MicroRNA-650 promotes melanoma metastasis via targeting inhibitor of growth family member 4

Abstract

Objective: This study aimed to evaluate the effects of microRNA-650 (miR-650) on melanoma metastasis and reveal the regulatory relationship between miR-650 and the inhibitor of growth family member 4 (ING4).

Methods: miR-650 expression was determined in human melanoma WM115 and A-375 cells. WM115 cells were transfected with miR-650 mimic or mimic control. The invasion and migration abilities of transfected WM115 cells were analyzed using Transwell and wound healing assays, respectively. Then, miR-650-overexpression lentivirus vector was constructed and transfected into WM115 cells. After injection into the mice, the number of micro-metastatic foci in the lung tissues was counted. A regulatory relationship between miR-650 and ING4 was identified in WM115 and A-375 cells.

Results: The miR-650 expression was upregulated in WM115 and A-375 cells. WM115 cells transfected with the miR-650 mimic exhibited higher invasive and migratory abilities than mock cells or cells transfected with negative control (NC). The number of micro-metastatic foci was significantly higher in mice injected with Lenti-miR-650 than that in those injected with mock or NC controls. Transfection with miR-650 mimic observably inhibited the expression of ING4 in WM115 and A-375 cells, whereas transfection with miR-650 inhibitor had the opposite effect. Dual-luciferase reporter gene assay showed that the miR-650 mimic inhibited the luciferase activity of ING4.

Conclusion: miR-650 promotes melanoma metastasis by downregulating ING4 expression.

Keywords: ING4; Melanoma; Metastasis; miR-650.

Fig. 1

qRT-PCR showed the relative expression of miR-650. A, The relative expression of miR-650 in normal HaCaT cells and human melanoma WM115 and A-375 cells. ***, p < 0.001 compared to HaCaT cells; ###, p < 0.001 compared to A-375 cells. B, The relative expression of miR-650 in WM115 cells after transfection. Mock, cells treated with lipofectamine 2000; NC, mimic negative control. The experiments were repeated three times. ***, p < 0.001 compared to mock or NC.

Fig. 2

The invasion of WM115 cells analyzed by transwell assay. A, Invasive cells under microscope; B, Quantization of invasive cells. Mock, cells treated with lipofectamine 2000; NC, mimic negative control. The experiments were repeated three times. The number of invasive cells was counted in 10 random fields of views at 200 × magnifications. **, p < 0.01 compared to mock or NC.

Fig. 3

The migration of WM115 cells analyzed by wound healing assay. A, Migrated cells under microscope; B, Quantization of migrated cells. Mock, cells treated with lipofectamine 2000; NC, mimic negative control. The experiments were repeated three times. *, p < 0.05 compared to mock or NC.

Fig. 4

The effects of miR-650 overexpression on the metastasis of melanoma in mice (N = 5 in each group). A, Relative expression of miR-650 in WM115 cells; B, The number of micrometastaic focus; C, HE staining of lung tissues (400 × ). Mock, cells treated with lipofectamine 2000; NC, mimic negative control. The number of micrometastatic focus in the lung of mice was counted in 5 random fields of views at 10 × magnifications. *, p < 0.05 compared to mock or NC.

Fig. 5

The regulatory effect of miR-650 on ING4. A, qRT-PCR showed the relative mRNA expression of ING4 in WM115 cells after transfection with miR-650 mimic or mimic NC; B, Western blot showed the protein expression of ING4 in WM115 cells after transfection with miR-650 mimic or mimic NC; C, Dual luciferase reporter gene assay confirmed the target relationship between miR-650 and ING4; D, qRT-PCR showed the miR-650 expression in WM115 cells after transfection with miR-650 inhibitor or inhibitor NC; E, qRT-PCR showed the relative mRNA expression of ING4 in WM115 cells after transfection with miR-650 inhibitor or inhibitor NC; F, Western blot showed the protein expression of ING4 in WM115 cells after transfection with miR-650 inhibitor or inhibitor NC; G, qRT-PCR showed the miR-650 expression in A-375 cells after transfection with miR-650 mimic, miR-650 inhibitor or their NC; H, qRT-PCR showed the relative mRNA expression of ING4 in A-375 cells after transfection with miR-650 mimic, miR-650 inhibitor or their NC; I: Western blot showed the protein expression of ING4 in A-375 cells after transfection with miR-650 mimic, miR-650 inhibitor or their NC. Mock, cells treated with lipofectamine 2000. The experiments were repeated three times. *, p < 0.05, and ***, p < 0.001 compared to mock or NC.