长链非编码生长停滞特异性蛋白6反义RNA1靶向miRNA-374a-3p调控高糖诱导的人肾小管上皮细胞损伤及纤维化分子机制的研究

Molecular mechanism of Lnc RNA DLX6-AS1 regulates human renal tubular epithelial cell damage and fibrosis induced by high glucose by targeting mi R-374a-3p

目的探讨长链非编码生长停滞特异性蛋白6反义RNA1(LncRNA DLX6-AS1)对高糖诱导的人肾小管上皮细胞HK-2损伤及纤维化的影响及其可能作用机制。方法高糖诱导HK-2细胞建立细胞损伤模型,实验分为正常对照(Con)组、高糖组(HG)、LncRNA DLX6-AS1小分子干扰RNA阴性对照(si-NC)+HG组(si-NC+HG组)、LncRNA DLX6-AS1小分子干扰RNA(si-LncRNA DLX6-AS1)+HG组(si-LncRNA DLX6-AS1+HG组)、miR-374a-3p寡核苷酸模拟物阴性对照mimic NC序列(miR-NC)+HG组(miR-NC+HG组)、miR-374a-3p寡核苷酸模拟物(miR-374a-3p mimics)+HG组(miR-374a-3p+HG组)、miR-374a-3p特异性寡核苷酸抑制剂阴性对照(anti-miR-NC)+si-LncRNA DLX6-AS1+HG组(anti-miR-NC+si-LncRNA DLX6-AS1+HG组)、miR-374a-3p特异性寡核苷酸抑制剂(anti-miR-374a-3p)+si-LncRNA DLX6-AS1+HG组(anti-miR-374a-3p+si-LncRNA DLX6-AS1+HG组)。qRT-PCR法检测LncRNA DLX6-AS1、miR-374a-3p表达,ELISA法检测TNF-α、IL-1β水平,双荧光素酶报告实验检测LncRNA DLX6-AS1和miR-374a-3p的靶向关系,Western blot法检测人平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(Fn)、I型胶原α1链(COL1a1)、COL3a1蛋白表达量。结果转染si-LncRNA DLX6-AS1或miR-374a-3p mimic可降低TNF-α、IL-1β水平和α-SMA、Fn、COL1a1、COL3a1蛋白水平(P<0.05)。LncRNA DLX6-AS1可靶向调节miR-374a-3p表达。共转染anti-miR-374a-3p与si-LncRNA DLX6-AS1可恢复转染si-LncRNA DLX6-AS1对HG诱导的HK-2细胞炎症及纤维化作用。结论干扰LncRNA DLX6-AS1表达可通过上调miR-374a-3p表达抑制高糖诱导的肾小管上皮细胞炎症反应及纤维化。

ObjectiveTo explore the effect of Lnc RNA DLX6-AS1 on the injury and fibrosis of human renal tubular epithelial cells HK-2 induced by high glucose and its possible mechanism.Methods HK-2 cells were induced by high glucose to establish a cell damage model.Experimental groups were as follows:Con group,HG group,si-NC+HG group,si-Lnc RNA DLX6-AS1+HG group,mi R-NC+HG group,mi R-374a-3p+HG group,anti-mi R-NC+si-Lnc RNA DLX6-AS1+HG group,anti-mi R-374a-3p+si-Lnc RNA DLX6-AS1+HG group.q RT-PCR method was used to detect the expression of Lnc RNA DLX6-AS1,mi R-374a-3p.ELISA method was used to measure the levels of TNF-αand IL-1β.The dual luciferase reporter experiment was used to evaluate the targeting relationship between Lnc RNA DLX6-AS1and mi R-374a-3p.Western blot method was used to test the protein expression ofα-SMA,Fn,COL1a1,and COL3a1.ResultsTransfection of si-Lnc RNA DLX6-AS1 or mi R-374a-3p mimic could reduce the levels of TNF-α,IL-1βand the protein levels ofα-SMA,Fn,COL1a1,COL3a1(P<0.05).Lnc RNA DLX6-AS1 could target the expression of mi R-374a-3p.Co-transfection of anti-mi R-374a-3p and si-Lnc RNA DLX6-AS1 could restore the effect of transfection of si-Lnc RNA DLX6-AS1 on the inflammation and fibrosis of HK-2 cells induced by HG.ConclusionInterfering with the expression of Lnc RNA DLX6-AS1 could inhibit the inflammatory response and fibrosis of renal tubular epithelial cells induced by high glucose by up-regulating the expression of mi R-374a-3p.