miR-142通过上调AKT2的表达促进角膜新生血管形成

miR-142 promotes corneal neovascularization formation via up-regulating AKT2

摘要:

目的：研究microRNA142（miR-142）对角膜新生血管形成的影响及其相关机制。方法：实验设置空白对照组、阴性对照组、过表达miR-142组以及干扰miR-142组，分别利用miR-142模拟物（mimic）和抑制物（inhibitor）过表达和干扰miR-142；CCK-8增殖实验检测miR-142对人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）增殖能力的影响；双荧光素酶报告基因验证AKT2是否为miR-142的直接靶基因；RT-PCR和Western blot实验检测miR-142对AKT2表达水平的影响。结果：CCK-8增殖实验结果显示空白对照组、阴性对照组和过表达miR-142组96 h的吸光度（absorbance，A）均值分别为872.21±44.20、842.16±49.54和1 407.91±16.58，差异具有统计学意义（F=102.8，P=0.000）。干扰miR-142后HUVECs增殖能力明显减弱（空白对照组=919.69±25.46，阴性对照组=856.76±36.22，干扰miR-142组=602.77±25.62；F=72.8，P=0.000）。双荧光素酶报告基因结果发现过表达miR-142后AKT2表达水平没有明显变化（对照组=0.36，过表达miR-142组=0.46，P=0.147）。RT-PCR结果显示过表达miR-142（0.408±0.046）后AKT2表达水平较阴性对照组（0.173±0.022）升高（P=0.018），干扰miR-142组（0.16±0.02）较阴性对照组（0.362±0.068）明显降低（P=0.037），差异均具有统计学意义。Western blot实验进一步验证了miR-142对AKT2基因的调控作用。进一步研究发现，shRNA干扰AKT2后miR-142促进HUVECs增殖的作用消失，对照组、过表达miR-142组、过表达miR-142+shRNA-AKT2组和shRNA-AKT2组的96 h吸光度值分别为981.80±75.40、1 357.81±47.56、806.39±49.70和921.59±40.38，差异具有统计学意义（F=40.97，P=0.000）。结论：miR-142通过上调AKT2的表达促进HUVECs增殖，从而促进角膜新生血管形成。

Abstract:

Objective：To study the impact of miR-142 on corneal neovascularization formation and related mechanism. Methods：Con－trol group，negative group，overexpression miR-142 group and knockdown miR-142 group were arranged. Mimic and inhibitor of miR-142 were used to overexpress or knock down miR-142，respectively. CCK-8 proliferation assay was performed to evaluate the im－paction of miR-142 on proliferative ability of human umbilical vein endothelial cells（HUVECs）. Dual-luciferase reporter gene system was used to verify whether AKT2 is the target of miR-142. RT-PCR and Western blot were used to detect effect of miR-142 on AKT2. Results：Results of CCK8 assay showed that absorbance（A） of control group，negative group and overexpression miR-142 group was 872.21±44.20，842.16±49.54，1 407.91±16.58，and the results had statistical significances（F=102.8，P=0.000）. Knockdown miR-142 significantly weakened the proliferation rate of HUVECs（control group=919.69±25.46，negative group=943.43±40.53，knockdown miR-142 group=602.77±25.62，F=72.8，P=0.000）. Consequence of dual-luciferase report gene system indicated that AKT2 wasn’t the target of miR-142（control group=0.36，overexpressed miR-142 group=0.46，P=0.147）. RT-PCR assay revealed that expression level of AKT2 was upregulated by overexpressing miR-142（0.408±0.046） than negative group（0.173±0.022）（P=0.018）；inhibition of miR-142 blocked the expression of AKT2（negative group=0.362±0.068，down-regulated miR-142 group=0.160±0.019，P=0.037）. Western blot assay verified the regulation of miR-142 on AKT2. Further study showed that the facilitation of miR-142 on proliferation of HUVECs was thoroughly blocked by shRNA-AKT2，results of control，overex－pressed miR-142，overexpressed miR-142+shRNA-AKT2 and shRNA-142 were 981.80±75.40，1 357.81±47.56，806.39±49.70，921.59±40.38，respectively，and the results had statistical significances（F=40.97，P=0.000）. Conclusion：miR-142 facilitates the proliferation of HUEVCs via up-regulating AKT2，suggesting that miR-142 could promote the formation of corneal neovascularization.