LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial-Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells
LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial-Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells
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LncRNA-MALAT1/miRNA-204-5p/Smad4 轴调节晶状体上皮细胞的上皮-间质转化、增殖和迁移
DOI：
10.1080/02713683.2020.1857778
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发表时间：
2020-12-18
影响因子：
2
通讯作者：
Zhao, Jiangyue
中科院分区：
文献类型：
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作者：
Peng, Cheng;Wang, Yuchi;Zhao, Jiangyue
摘要目的：后囊膜混浊（PCO）是白内障术后常见的并发症，主要源于人晶状体上皮细胞（LECs）上皮-间质转化（EMT）、增殖和迁移。本研究旨在探讨长链非编码RNA转移相关肺腺癌转录本1 (MALAT1)/miR-204-5p/Smad4轴是否参与LECs的EMT。材料和方法：用tgf - β 2 （10 ng/mL）培养和诱导lec。将抗MALAT1的SiRNA （Si-MALAT1）转染到lec中，抑制MALAT1的表达。为了过表达或敲低miR-204-5p，将miR-204-5p模拟物（miR-204-5p模拟物）和anti-miR-204-5p （miR-204-5p抑制剂）转染到LECs中。我们使用RNA FISH来确定MALAT1的位置。RT-qPCR分析MALAT1和miR-204-5p的RNA水平。此外，用Western blot分析Smad4靶蛋白水平、上皮分化和间充质标志物。我们用EdU标记法检测细胞增殖，用Transwell法分析细胞迁移。在lec中进行双荧光素酶报告基因试验，以验证miRNA-204-5p是否受到MALAT1的负调控，Smad4是miR-204-5p的直接靶点。结果：在PCO标本中，MALAT1表达上调。MALAT1在tgf - β 2诱导的lec中过表达，敲低MALAT1可减弱tgf - β 2诱导的EMT。此外，MALAT1的上调与miR-204-5p的下调和Smad4的上调相关。重要的是，MALAT1被发现位于LECs的细胞质中。此外，荧光素酶报告基因检测证实MALAT1可以负调控miR-204-5p的表达，进而调控其直接靶点Smad4。最后，MALAT1的敲低可以抑制lec的EMT、增殖和迁移；然而，这些可以通过anti-miR-204-5p逆转。结论：我们的研究结果表明MALAT1可能作为ceRNA通过“海绵”miR-204-5p和靶向Smad4来调节LECs的EMT、增殖和迁移，并作为预防PCO的有希望的治疗靶点。
Purpose: 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.Materials and methods: LECs were cultured and induced with TGF-beta 2 (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.Results: The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-beta 2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-beta 2 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.Conclusions: 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.