Clinico-pathological association of delineated miRNAs in uveal melanoma with monosomy 3/Disomy 3 chromosomal aberrations

PURPOSE: To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM) tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort. METHODS: Based on chromosomal 3 aberration, UM (n = 86) were grouped into monosomy 3 (M3; n = 51) and disomy 3 (D3; n = 35) by chromogenic in-situ hybridisation (CISH). The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE) UM samples (n = 6) using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86) and mRNAs were validated in frozen UM (n = 10) by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52). RESULTS: Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0). Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134) were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated. CONCLUSION: UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness

Significantly dys-regulated miRNAs and pathways in UM.

<p>A: Trendline graph shows the absolute expression of key miRNAs identified by ANOVA and SAM. The differentially expressed miRNAs indicates the discriminating profiles in M3 and D3 UM tumors. The blue line indicates the miRNA expression in D3 UM tumors while red line indicates the miRNA expression in M3 tumors. B: Hierarchical cluster shows key miRNAs subjected to unsupervised analysis. The differentially expressed miRNAs indicates the co-expression pattern across M3 and D3 UM tumors. The green colour indicates the down-regulated miRNAs while the red colour indicates the up-regulated miRNAs in M3 and D3 UM tumors. C: Analysis of key gene ontology (GO) and Pathway Enrichment by the differentially expressed miRNAs. A few of the pathways (i) and GO (ii and iii) targeted by these differentially expressed miRNAs using miRTarbase and PANTHER data base with p-value cut-off ≤0.05 along with Boneferroni FDR correction.</p

Graphical representation of metastasis-free survival was analysed by Kaplan-Meier analysis.

<p>The cumulative proportion surviving at end of the interval (60 months) is represented in percentage. Follow up of 52 patients (2–5 years of duration) were considered in the study.</p

Unsupervised and supervised analysis of miRNA expression in UM.

<p>A: Graphical representation of the unsupervised analysis: principal component analysis (PCA) for the UM tumor tissues: M3 (n = 3), and D3 (n = 3) showing the clustering of samples correspondingly in to two distinct groups (M3 and D3). Blue colored triangle denotes M3 UM tumors while red colored spheres denotes D3 UM tumors. B: Supervised analysis: The significance analysis of microarray (SAM) plot reveals the de regulated miRNAs between the monosomy and disomy formalin fixed UM primary tumors. The false discovery rate (FDR) has set at Q = 0.78%. The list of miRNAs with Q = 0.0 has been considered for the validation by qRT-PCR. C: Venn diagram on intersection of miRNAs obtained from ANOVA and SAM. <i>miR-134</i>, <i>miR-1238</i>, <i>miR-149*</i> are the common select miRNAs observed from this study.</p

Network interaction between the dysregulated miRNAs and their gene targets in UM.

<p>A: D3 UM tumor; B: M3 UM tumor. The network is drawn with the expressions obtained by qRT-PCR using cytoscape v.2.8. Green colour indicates the down-regulation of miRNAs/ genes; red colour indicates the up-regulation of miRNAs/ genes; Yellow colour indicates varied miRNA/ gene expression.</p