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

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

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

A Multi-Atlas Labeling Approach for Identifying Subject-Specific Functional Regions of Interest

<div><p>The functional region of interest (fROI) approach has increasingly become a favored methodology in functional magnetic resonance imaging (fMRI) because it can circumvent inter-subject anatomical and functional variability, and thus increase the sensitivity and functional resolution of fMRI analyses. The standard fROI method requires human experts to meticulously examine and identify subject-specific fROIs within activation clusters. This process is time-consuming and heavily dependent on experts’ knowledge. Several algorithmic approaches have been proposed for identifying subject-specific fROIs; however, these approaches cannot easily incorporate prior knowledge of inter-subject variability. In the present study, we improved the multi-atlas labeling approach for defining subject-specific fROIs. In particular, we used a classifier-based atlas-encoding scheme and an atlas selection procedure to account for the large spatial variability across subjects. Using a functional atlas database for face recognition, we showed that with these two features, our approach efficiently circumvented inter-subject anatomical and functional variability and thus improved labeling accuracy. Moreover, in comparison with a single-atlas approach, our multi-atlas labeling approach showed better performance in identifying subject-specific fROIs.</p></div

Influence of forest parameters on fROI labeling results.

<p>Each element in the matrix represents an averaged Dice accuracy across all fROIs from the RFs trained with the specific pair of maximum tree depth and tree number in the forest.</p

Effect of atlas selection on labeling accuracy measured by Dice’s coefficient.

<p>The labeling accuracy for the rOFA and rpFFA was computed with a different number of top-ranked and randomly selected atlases. SS, similarity based selection; RS, random selection.</p

Relationship between average Dice accuracy obtained by individual atlases and their corresponding ranks.

<p>The rank for an atlas was assigned by computing the activation pattern similarity between the atlas and the target image. Dice’s index was iteratively computed with each subject as the target image and then averaged across all atlases (N = 202).</p

Effect of atlas encoding scheme on labeling accuracy measured by Dice’s coefficient.

<p>The RF encoding scheme showed better accuracy than the image-based encoding scheme in labeling the rOFA and rpFFA (paired t-test p < 0.00001).</p