巩膜重塑的潜在研究靶点：miR-29a 对巩膜成纤维细胞的作用。

A Potential Research Target for Scleral Remodeling: Effect of MiR-29a on Scleral Fibroblasts.

机构信息

Department of Ophthalmology, Yongchuan Hospital of Chongqing Medical University, Chongqing, China,

Department of Ophthalmology, Yongchuan Hospital of Chongqing Medical University, Chongqing, China.

出版信息

Ophthalmic Res. 2022;65(5):566-574. doi: 10.1159/000525189. Epub 2022 May 23.

DOI:10.1159/000525189

PMID:35605595

Abstract

INTRODUCTION

The purpose of this study was to determine whether miR-29a regulates cell survival and apoptosis and the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MMP-2, and collagen I in scleral fibroblasts.

METHODS

We transfected scleral fibroblasts with the miR-29a mimic and inhibitor. The effects of miR-29a on cell proliferation and apoptosis were determined using the CCK-8 assay and flow cytometry, respectively. Quantitative polymerase chain reaction (qPCR) was used to determine whether miR-29a regulates the mRNA levels of PTEN, MMP-2, and collagen I. The protein expression of PTEN, MMP-2, and collagen I was also assessed by western blot analysis.

RESULTS

The results of CCK-8 showed that, at 0, 24, 48, and 72 h after transfection, the relative optical density values in the mimic group were 0.233 ± 0.005, 0.380 ± 0.008, 0.650 ± 0.040, and 0.906 ± 0.032, and in the inhibitor group were 0.272 ± 0.011, 0.393 ± 0.029, 0.597 ± 0.059, and 0.950 ± 0.101, respectively. The flow cytometry results showed that the apoptosis rates of each group were as follows: the mimic group (0.043 ± 0.007), the NC group (0.040 ± 0.006), the inhibitor group (0.032 ± 0.003), the inhibitor NC group (0.027 ± 0.010), the lipofectamine group (0.027 ± 0.005), and the blank group (0.031 ± 0.009). The qPCR results indicated that in the mimic group, PTEN (0.795 ± 0.182, p = 0.2783), MMP-2 (0.621 ± 0.105, p = 0.0033), and COL1A1 (0.271 ± 0.100, p = 0.0002) expression decreased, whereas in the inhibitor group, PTEN (1.211 ± 0.100, p = 0.2614), MMP-2 (1.161 ± 0.053, p = 0.1190), and COL1A1 (1.7040 ± 0.093, p = 0.0003) increased. Western blot analysis showed that in the mimic group, the expression of PTEN (0.392 ± 0.039, p < 0.0001), MMP-2 (0.577 ± 0.017, p < 0.0001), and COL1A1 (0.072 ± 0.006, p < 0.0001) protein decreased, whereas in the inhibitor group, PTEN (1.043 ± 0.042, p = 0.9413), MMP-2 (1.397 ± 0.075, p = 0.0002), and COL1A1 (1.935 ± 0.081, p < 0.0001) expression increased.

CONCLUSION

MiR-29a inhibits the expression of PTEN, MMP-2, and collagen I on scleral fibroblasts, which may provide a basis studies in sclera.

摘要

简介

本研究旨在确定 miR-29a 是否调节巩膜成纤维细胞的细胞存活和凋亡以及磷酸酶和张力蛋白同源物缺失的第 10 号染色体（PTEN）、MMP-2 和胶原 I 的表达。

方法

我们用 miR-29a 模拟物和抑制剂转染巩膜成纤维细胞。通过 CCK-8 测定和流式细胞术分别确定 miR-29a 对细胞增殖和凋亡的影响。定量聚合酶链反应（qPCR）用于确定 miR-29a 是否调节 PTEN、MMP-2 和胶原 I 的 mRNA 水平。Western blot 分析还评估了 PTEN、MMP-2 和胶原 I 的蛋白表达。

结果

CCK-8 的结果表明，转染后 0、24、48 和 72 小时，模拟组的相对光密度值分别为 0.233 ± 0.005、0.380 ± 0.008、0.650 ± 0.040 和 0.906 ± 0.032，抑制剂组分别为 0.272 ± 0.011、0.393 ± 0.029、0.597 ± 0.059 和 0.950 ± 0.101。流式细胞术结果显示，各组的凋亡率如下：模拟组（0.043 ± 0.007）、NC 组（0.040 ± 0.006）、抑制剂组（0.032 ± 0.003）、抑制剂 NC 组（0.027 ± 0.010）、脂质体组（0.027 ± 0.005）和空白组（0.031 ± 0.009）。qPCR 结果表明，在模拟组中，PTEN（0.795 ± 0.182，p = 0.2783）、MMP-2（0.621 ± 0.105，p = 0.0033）和 COL1A1（0.271 ± 0.100，p = 0.0002）表达降低，而在抑制剂组中，PTEN（1.211 ± 0.100，p = 0.2614）、MMP-2（1.161 ± 0.053，p = 0.1190）和 COL1A1（1.7040 ± 0.093，p = 0.0003）增加。Western blot 分析表明，在模拟组中，PTEN（0.392 ± 0.039，p < 0.0001）、MMP-2（0.577 ± 0.017，p < 0.0001）和 COL1A1（0.072 ± 0.006，p < 0.0001）蛋白表达降低，而在抑制剂组中，PTEN（1.043 ± 0.042，p = 0.9413）、MMP-2（1.397 ± 0.075，p = 0.0002）和 COL1A1（1.935 ± 0.081，p < 0.0001）表达增加。