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.
Highlights
MicroRNAs are non-protein-coding, short (20–25) nucleotide RNAs that regulate expression of at least 30% of all genes.[1,2] MiRNA-protein complexes bind to the 3’-untranslated region of their target mRNAs, leading to inhibition of translation or destabilization of the target mRNAs.[2,3,4] miR-34a, a gene product of chromosomal locus 1p36.23, is a tumorPLOS ONE | DOI:10.1371/journal.pone.0168320 December 15, 2016LGR4 Is a Direct Target of MiR-34a suppressor in many types of tumors.[5]
Ki67 positive cells were 39 ± 16% and 26 ± 15% in LGR4 siRNA and miR-34a transfected cells, respectively, whereas they were 85± 8% and 81 ± 3%, respectively, in the mock and negative control (NC) groups (Fig 2D). These results show that there is a direct relationship between decreases in LGR4 expression and ARPE-19 cell proliferation
We have shown that miR-34a modulates proliferation and migration of ARPE-19 cells through changes c-Met expression.[8] miR-34a upregulation suppressed c-Met expression which led to declines in ARPE-19 cell proliferation and migration
Summary
LGR4 Is a Direct Target of MiR-34a suppressor in many types of tumors.[5] This miRNA is the focus of many research endeavors to better understand regulatory mechanisms of cell proliferation and migration. One of the findings showed that the miR-34 family are targets of the p53 oncogene family.[6] In mammals, the miR-34 family contains three processed miRNAs with miR-34a being ubiquitously expressed.[5] The other two, miR-34b and miR-34c share a common primary transcript and are mainly expressed in the lungs.[5] miR-34a expression is pronounced in the CNS system and implicated in controlling cell proliferation, cell cycle arrest and senescence. The other two, miR-34b and miR-34c share a common primary transcript and are mainly expressed in the lungs.[5] miR-34a expression is pronounced in the CNS system and implicated in controlling cell proliferation, cell cycle arrest and senescence. [5,7] The identified target genes of miR-34a include silent information regulator 1 (SIRT1), Bcl-2, CD44, c-Met, various cyclins and CDKs, and the proto-oncoproteins MYC, MYCN amongst numerous others.[5,8]
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