天津医药 ›› 2023, Vol. 51 ›› Issue (2): 131-138.doi: 10.11958/20220849

• 细胞与分子生物学 • 上一篇    下一篇

LncRNA OIP5-AS1调节miR-25-3p/SOX4轴对高糖诱导的人肾小管上皮细胞生物学过程的影响

杨娟(), 张厚芬, 吴松, 陈莹, 罗华荣()   

  1. 中国贵航集团三0二医院肾内、内分泌科(邮编561000)
  • 收稿日期:2022-05-27 修回日期:2022-08-09 出版日期:2023-02-15 发布日期:2023-02-24
  • 通讯作者: E-mail:h3442s@163.com
  • 作者简介:杨娟(1982),女,副主任医师,主要从事糖尿病及并发症诊治方面研究。E-mail:yangjuan8212@163.com
  • 基金资助:
    安顺市科技计划项目(安市科社[2021]45号)

Effects of lncRNA OIP5-AS1 regulating miR-25-3p/SOX4 axis on the biological process of human renal tubular epithelial cells induced by high glucose

YANG Juan(), ZHANG Houfen, WU Song, CHEN Ying, LUO Huarong()   

  1. Department of Nephrology Endocrinology, 302 Hospital of China Guihang Group, Anshun 561000, China
  • Received:2022-05-27 Revised:2022-08-09 Published:2023-02-15 Online:2023-02-24
  • Contact: E-mail:h3442s@163.com

摘要:

目的 探讨长链非编码RNA Opa相互作用蛋白5-反义转录物1(lncRNA OIP5-AS1)对高糖诱导的人肾小管上皮细胞增殖、凋亡和氧化应激损伤的影响及分子机制。方法 体外培养人肾皮质近曲小管上皮细胞HK-2,分为正常葡萄糖组(NG组)、高糖组(HG组)、HG+si-NC组、HG+si-OIP5-AS1组、HG+miR-NC组、HG+miR-25-3p组、HG+si-OIP5-AS1+inhibitor-NC组、HG+si-OIP5-AS1+miR-25-3p inhibitor组。转染48 h后,实时荧光定量PCR(qPCR)检测细胞中lncRNA OIP5-AS1、miR-25-3p和性别决定区Y框蛋白4(SOX4)mRNA水平;CCK-8法检测细胞活力;检测细胞培养上清液中乳酸脱氢酶(LDH)活性;流式细胞术分析细胞凋亡情况;检测细胞中丙二醛(MDA)水平和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;DCFH-DA荧光探针检测细胞内活性氧(ROS)的产生;Western blot实验检测细胞中SOX4、B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)和裂解的Caspase-3(Cleaved-Caspase-3)蛋白表达。双荧光素酶报告基因实验确认miR-25-3p与lncRNA OIP5-AS1和SOX4的靶向关系。结果 与NG组相比,HG组细胞中lncRNA OIP5-AS1和SOX4 mRNA和蛋白表达水平升高,miR-25-3p水平降低(P<0.05);敲低lncRNA OIP5-AS1可显著下调SOX mRNA和蛋白水平,上调miR-25-3p水平,增加HK-2细胞活力和SOD、CAT活性以及Bcl-2蛋白水平,降低细胞凋亡率、LDH活性、MDA、ROS水平、Bax蛋白水平及Cleaved-Caspase-3/Caspase-3比值(P<0.05);上调miR-25-3p表达与敲低lncRNA OIP5-AS1的作用一致;在敲低lncRNA OIP5-AS1的基础上,下调miR-25-3p可明显减弱lncRNA OIP5-AS1敲低对高糖诱导的HK-2细胞氧化应激损伤的保护作用(P<0.05)。双荧光素酶报告基因实验显示lncRNA OIP5-AS1和miR-25-3p,以及miR-25-3p和SOX4之间存在结合位点。结论 lncRNA OIP5-AS1可能通过miR-25-3p/SOX4轴促进高糖诱导的HK-2细胞损伤。

关键词: 糖尿病肾病, 肾小管, 上皮细胞, 细胞凋亡, 细胞增殖, miR-25-3p, OIP5-AS1, 性别决定区Y框蛋白4

Abstract:

Objective To investigate the impact and molecular mechanism of long non-coding RNA Opa-interacting protein 5-antisense transcript 1 (lncRNA OIP5-AS1) on the proliferation, apoptosis and oxidative stress damage of human renal tubular epithelial cells induced by high glucose. Methods Human renal cortical proximal tubule epithelial cells HK-2 were cultured in vitro. HK-2 cells were transfected with lncRNA OIP5-AS1 small interfering RNA (si-OIP5-AS1), miR-25-3p mimic, miR-25-3p inhibitor and their negative controls si-NC, miR-NC and inhibitor-NC. Cells were divided into the normal glucose group (NG group), the high glucose group (HG group), the HG+si-NC group, the HG+si-OIP5-AS1 group, the HG+miR-NC group, the HG+miR-25-3p group, the HG+si-OIP5-AS1+inhibitor-NC group and the HG+si-OIP5-AS1+miR-25-3p inhibitor group. Forty-eight hours after transfection, real-time quantitative PCR (qPCR) was performed to detect levels of lncRNA OIP5-AS1, miR-25-3p and sex-determining region Y-box protein 4 (SOX4) mRNA in cells. CCK-8 assay was performed to detect cell viability and lactate dehydrogenase (LDH) activity in the cell culture supernatant. Flow cytometry was performed to analyze apoptosis, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities in cells. DCFH-DA fluorescent probe was implemented to detect intracellular reactive oxygen species (ROS) production. Western blot experiment was performed to detect the protein expression of SOX4, B-cell lymphoma factor 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 (Caspase-3) and cleaved-Caspase-3 (Cleaved-Caspase-3) in cells. Dual luciferase reporter assay confirmed the targeting relationship between lncRNA OIP5-AS1 and miR-25-3p, and between miR-25-3p and SOX4. Results Compared with the NG group, the expression levels of lncRNA OIP5-AS1 and SOX4 were significantly increased in the HG group, and the level of miR-25-3p was significantly decreased (P<0.05). Knockdown of lncRNA OIP5-AS1 was able to significantly down-regulate SOX mRNA and protein levels, and up-regulate miR-25-3p level, increase HK-2 cell viability, SOD, CAT activities and Bcl-2 protein level, and reduce apoptosis rate, LDH activity, MDA, ROS levels, Bax protein level and Cleaved-Caspase-3/Caspase-3 ratio (P<0.05). The up-regulating miR-25-3p expression was consistent with that of knocking down lncRNA OIP5-AS1. On the basis of knockdown of lncRNA OIP5-AS1, down-regulation of miR-25-3p significantly attenuated the protective effect of lncRNA OIP5-AS1 knockdown on high glucose-induced oxidative stress damage in HK-2 cells (P<0.05). Dual luciferase reporter assay showed binding sites between lncRNA OIP5-AS1 and miR-25-3p, as well as between miR-25-3p and SOX4. Conclusion The lncRNA OIP5-AS1 may promote high glucose-induced HK-2 cell damage through the miR-25-3p/SOX4 axis.

Key words: diabetic nephropathies, kidney tubules, epithelial cells, apoptosis, cell proliferation, miR-25-3p, OIP5-AS1, sex-determining region Y-box 4

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