circTMBIM6抑制组,分别转染si-NC+miR-NC阴性对照、si-NC+miR-19a-3p inhibitor和si-circTMBIM,采用RT-PCR法和Westernblotting法分别检测PPARαmRNA和蛋白表达,进一步验证circTMBIM6通过miR-19a-3p调控PPARα的表达。采用CCK-8实验检测各组细胞增殖抑制率,观察肾癌细胞舒尼替尼耐药中circTMBIM6与miR-19a-3p/PPARα轴的调控作用。结果与正常肾癌细胞相比,舒尼替尼耐药的肾癌细胞circTMBIM6表达均升高(P均<0.01)。生物信息学分析预测显示,miR-19a-3p分别与circTMBIM6的3'UTR以及PPARαmRNA的3'UTR具有潜在互补结合位点。双荧光素酶报告基因活性检测显示,在circTMBIM6野生型3'UTR中,miRObjective To investigate the expression level of circTMBIM6 in sunitinib-resistant renal carcinoma(RCC)cells and to explore its relationship with miR-19a-3p/PPARαaxis.Methods The expression of TMBIM6 in suni⁃tinib-resistant RCC cell lines A498,Caci-1 and 780-O was detected by qRT-PCR.The STARBASE and Target scan data⁃bases were used to predict the complementary binding of miR-19a-3p to the 3'untranslated region(3'UTR)of circTMBIM6 mRNA and the 3'UTR of PPARαmRNA.The wild-type and mutant circTMBIM6 mRNA 3'UTR double fluorescence re⁃porter plasmids containing binding sites were constructed.786-O cells were co-transfected with circTMBIM6-WT,circTM⁃BIM6-MUT,miR-19a-3p mimics,or miR-NC negative control.At the same time,PPARα-WT,PPARα-MUT and miR-19a-3p mimics or miR-NC negative control were co-transfected into 786-O cells to construct the wild-type and mutant PPARαmRNA 3'UTR double fluorescence reporter plasmids.The effects of miR-19a-3p on the activities of circTMBIM63'UTR and PPARαmRNA 3'UTR luciferase reporter gene were detected.The si-circTMBIM6 and si-NC+miR-NC nega⁃tive control were co-transfected into A498,Caci-1 and 780-O cells.The three types of renal carcinoma cells were divided into the si-circTMBIM6(down-regulated group)and si-NC+miR-NC group(control group).The expression of miR-19a-3p in the above three cell lines was detected by qPCR.These three kinds of cells were divided into the miR-19a-3p overex⁃pression group,miR-19a-3p inhibitor group,and control group,which were transfected with miR-19a-3p mimics,miR-19a-3p inhibitor and si-NC+miR-NC negative control,respectively.The expression of PPARαmRNA and protein was de⁃tected by qRT-PCR and Western blotting.These three kinds of renal carcinoma cells were divided into the negative control group,miR-19a-3p inhibitor group,and circTMBIM6 inhibitor group,which were transfected with si-NC+miR-NC nega⁃tive control,si-NC+miR-19a-3p inhibitor,and si-circTMBIM,respectively.RT-PCR and Western blotting were used to detect the expression levels o

关 键 词：肾肿瘤 舒尼替尼耐药 circTMBIM6 miR-19a-3p PPARΑ 

分 类 号：R737.11[医药卫生—肿瘤]