LGR4 Is a Direct Target of MicroRNA-34a and Modulates the Proliferation and Migration of Retinal Pigment Epithelial ARPE-19 Cells

Abstract

The pathology of proliferative vitreoretinopathy and proliferative diabetic retinopathy is linked to proliferation, migration, and adhesion of the retinal pigment epithelium. MicroRNA-34a (miR-34a) expression modulates changes in proliferation and migration of retinal pigment epithelial cell line ARPE-19. In this study, we determined that miR-34a interacts with LGR4, identified by bioinformatics using TargetScan Human 5.0, to affect these changes. Double luciferase gene reporter assay confirmed miR-34a involvement in mediating control. miR-34a mimic transfection decreased LGR4 expression. Western blot analysis documented corresponding protein expression inhibition. MTS, Ki67 immunostaining, scratch and transwell testing, along with attachment assay showed that miR-34a upregulation inhibited ARPE-19 cell proliferation, migration and attachment partly through downregulation of LGR4 protein expression. Western blot analysis revealed that both miR-34a upregulation and LGR4 downregulation induced declines in E2F1, p-CDC2, CDK2, CDK4 and CDK6 protein expression. Taken together, miR-34a gene expression upregulation inhibits ARPE-19 cell proliferation, migration and adhesion partly by suppressing LGR4 expression. These results substantiate earlier indications that both miR-34a and LGR4 are potential drug targets to prevent fibrosis in a clinical setting.

The Interplay of MicroRNA-34a, LGR4, EMT-Associated Factors, and MMP2 in Regulating Uveal Melanoma Cells

“…29 We also showed that LGR4 is a direct target of miR-34a in RPE cells. 13 Here we showed that transfection of miR-34a to M17 and SP6.5 cells decreased…”

“…One of the microRNAs of particular interest is microRNA-34a (miR-34a), which is a gene product from chromosomal locus 1p36.22 that is also a tumor suppressor in many types of tumor. [13][14][15] This has been implicated previously in normal biological processes such as cell proliferation, inhibition, cell cycle arrest, and senescence.…”

“…We previously identified a novel miR-34a target, LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) in retinal pigment epithelium cells. 13 LGR4, also known as GPR48 (G protein-coupled receptor 48), has been implicated in embryonic development, cell motility, and tumor metastasis. 14,[16][17][18][19] LGR4 was found to be necessary for prostate cancer metastasis epithelial-mesenchymal transition (EMT).…”

Downregulation of circulating miR-320a and target gene prediction in patients with diabetic retinopathy

“…TSC-mTOR pathway may result in the development of metabolic diseases and DM complications [37]. Curiously, the CDK6 gene was reported as an inductor of pancreatic β-cell replication and human islets proliferation by Fiaschi-taesch et al [38] The CDK6 is still suppressed indirectly by upregulation miRNAs in DM and complications of the disease [39,40].…”

Diagnostic Importance of Mirna in Diabetic Retinopathy

“…MiR 15 b and miR 16 show downregulation under hyperglycemic conditions and play a significant role in inhibiting insulin resistance and preventing the apoptosis under such conditions rendering protection to HRECs (68). In addition to their regulatory roles regarding insulin resistance, these mRNA also possess inhibitory roles in proinflammatory signaling hence preventing retinal leukostatis in DR that is illustrated by Ye et al Some inferences drawn by various conclusions state that proinflammatory signals such as IL-1β, TNFα and NFκB in HRECs cells under HG c o n d i t i o n s a r e s i g n i f i c a n t l y r e d u c e d b y overexpression of miR15 a and miR16 (69). Wang et al elucidated dual action of miR15 in DR that is antii n f l a m m a t o r y a n d a n t i a n g i o g e n i c ( 7 0 ) .…”