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.

Fig 1. LGR4 is a direct target of miR-34a.

(A) HEK293 cells were co-transfected with miR-34a, pMIR-LGR4 3’-UTR or pMIR-LGR4 3’-UTR-Mut, along with a pRL-SV40 reporter plasmid. After 24 hours, the luciferase activity was measured. Values are presented as relative luciferase activity after normalized to Renilla luciferase activity. Results are expressed as mean ± SD. n = 3, *p < 0.05. (B) ARPE-19 cells were transfected with either miR-34a or a negative control (NC). Cell lysates were prepared and used for Western blot analysis of LGR4. β-actin was probed as loading control. The band intensity was measured with ImageJ software. Images are representative of at least three independent experiments.

Fig 2. Knockdown of LGR4 inhibits ARPE-19 cell proliferation.

Either a miR-34a mimic, LGR4 specific siRNA or a negative control (NC) was transfected into ARPE-19 cells. (A) Western blot analysis evaluated knockdown efficiency. The band intensity was measured with ImageJ software and normalized to the expression level in mock-treated cells. (B) MTS assay measured cell proliferation rates. Data at each time point were expressed as mean ± SD of the results obtained from three independent assays each performed in triplicates. (C) Ki67 immunostaining identified proliferating cells 48 hours after transfection. Images are representative of those obtained in three independent experiments. Scale bar: 200 μm. (D) Ki67 positive cells were expressed as percentages based on the number of Ki67 positive cells in three visual fields divided by the number of DAPI positive cells. Results are expressed as mean ± SD. n = 3, *p < 0.05.

Fig 3. Downregulation of LGR4 decreases ARPE-19 cell migration.

ARPE-19 cells were transfected with LGR4 specific siRNA or a scrambled negative control (NC). Transwell (A) and in vitro scratch assays (B) were performed to evaluate the ARPE-19 cell migration. The number of cells that migrated in the transwell assay was quantified by counting the number of cells appearing in the lower chamber in five independent vision fields with a 20X microscope objective. (C) For in vitro scratch assay, cells that migrated into the gap were counted at 48 hours and 72 hours as indicated. Results were expressed as mean ± SD (n = 3, *p < 0.05). All images are representative of at least three independent experiments. Scale bar: 100 μm for transwell assay, and 200 μm for in vitro scratch assay.

Fig 4. miR-34a expression or LGR4 knockdown attenuates ARPE-19 adhesion.

Forty-eight hours after transfection with either a miR-34a, a LGR4 specific siRNA or a negative control (NC), ARPE-19 cells were harvested and re-seeded for 2 hours, and then unattached cells were removed by rinsing with PBS. (A) The phase-contrast images of attached cells were captured with an attached camera. Images represent those obtained in three independent experiments. Scale bar: 100 μm. (B) Attached cells were quantitated with the MTS assay. Data were expressed as mean ± SD of results from three independent experiments with each repeated in triplicates. *p < 0.05.

Fig 5. Knockdown of LGR4 downregulates the expression of cell cycle-related molecules reminiscent of miR-34a mimic transfection.

(A) After transfection with either LGR4 specific siRNA or miR-34a mimic, ARPE-19 cell lysates were prepared and probed with anti-LGR4, E2F1, p-CDC2, CDK2, CDK4 and CDK6 antibodies. β-actin was probed as a loading control. These results are representative of at least three independent experiments. (B) The band intensity was measured with ImageJ software. The densitometric ratio between proteins of interest and β-actin was calculated and then normalized to the expression level in mock-treated cell. Results were expressed as mean ± SD (n = 3, *p < 0.05).