miR-17-5p对小梁网细胞外基质蛋白表达的调控作用及其机制

目的　探讨miR-17-5p对小梁网细胞外基质（ECM）蛋白表达的调控作用及其机制。方法　收集原发性开角型青光眼（POAG）患者和正常小梁网组织，利用RT-PCR和Western blot分别检测miR-17-5p和Smad3表达；将人小梁网细胞（HTMCs）分为miR-17-5p mimics和miR-17-5p NC组，分别转染载有miR-17-5p mimics组和miR-17-5p NC的载体，Western blot检测HTMCs中ECM蛋白纤连蛋白（FN）、胶原蛋白I（CoL-I）、α-平滑肌肌动蛋白（α-SMA）的表达；靶基因预测库预测miR-17-5p靶基因，荧光素酶报告载体联合Western blot鉴定靶基因。HTMCs中共转染miR-17-5p mimics和Smad3过表达载体，Western blot检测各转染组细胞中Smad3、FN、CoL-I、α-SMA的蛋白表达。结果　正常小梁网组织中miR-17-5p表达量为1.16±0.11，高于POAG小梁网组织的0.19±0.04，差异有统计学意义（t=10.641,P<0.001）；正常小梁网组织中Smad3蛋白表达量为0.31±0.06，低于POAG小梁网组织的0.86±0.07，差异有统计学意义（t=8.105,P<0.001）。miR-17-5p mimics组HTMCs中FN、CoL-I和α-SMA蛋白表达均低于miR-17-5p NC组，差异均有统计学意义（均为P<0.001）；靶基因预测库预测及荧光素酶报告载体联合Western blot鉴定证实Smad3为miR-17-5p下游靶基因；miR-17-5p+Smad3转染组HTMCs中Smad3、FN、CoL-I和α-SMA蛋白表达均高于miR-17-5p+vector转染组，差异均有统计学意义(均为P<0.001)。结论　miR-17-5p 能抑制HTMCs的ECM蛋白表达，这一过程可能是通过靶向抑制Smad3表达而实现的。

miR-17-5p targets Smad3 to regulate extracellular matrix protein expression in trabecular meshwork cells

Objective To investigate the regulatory effect of miR-17-5p on the expression of extracellular matrix protein in trabecular meshwork and its possible mechanism.Methods Trabecular meshwork tissues were collected from patients with primary open angle glaucoma(POAG)and those with no history of eye disease.RT-PCR and Western Blot were used to detect miR-17-5p and Smad3 expression.Human trabecular meshwork cells(HTMCs)were divided into miR-17-5p mimics group and miR-17-5p NC group,and transfected with plasmids carrying miR-17-5p mimics group and miR-17-5p NC.Western blot were used to detect HTMCs extracellular matrix(ECM)related proteins such as fibronectin(FN),collagen I(CoL-I),Laminin.The target gene prediction library predicted the miR-17-5p target gene,and the luciferase reporter vector combined with Western blot identified the target gene;HTMCs were co-transfected with miR-17-5p mimics and Smad3 overexpression vectors.Western blot detected the expression of Smad3,FN,CoL-I,α-SMA in each transfection group.Results The expression level of miR-17-5p in normal trabecular meshwork tissue was 1.16±0.11,which was higher than that in POAG trabecular meshwork tissue(0.19±0.04),and the difference was statistically significant(t=10.641,P<0.001).The expression of Smad3 protein in normal trabecular meshwork tissue was 0.31±0.06,which was lower than that in POAG trabecular meshwork tissue(0.86±0.07),and the difference was statistically significant(t=8.105,P<0.001).The protein expression of FN,CoL-I andα-SMA in HTMCs of miR-17-5p mimics group was lower than that of miR-17-5p NC group,and the difference was statistically significant(all P<0.001).Target gene prediction library prediction and luciferase reporter vector combined with Western blot identification confirmed that Smad3 was the downstream target gene of miR-17-5p.The expression of Smad3,FN,CoL-I andα-SMA protein in HTMCs of miR-17-5p+Smad3 transfection group was higher than that of miR-17-5p+vector transfection group,and the difference was statistically significant(all P<0.001).Conclusion miR-17-5p can inhibit the expression of HTMCs extracellular matrix,and this process may be achieved by targeted inhibition of Smad3 expression.