抗VEGF治疗对PDR患者玻璃体液中TGF-β及相关microRNA水平的影响

The effect of anti-VEGF therapy on the expression levels of TGF-βand related microRNAs in the vitreous of patients with proliferative diabetic retinopathy

目的探讨抗血管内皮生长因子(VEGF)治疗对增生型糖尿病视网膜病变(PDR)患者玻璃体液内转化生长因子β(TGF-β)水平以及相关microRNA表达水平的影响。方法横断面研究。纳入2020年6月至2021年2月于郑州大学第一附属医院眼科行玻璃体切除术的眼底病患者67例(67只眼), 年龄(54.37±11.70)岁, 男性38例, 女性29例。其中PDR患者45例, 未经治疗者(29例)为患病组, 玻璃体腔注射抗VEGF药物治疗后7 d者(16例)为治疗组;其余22例特发性黄斑裂孔患者为对照组。采用定量反转录聚合酶链式反应检测36例患者(每组12例)玻璃体液内microRNA(hsa-miR-24-3p和hsa-miR-197-3p)水平, 采用酶联免疫吸附试验检测67例患者玻璃体液内TGF-β及VEGF-A水平。采用RNAhybrid、miRanda及TargetScan7.2数据库预测hsa-miR-24-3p和hsa-miR-197-3p的靶基因, 对靶向mRNA进行信号通路的富集分析。应用单因素方差分析比较3个组间生长因子以及microRNA水平, 组间两两比较采用最小显著差法, 生长因子浓度与microRNA相对表达量之间的相关分析使用Person相关性检验。结果对照组玻璃体液内VEGF-A、TGF-β、hsa-miR-24-3p、hsa-miR-197-3p表达水平分别为(158.15±17.72)pg/ml、(640.47±24.80)pg/ml、0.81±0.11、1.07±0.15, 患病组分别为(1047.54±26.61)pg/ml、(3553.17±92.61)pg/ml、8.50±2.33、12.23±3.38, 治疗组分别为(778.10±27.73)pg/ml、(3376.02±78.83)pg/ml、4.54±0.67、3.90±0.65。其中患病组各项指标较对照组均显著升高(F=355.581, 440.538, 7.546, 7.546;P均<0.05);治疗组玻璃体液内VEGF-A、hsa-miR-24-3p、hsa-miR-197-3p表达水平较患病组显著降低(P均<0.05), TGF-β浓度也降低, 但差异无统计学意义(P>0.05);36例患者玻璃体液内VEGF-A浓度与hsa-miR-24-3p、hsa-miR-197-3p表达水平呈正相关(r=0.48, 0.51;P<0.05), TGF-β浓度也与两者呈正相关(r=0.40, 0.42;P<0.05)。信号通路富集分析结果表明, hsa-miR-24-3p、hsa-miR-197-3p的靶向mRNA在VEGF及TGF-β信号传导通路上显著富集。结论对于PDR患者, hsa-miR-24-3p、hsa-miR-197-3p是VEGF-A、TGF-β的相关microRNA, 并可能是潜在的危险因子, 抗VEGF治疗可显著降低PDR患者玻璃体液内TGF-β相关microRNA(即hsa-miR-24-3p、hsa-miR-197-3p)的表达水平, 但无法有效降低玻璃体液内TGF-β水平, 提示联合抗TGF治疗可能对延缓PDR进展有一定意义。深入探索并验证hsa-miR-24-3p和hsa-miR-197-3p的靶向mRNA在VEGF及TGF-β信号传导通路富集的部分, 可为研究PDR提供新的思路及方向。(中华眼科杂志, 2021, 57:922-929)

Objective To investigate the effect of anti-vascular endothelial growth factor(VEGF)therapy on the expression levels of transforming growth factor-beta(TGF-β)and its related microRNAs in the vitreous of patients with proliferative diabetic retinopathy(PDR).Methods This cross-sectional study included 67 patients(67 eyes),38 males and 29 females,aged(54.37±11.70)years,who underwent vitrectomy from June 2020 to February 2021.There were 45 PDR patients(45 eyes),including 29 patients(29 eyes)without anti-VEGF therapy in the disease group and 16 patients(16 eyes)who were admitted at 7 days after anti-VEGF therapy in the treatment group.The other 22 idiopathic macular hole patients(22 eyes)were in the negative control group.The microRNA(hsa-miR-24-3p and hsa-miR-197-3p)levels in the vitreous of 36 patients(12 cases randomly chosen from each group)were detected by quantitative reverse transcription polymerase chain reaction.The levels of TGF-βand VEGF-A in the vitreous of 67 patients were detected by enzyme-linked immunosorbent assay.Target gene prediction of hsa-miR-24-3p and hsa-miR-197-3p was performed on RNAhybrid,miRanda and TargetScan7.2 databases,and pathway enrichment analyses were conducted for all target mRNAs.One-way ANOVA was used to compare the levels of growth factors and microRNAs among the three groups,and the least significant difference method was used for multiple comparisons between groups.Pearson correlation test was used to analyze the correlation between growth factors and microRNAs.Results The expression levels of VEGF-A,TGF-β,hsa-miR-24-3p and hsa-miR-197-3p were(158.15±17.72)pg/ml,(640.47±24.80)pg/ml,0.81±0.11 and 1.07±0.15 in the control group,(1047.54±26.61)pg/ml,(3553.17±92.61)pg/ml,8.50±2.33 and 12.23±3.38 in the disease group,and(778.10±27.73)pg/ml,(3376.02±78.83)pg/ml,4.54±0.67 and 3.90±0.65 in the treatment group,respectively.All indicators were significantly higher in the disease group than those in the control group(F=355.581,440.538,7.546 and 7.546;all P<0.05).The expression levels of VEGF-A,hsa-miR-24-3p and hsa-miR-197-3p in the treatment group were significantly lower than those in the disease group(all P<0.05).The concentration of TGF-βwas not statistically significantly lower in the treatment group compared to the disease group.The concentrations of VEGF-A and TGF-βwere significantly positively correlated with the expression levels of hsa-miR-24-3p and hsa-miR-197-3p in the vitreous of randomly chosen 36 patients(r=0.48,0.51,0.40 and 0.42;all P<0.05).Pathway enrichment analysis showed that some target mRNAs of hsa-miR-24-3p and hsa-miR-197-3p were involved in VEGF and TGF-βsignal pathways.Conclusions In the vitreous of patients with PDR,hsa-miR-24-3p and hsa-miR-197-3p were positively related to VEGF-A and TGF-β,and may be potential risk factors.Anti-VEGF treatment can significantly reduce the expression level of TGF-β-related microRNAs,namely hsa-miR-24-3p and hsa-miR-197-3p,but cannot effectively reduce the concentration of TGF-β,suggesting that combined anti-TGF treatment may be beneficial for delaying the progression of PDR.Furthermore,it may be a new research direction of PDR to validate the target mRNAs of hsa-miR-24-3p and hsa-miR-197-3p involved in VEGF and TGF-βsignal pathways.(Chin J Ophthalmol,2021,57:922-929)