信号转导及转录激活因子4(STAT4)介导的circ_0001879表达对高糖条件下人视网膜微血管内皮细胞增殖和凋亡的影响

Effects of signal transducer and activator of transcription 4 mediated circ_0001879 on proliferation and apoptosis of human retinal endothelial cells induced by high glucose

目的探讨信号转导及转录激活因子4(STAT4)介导的circ_0001879表达对高糖处理的人视网膜微血管内皮细胞(HREC)增殖和凋亡的影响。方法将HREC分为正常组(5.5 mmol·L^(-1)葡萄糖)、高糖组(25.0 mmol·L^(-1)葡萄糖)和甘露醇对照组(5.5 mmol·L^(-1)葡萄糖+19.5 mmol·L^(-1)甘露醇),继续培养。将高糖组细胞进一步分组,进行相应转染处理。采用实时荧光定量PCR检测各组细胞circ_0001879、miR-338和多效生长因子(PTN)的表达。通过MTT、流式细胞术分析circ_0001879调控miR-338/PTN对HREC增殖和凋亡的影响。采用双荧光素酶报告实验确定miR-338和circ_0001879、miR-338和PTN之间的相互作用;ChIP实验验证STAT4与circ_0001879的结合。结果与甘露醇对照组HREC中circ_0001879(1.21±0.13)、miR-338(0.99±0.08)和PTN(1.12±0.11)的表达相比,高糖组HREC中circ_0001879(2.93±0.21)和PTN(3.62±0.33)的表达显著升高,而miR-338(0.44±0.05)的表达降低,差异均有统计学意义(均为P<0.05)。通过MTT和流式细胞术检测各组细胞增殖活力和凋亡率,结果显示,过表达miR-338能够抑制高糖处理的HREC的增殖活力,促进HREC凋亡,而敲减miR-338的表达则作用相反;miR-inhibitor对高糖处理的HREC的作用能够被si-circ部分抑制。双荧光素酶报告实验结果显示,PTN是miR-338的一个靶基因,且其表达受miR-338和circ_0001879的影响。ChIP实验结果显示,STAT4与circ_0001879启动子区域的B1、B3结合,STAT4能够与circ_0001879的启动子结合并且促进circ_0001879的转录。结论STAT4能够激活circ_0001879的转录,circ_0001879进一步通过调节miR-338/PTN轴促进高糖条件下HREC增殖,抑制细胞凋亡。

Objective To explore the effects of signal transducer and activator of transcription 4(STAT4)mediated circ_0001879 on proliferation and apoptosis of human retinal endothelial cells(HREC)treated by high glucose(HG).Methods HRECs were divided into the normal group(5.5 mmol·L^(-1) glucose),HG group(25.0 mmol·L^(-1) glucose),and mannitol control group(5.5 mmol·L^(-1) glucose+19.5 mmol·L^(-1) mannitol).Cells in the HG group were further grouped and transfected accordingly.The expression levels of circ_0001879,miR-338 and pleiotrophin(PTN)were detected by real-time quantitative polymerase chain reaction.The effects of circ_0001879 regulated miR-338/PTN on HREC proliferation and apoptosis were analyzed by MTT and flow cytometry.The interaction between miR-338 and circ_0001879,miR-338 and PTN was determined by the dual-luciferase reporter assay.The binding of STAT4 to circ_0001879 was verified by ChIP assay.Results Compared with the mannitol control group circ_0001879(1.21±0.13),miR-338(0.99±0.08),and PTN(1.12±0.11),the expression of circ_0001879(2.93±0.21)and PTN(3.62±0.33)in the HG group was significantly increased,while the expression of miR-338(0.44±0.05)was significantly decreased(all P<0.05).MTT and flow cytometry were used to detect the proliferation activity and apoptosis rate of HRECs in each group,and the results showed that overexpression of miR-338 could inhibit proliferation activity of HG-treated HRECs and promote their apoptosis,while knockdown of miR-338 expression revealed an opposite effect.The effects of miR-inhibitor on HG-treated HRECs could be partially inhibited by si-circ.The dual-luciferase reporter assay results showed that PTN was a target gene of miR-338,and its expression was affected by miR-338 and circ_0001879.ChIP assay results showed that STAT4 bound to B1 and B3 in the promoter region of circ_0001879,and STAT4 could bind to the promoter of circ_0001879 to promote the transcription of circ_0001879.Conclusion STAT4 can activate the transcription of circ_0001879.circ_0001879 further promotes HREC pro-liferation in HG and inhibits their apoptosis by regulating the miR-338/PTN axis.