LncRNA HAGLR靶向miR-625-5p对脂多糖诱导的视网膜色素上皮细胞凋亡和炎症因子表达的影响

Effect of long-chain non-coding ribonucleic acid HAGLR targeting micro ribonucleic acid-625-5p on lipopolysaccharide-induced apoptosis and inflammatory factor expression in retinal pigment epithelial cells

目的 探讨长链非编码RNA HAGLR(LncRNA HAGLR)是否可通过靶向调控微小RNA-625-5p(miR-625-5p)表达而影响脂多糖(LPS)诱导的视网膜色素上皮(RPE)细胞凋亡、炎症因子表达,为揭示视网膜病变机制奠定实验基础。方法 将LPS诱导人视网膜色素上皮细胞(ARPE-19)设为LPS组,正常培养的ARPE-19细胞设为Con组。实时荧光定量聚合酶链反应(qRT-PCR)检测LncRNA HAGLR、miR-625-5p表达水平。根据转染物不同分为LPS+sh-NC组、LPS+sh-HAGLR组、LPS+miR-NC组、LPS+miR-625-5p组、LPS+sh-HAGLR+anti-miR-NC组、LPS+sh-HAGLR+anti-miR-625-5p组。采用流式细胞术检测细胞凋亡率;ELISA法检测白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)水平;验证LncRNA HAGLR、miR-625-5p靶向关系;Western blot检测活化的半胱氨酸天冬氨酸特异性蛋白酶3(cleaved-Caspase 3)、cleaved-Caspase 9蛋白水平。结果 与Con组比较,LPS组LncRNA HAGLR表达水平升高,miR-625-5p表达水平降低(均为P<0.05),细胞凋亡率,cleaved-Caspase 3、cleaved-Caspase 9蛋白水平,IL-6、IL-1β水平均升高(均为P<0.05)。与LPS+sh-NC组比较,LPS+sh-HAGLR组细胞凋亡率,cleaved-Caspase 3、cleaved-Caspase 9蛋白水平,IL-6、IL-1β水平均降低(均为P<0.05);LncRNA HAGLR可负向调控miR-625-5p表达水平(P<0.05)。与LPS+miR-NC组比较,LPS+miR-625-5p组miR-625-5p表达水平升高,细胞凋亡率,cleaved-Caspase 3、cleaved-Caspase 9蛋白水平,IL-6、IL-1β水平均降低(均为P<0.05)。与LPS+sh-HAGLR+anti-miR-NC组比较,LPS+sh-HAGLR+anti-miR-625-5p组miR-625-5p表达水平降低,细胞凋亡率,cleaved-Caspase 3、cleaved-Caspase 9蛋白水平,IL-6、IL-1β水平均升高(均为P<0.05)。结论 干扰LncRNA HAGLR表达可通过靶向调控miR-625-5p表达抑制细胞凋亡、炎症因子表达,以减轻LPS诱导的ARPE-19细胞损伤。

Objective To investigate whether long-chain non-coding ribonucleic acid HAGLR(LncRNA HAGLR)can affect lipopolysaccharide(LPS)-induced apoptosis and expression of inflammatory factors of retinal pigment epithelium(RPE)cells by targeted regulation of the expression of micro ribonucleic acid-625-5p(miR-625-5p),so as to lay an experimental foundation for revealing the mechanism of retinopathy.Methods LPS-induced human retinal pigment epithelial(ARPE-19)cells were set as the LPS group,and normally cultured ARPE-19 cells were assigned to the Con group.Quantitative real-time polymerase chain reaction was used to detect the expression levels of LncRNA HAGLR and miR-625-5p.Based on different transfection reagents,the cells were divided into the LPS+sh-NC group,LPS+sh-HAGLR group,LPS+miR-NC group,LPS+miR-625-5p group,LPS+sh-HAGLR+anti-miR-NC group,and LPS+sh-HAGLR+anti-miR-625-5p group.Flow cytometry was used to detect apoptosis rate;enzyme-linked immunosorbent assay was used to detect the levels of interleukin-6(IL-6)and interleukin-1β(IL-1β);the targeting relationship between LncRNA HAGLR and miR-625-5p was verified;Western blot was used to detect the protein levels of activated cysteinyl aspartate specific proteinase 3 and 9(cleaved-Caspase 3 and cleaved-Caspase 9).Results Compared with the Con group,the LPS group showed an increase in the expression level of LncRNA HAGLR and a decrease in the expression level of miR-625-5p(both P<0.05),and there were increases in apoptosis rate,protein levels of cleaved-Caspase 3 and cleaved-Caspase 9,and levels of IL-6 and IL-1β(all P<0.05).Compared with the LPS+sh-NC group,the LPS+sh-HAGLR group showed decreases in apoptosis rate,protein levels of cleaved-Caspase 3 and cleaved-Caspase 9,and levels of IL-6 and IL-1β(all P<0.05);LncRNA HAGLR could negatively regulate the expression level of miR-625-5p(P<0.05).Compared with the LPS+miR-NC group,the LPS+miR-625-5p group showed an increase in the expression level of miR-625-5p and decreases in apoptosis rate,protein levels of cleaved-Caspase 3 and cleaved-Caspase 9,and levels of IL-6 and IL-1β(all P<0.05).Compared with the LPS+sh-HAGLR+anti-miR-NC group,the LPS+sh-HAGLR+anti-miR-625-5p group showed a decrease in the expression level of miR-625-5p and increases in apoptosis rate,protein levels of cleaved-Caspase 3 and cleaved-Caspase 9,and levels of IL-6 and IL-1β(all P<0.05).Conclusion Interference on LncRNA HAGLR expression can realize the targeted regulation of miR-625-5p expression to inhibit the apoptosis and inflammatory factor expression,reducing LPS-induced injury of ARPE-19 cells.