LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells

<b>Purpose</b>: Posterior capsular opacification (PCO), a common complication after cataract surgery, primarily originated from the epithelial–mesenchymal transition (EMT), proliferation, and migration of human lens epithelial cells (LECs). This study aimed to explore whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-204-5p/Smad4 axis are involved in EMT of LECs. LECs were cultured and induced with TGF-β2 (10 ng/mL). SiRNA against MALAT1 (Si‐MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR‐204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-β2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-β2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by “sponging” miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

<b>研究目的</b>：后囊膜混浊（Posterior capsular opacification, PCO）是白内障术后常见并发症，其核心致病机制与人晶状体上皮细胞（human lens epithelial cells, LECs）的上皮间质转化（epithelial–mesenchymal transition, EMT）、增殖及迁移密切相关。本研究旨在探讨长链非编码RNA转移相关肺腺癌转录本1（long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1, MALAT1）/miR-204-5p/Smad4通路是否参与LECs的EMT进程。本研究体外培养LECs并以10 ng/mL的转化生长因子-β2（transforming growth factor-β2, TGF-β2）进行诱导，将靶向MALAT1的小干扰RNA（Si-MALAT1）转染至LECs以敲低其表达；为实现miR-204-5p的过表达或敲低，分别将miR-204-5p模拟物与miR-204-5p抑制剂转染至LECs。本研究采用RNA荧光原位杂交（RNA FISH）鉴定MALAT1的细胞定位，通过实时定量聚合酶链反应（RT-qPCR）分析MALAT1与miR-204-5p的RNA水平，利用蛋白质免疫印迹（Western blot）检测Smad4靶蛋白、上皮分化标志物及间质标志物的表达水平；采用EdU标记法检测细胞增殖能力，通过Transwell实验分析细胞迁移能力；在LECs中开展双荧光素酶报告基因实验，以验证miR-204-5p是否受MALAT1负向调控，且Smad4为miR-204-5p的直接靶基因。研究发现，MALAT1在PCO标本中表达上调，在TGF-β2诱导的LECs中亦呈过表达状态，而敲低MALAT1可缓解TGF-β2诱导的EMT；此外，MALAT1的表达上调与miR-204-5p的表达下调及Smad4的表达上调显著相关。值得注意的是，MALAT1定位于LECs的细胞质中；进一步的双荧光素酶报告基因实验证实，MALAT1可负向调控miR-204-5p的表达，进而调控其直接靶基因Smad4。最终实验结果显示，敲低MALAT1可抑制LECs的EMT、增殖与迁移，但该效应可被anti-miR-204-5p逆转。本研究揭示，MALAT1可作为内源竞争RNA（competing endogenous RNA, ceRNA）通过“海绵吸附”miR-204-5p并靶向调控Smad4，进而参与LECs的EMT、增殖与迁移过程，有望成为预防PCO的潜在治疗靶点。