长链非编码核糖核酸LINC00261通过miR-148b-3p/PTEN途径对高糖环境中HK-2细胞的保护作用

Protective effect of long noncoding ribonucleic acid LINC00261 on HK-2 cells in high glucose environment via miR-148b-3p/PTEN pathway

目的探讨长链非编码RNA(lncRNA)LINC00261在糖尿病肾病中的表达,及其可能通过微小核糖核酸miR-148b-3p/PTEN途径对高糖环境中HK-2细胞的保护作用。方法选择2016年3月至2018年5月本院的19例糖尿病肾病患者和23例健康对照者,采集血样,通过实时定量PCR(qRT-PCR)测定LINC00261和miR-148b-3p的表达。在细胞实验中,将HK-2肾小管上皮细胞分为7组:正常葡萄糖组(5.5 mmol/L培养,NG组)、高葡萄糖糖组(30.0 mmol/L培养,HG组)、其余5组也均为高葡萄糖培养:空质粒转染pcDNA(HG+pcDNA组)、转染LINC00261(HG+pcDNA-LINC00261组)、转染阴性对照anti-miR-NC(HG+anti-miR-NC组)、转染抑制剂anti-miR-148b-3p(HG+anti-miR-148b-3p组)、LncRNA和miRNA同时转染(HG+pcDNA-LINC0026+miR-148b-3p组)。应用Western印迹、细胞计数试剂盒8和流式细胞术分别检测PTEN蛋白表达、细胞增殖与凋亡。测超氧化物歧化酶(SOD)试剂盒和丙二醛(MDA)试剂盒分别检测SOD活性和MDA含量。双荧光素酶报告实验用于LINC00261、miR-148b-3p、PTEN的相互关系鉴定。结果与健康对照者比较,糖尿病肾病患者的LINC00261表达明显降低,而miR-148b-3p表达则明显增高(P<0.05)。过表达LINC00261或抑制miR-148b-3p后,高糖环境中HK-2细胞的miR-148b-3p表达、细胞凋亡及MDA含量均下降,而PTEN蛋白表达、细胞增殖及SOD活性均增高(P<0.05)。结论LINC00261可能通过调控miR-148b-3p/PTEN途径,促进高糖环境中HK-2肾小管上皮细胞增殖、降低氧化应激、减少细胞凋亡。

Objective To investigate the expression of long noncoding RNA(lncRNA)LINC00261 in diabetic nephropathy and its possible protective effect on HK-2 cells in high glucose environment through the microRNA miR-148 b-3 p/PTEN pathway.Methods From March 2016 to May 2018,19 patients with diabetic nephropathy and 23 healthy controls from our hospital were selected.The blood samples were collected,and the expressions of LINC00261 and miR-148 b-3 p were determined by quantitative real-time PCR(qRT-PCR).In cell experiments,the HK-2 renal tubular epithelial cells were divided into 7 groups:NG group with normal glucose(5.5 mmol/L),HG group with high glucose(30.0 mmol/L),and the other 5 groups were all with high glucose:pcDNA group(empty plasmid transfection),LINC00261 group(pcDNA-LINC00261 transfection),anti-miR-NC group(negative control transfection),anti-miR-148 b-3 p group(inhibitor transfection),and LINC0026+miR-148 b-3 p group(transfection of pcDNA-LINC00261 and miR-148 b-3 p).The expression of PTEN protein,cell proliferation,and apoptosis were detected by western blotting,cell counting kit 8,and flow cytometry,respectively.Superoxide dismutase(SOD)kit and malondialdehyde(MDA)kit were used to detect SOD activity and MDA content,respectively.The dual luciferase reporter assay was used to identify the relationship among LINC00261,miR-148 b-3 p,and PTEN.Results Compared with healthy controls,the expression of LINC00261 in patients with diabetic nephropathy was significantly decreased,while the expression of miR-148 b-3 p was apparently increased(P<0.05).After overexpression of LINC00261 or inhibition of miR-148 b-3 p,the expression of miR-148 b-3 p,apoptosis,and MDA of HK-2 cells in high glucose environment were all decreased,but the PTEN protein expression,cell proliferation,and SOD activity were all increased(P<0.05).Conclusion LINC00261 may promote the proliferation,relieve oxidative stress,and decrease apoptosis of HK-2 renal tubular epithelial cells in high glucose environment through regulating the miR-148 b-3 p/PTEN pathway.