MiR-144 Inhibits Uveal Melanoma Cell Proliferation and Invasion by Regulating c-Met Expression

<div><p>MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.</p></div

Overexpression of miR-144inhibited proliferation and invasion of uveal melanoma cells (A) qRT–PCR analysis of miR-144 expression in MUM-2B cells which was transfected miR-144 mimics, inhibitors, scramble or control.

<p>(B) The CCK-8 proliferation assay showed that miR-144 mimics can inhibit the proliferation of the MUM-2B cells. Meanwhile, miR-144 inhibitor increased the proliferation of the MUM-2B cells. (C) Invasion analysis of MUM-2B cells after treatment withmiR-144 mimics, inhibitors or scramble or control; the relative ratio of invasive cells per field is shown below, *p<0.05, ** p<0.01, and ***p<0.001.</p

c-Met is a critical downstream target of miR-144 (A) Targetscan analysis using available algorithms indicated that c-Met is a theoretical target gene of miR-144.

<p>(B) Luciferase reporter gene assays showed that ectopic of miR-144 remarkably reduced luciferase activity in the c-Met wild-type reporter gene but not the mutant c-Met 3’UTR.(C) qRT-PCR analysis of c-Met expression in the MUM-2B cells which was transected miR-144 mimics, inhibitors, scramble or control. GAPDH was used as internal control. (D) Western blot analysis has shown that miR-144 mimic inhibited the protein expression of c-Met in MUM-2B cells. GAPDH was also detected as a loading control. ***p<0.001.</p

Inhibition of c-Met inhibits uveal melanoma cell proliferation and invasion (A) Western blotting analysis was performed to examine the effects of siRNA-c-Met on the expression of c-Met.

<p>GAPDH was also detected as a loading control. (B) The cell growth in MUM-2B cells co-transfected with either siRNA-c-Met, siRNA-c-Met and miR-144 inhibitor or control using CCK-8 proliferation assay. (C) The cell invasive in MUM-2B cells co-transfected with either siRNA-c-Met, siRNA-c-Met and miR-144 inhibitor or control using invasion assay. *p<0.05, ** p<0.01, and ***p<0.001.</p

The expression ofmiR-144 was downregulated in uveal melanoma cells and tissues (A) qRT–PCR analysis of miR-144expression in uveal melanoma cell lines (MUM-2B, C918, MUM-2C and OCM-1A) and one human melanocyte cell line (D78).

<p>The level of miR-144 expression was normalized to U6. (B) qRT–PCR analysis of miR-144expression in 5human uveal melanoma tissues and 5 normal uvea tissues. The level of miR-144 expression was normalized to U6.***p<0.001.</p