目的：探讨微小RNA-96-5p(miR-96-5p)对高糖诱导的大鼠视网膜血管内皮细胞增殖和凋亡的影响及其作用机制。

方法：体外培养SD大鼠视网膜血管内皮细胞(RRVEC)进行实验，将细胞分为对照组(NG)、高糖组(HG)，收集高糖诱导的细胞，分别或共同转染miR-96-5p模拟物(mimic)、无义miRNA(miR-NC)、FOXO4 siRNA(si-FOXO4)、FOXO4阴性对照序列(si-NC)、pcDNA-FOXO4、pcDNA。分别采用qRT-PCR与Western blotting检测miR-96-5p与FOXO4的表达； MTT法检测细胞增殖活性； 流式细胞仪检测细胞凋亡率； 双荧光素酶报告实验验证miR-96-5p的靶基因； Western blotting检测细胞中CyclinD1、p21、p27、Bcl-2、Bax、cleaved-caspased-3蛋白表达。

结果：高糖处理后，RRVEC中miR-96-5p、CyclinD1、Bcl-2表达水平均显著降低，而FOXO4、p21、p27、Bax、cleaved-caspased-3表达水平显著升高，抑制细胞增殖活性，促进细胞凋亡； miR-96-5p过表达与抑制FOXO4表达后CyclinD1、Bcl-2表达水平显著升高，抑制p21、p27、Bax、cleaved-caspased-3表达，增强细胞增殖能力，抑制细胞凋亡； 双荧光素酶报告实验证明，FOXO4是miR-96-5p的靶基因； FOXO4过表达可逆转miR-96-5p过表达对高糖诱导的RRVEC增殖和凋亡的作用。

结论：miR-96-5p通过靶向FOXO4以抑制高糖诱导的大鼠视网膜血管内皮细胞凋亡并促进细胞增殖。

METHODS: SD rat retinal vascular endothelial cells(RRVEC)were cultured and the RRVEC was divided into control group(NG)and high glucose group(HG). The high glucose-induced RRVECs were harvested separately or co-transfected with miR-96-5p mimic, miR-NC, si-FOXO4, si-NC. The expression of miR-96-5p and FOXO4 was detected by qRT-PCR and Western blotting, respectively. MTT assay was used to detect the proliferation activity. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter assay validated the target gene of miR-96-5p. Western blotting was used to detect the expression of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspased-3.

RESULTS:The expression levels of miR-96-5p, CyclinD1 and Bcl-2 in RRVEC were significantly decreased after high glucose treatment, and the expression levels of FOXO4, p21, p27, Bax and cleaved-caspased-3 were significantly increased, inhibiting cell proliferation activity, but promoting apoptosis. Overexpression of miR-96-5p and inhibition of FOXO4 expression increased the expression levels of CyclinD1 and Bcl-2, inhibited the expression of p21, p27, Bax, cleaved-caspased-3, enhanced cell proliferation and inhibited apoptosis. Dual luciferase reporter assay demonstrated that FOXO4 was a target gene for miR-96-5p. Overexpression of FOXO4 reversed the effect of miR-96-5p overexpression on high glucose-induced proliferation and apoptosis of RRVEC.

CONCLUSION:miR-96-5p inhibits high glucose-induced apoptosis of rat retinal vascular endothelial cells and promotes cell proliferation by targeting FOXO4.