摘要
目的 探讨LINC00052通过靶向miR-148b-3p影响高糖诱导的肾小管上皮细胞增殖、凋亡及上皮间质转化(EMT)的分子机制.方法 将肾小管上皮细胞HK-2分为对照组(NC 组)、高糖组(HG 组)、pcDNA-LINC00052+HG 组、pcDNA-NC+HG 组、anti-miR-148b-3p+HG 组、anti-miR-NC+HG 组、miR-148b-3p+pcDNA-LINC00052+HG 组、miR-NC+pcDNA-LINC00052+HG组;采用实时荧光定量PCR(qRT-PCR)检测LINC00052和miR-148b-3p的表达水平;蛋白质印迹法(Western blot)检测E钙黏蛋白(E-Cadherin)、神经钙黏蛋白(N-Cadherin)、波形蛋白(vimentin)蛋白表达;CCK-8检测细胞活性;流式细胞术检测细胞凋亡;双荧光素酶报告实验分为:miR-NC+LINC00052-WT 组、miR-148b-3p+LINC00052-WT 组、miR-NC+LINC00052-MUT 组、miR-148b-3p+LINC00052-MUT 组;将 LINC00052 过表达载体、抑制表达载体及其阴性对照转染至HK-2细胞中,分为:pcDNA-NC组、pcDNA-LINC00052组、si-NC组、si-LINC00052组,检测LINC00052和miR-148b-3p的靶向关系.结果 与NC组比较,HG组的细胞活性、E-Cadherin 和 LINC00052 表达水平降低(均 P<0.001),miR-148b-3p、N-Cadherin 和 vimentin表达水平、细胞凋亡率升高(均P<0.001).与pcDNA-NC+HG组比较,pcDNA-LINC00052+HG组的miR-148b-3p、N-Cadherin和vimentin表达水平以及细胞凋亡率降低,E-Cadherin表达水平和细胞活性升高(均P<0.001).与 anti-miR-NC+HG 组比较,anti-miR-148b-3p+HG 组的 miR-148b-3p、N-Cadherin和vimentin表达水平以及细胞凋亡率降低,E-Cadherin表达水平和细胞活性升高(均P<0.001).与pcDNA-NC组比较,pcDNA-LINC00052组的LINC00052表达水平升高,miR-148b-3p表达水平降低(均P<0.001);与si-NC组比较,si-LINC00052组的LINC00052表达水平降低,miR-148b-3p 表达水平升高(均P<0.001).与 miR-NC+pcDNA-LINC00052+HG 组比较,miR-148b-3p+pcDNA-LINC00052+HG 组 miR-148b-3p、N-Cadherin 和 vimentin 表达水平以及细胞凋亡率升高,E-Cadherin表达水平以及细胞活性降低(均P<0.001).结论 LINC00052通过靶向miR-148b-3p抑制高糖诱�
Objective To explore the molecular mechanism of LINC00052 on the proliferation,apoptosis and epithelial-mesenchymal transition(EMT)of renal tubular epithelial cellss induced by high glucose by targeting miR-148b-3p.Methods The renal tubular epithelial cells HK-2 were divided into control group(NC group),high glucose group(HG group),peDNA-LINCO0052+HG group,pcDNA-NC+HG group,anti-miR-148b-3p+HG group,anti-miR-NC+HG group,miR-148b-3p+pcDNA-LINC00052+HG group,miR-NC+pcDNA-LINC00052+HG group;real-time fluorescent quantitative PCR(qRT-PCR)detected the expression of LINC00052 and miR-148b-3p level;the protein expressions of E-Cadherin,N-Cadherin and vimentin were detected by Western blot.CCK-8 was used to detected cell viability.Apoptosis was detected by flow cytometry.The dual luciferase reporter experiment was divided into:miR-NC+LINC00052-WT group,miR-148b-3p+LINC00052-WT group,miR-NC+LINC00052-MUT group,miR-148b-3p+LINC00052-MUT group.The LINC00052 overexpression vector,inhibition expression vector and their negative control were transfected into HK-2 cells and divided into pcDNA-NC group,pcDNA-LINC00052 group,si-NC group and si-LINCO0052 group,and the targeting relationship between LINC00052 and miR-148b-3p was detected.Results Compared with the NC group,the cell viability,the expression levels of E-Cadherin and LINCO0052 in the HG group were decreased(all P<0.001),the expression levels of miR-148b-3p,N-Cadherin and vimentin,and the apoptosis rate were increased(all P<0.001).Compared with the pcDNA-NC+HG group,the expression levels of miR-148b-3p,N-Cadherin,vimentin and apoptosis rate were decreased,and the expression level of E-Cadherin and cell activity were increased in the pcDNALINC00052+HG group(all P<0.001).Compared with the anti-miR-NC+HG group,the expression levels of miR-148b-3p,N-Cadherin and vimentin and the apoptosis rate were decreased,and the expression level of E-Cadherin and the cell activity were increased in the anti-miR-148b-3p+HG group(all P<0.001).Compared with the pcDNA-NC group,the expressio
作者
庞紫蕊
张林波
刘宋芳
Pang Zirui;Zhang Linbo;Liu Songfang(Department of Endocrinology,Ankang Central Hospital,Ankang 725000,China;Department of Nephrology,Yulin First Hospital,Yulin 719000,China;Department of Endocrinology,Xi'an Ninth Hospital,Xi'an 710000,China)
出处
《国际泌尿系统杂志》
2023年第2期317-321,共5页
International Journal of Urology and Nephrology
基金
陕西省社会发展科技攻关项目(2016SF-228)。
