Oncogenic microRNA 17-92 cluster is regulated by epithelial cell adhesion molecule and could be a potential therapeutic target in retinoblastoma.

PURPOSE: Several miRNAs have been reported as candidate oncogenes and tumor suppressors, which are involved in the pathways specifically altered during tumorigenesis or metastasis. The miR 17-92 cluster located in 13q31 locus might contribute to retinoblastoma (RB) oncogenesis as 13q31 is amplified often in RB. We attempted to identify the factors involved in the regulation of miR 17-92 cluster in RB. METHODS: Real-time quantitative reverse transcriptase PCR was performed to study the expression of the miR 17-92 cluster in primary RB tumors and in Y79 cells after epithelial cell adhesion molecule (EpCAM) silencing. EpCAM was silenced using siRNA and confirmed by western blotting. The Y79 cells were transfected with individual and mixed antagomirs and studied the cell viability by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion by matrigel analysis and caspase-3 expression by flow cytometry. RESULTS: The relative expression of miR 17-92 cluster, compared to that of a normal retina, ranged from 25 to 220 fold (p<0.0001), miR-18 being highly expressed in RB. Post EpCAM silencing resulted in a significant decrease (p<0.01) in the expression of the miR 17-92 cluster by 4 to eightfold in Y79 cells. Y79 cells transfected with an antagomirs mix (all 5 miRNAs) showed decreased cell viability (p<0.001) and cell invasion (p<0.001). Similarly, Y79 cells treated with antagomirs mix showed increased expression of caspase-3 (p<0.001), which confirms the anti-proliferative effect of antagomirs. CONCLUSIONS: This study has showed varied expression of the miR17-92 cluster in primary RB tumors. EpCAM influences miR 17-92 cluster expression in retinoblastoma. In addition, we showed that the miR 17-92 cluster plays a role in RB cell proliferation and invasion. Therefore, targeting the miRNA 17-92 cluster may be beneficial for controlling Y79/RB cell proliferation and invasion.

Hypermethylation of adenomatosis polyposis coli-2 and its tumor suppressor role in retinoblastoma.

PURPOSE: Retinoblastoma (RB) is a progressive eye cancer of infancy and childhood. Hypermethylation, epigenetic silencing of genes is one of the key events in tumorigenesis. The purpose of this study is to investigate hypermethylation of adenomatosis polyposis coli homologue, APC-2 and possible interaction of APC-2 with Wnt signaling ß-catenin protein in Retinoblastoma. METHODOLOGY: Primary RB tumor samples and cell line were used for the study. DNA isolation, bisulfite conversion, methylation specific PCR and DNA sequencing analysis of PCR products were performed to identify CpG islands and methylation in primary RB tumor samples (n = 30). Chemical demethylation and retrieval of APC-2 expression was studied using 5-Azacytidine (5'-AZC). Flow cytometry, immunofluorescence, western blot were performed for APC-2 expression analysis in demethylated Y79 cells. Co-localization study was conducted to understand the interaction between APC-2 and ß-catenin. RESULTS: APC-2 gene was methylated and down regulated in primary RB tumors. We observed that 70% of RB tumors (21/30) showed positivity with APC-2 methylation. The RB Y79 cells after treatment with demethylating agent 5'-AZC retrieved APC-2 expression, which was confirmed by immunofluorescence and Western blot. Flow cytometry showed APC-2 expression of 29.22% in 5'-AZC treated cells. Co-localization study showed interaction of APC-2 and RB upregulated ß-catenin in Y79 cells. CONCLUSION: We report that APC-2 gene is hypermethylated in both RB tumor samples and Y79 cells. Reduced APC-2 lead to increased Wnt signaling pathway protein, ß-catenin suggesting tumor suppressive role of APC-2 gene.

A comparative fluorescent beacon-based method for serum microRNA quantification.

Circulating serum microRNAs (miRNAs) are promising biomarkers for disease diagnosis. The quantification of the serum miRNA copy number is a challenge due to the presence of low levels in the serum. Here, we report on a direct measurement of the miRNA copy number from human serum using a locked nucleic acid (LNA) modified beacon probe with a single step using fluorescence spectroscopy and microscopy. We had used a minimum volume of 0.1 µL healthy human serum and retinoblastoma serum to show the biological variation of the miRNA copy number.

A focus on microfluidics and nanotechnology approaches for the ultra sensitive detection of microRNA.

MicroRNAs are small RNAs that are deregulated under disease conditions. This allows them to be used as biomarkers for disease diagnosis. Recently, such microRNAs are serving as non- invasive blood based biomarkers due to ease of detection, and high stability at room temperature in biofluids. In view of these advancements in biomarker research, efficient miRNA detection at the femtomolar level is important. MiRNA detection techniques to date include Northern blotting, real time PCR and microarray technology. While these are efficient techniques, they cannot detect very low miRNA levels, and have questionable specificity. This review describes nanotechnology, microfluidics and liquid phase miRNA detection techniques that are highly sensitive and specific. With some sophistication, some of these techniques can be used as point of care devices to rapidly detect low level miRNA in patient clinical samples to aid disease diagnosis and prognosis.

EpCAM knockdown alters microRNA expression in retinoblastoma--functional implication of EpCAM regulated miRNA in tumor progression.

The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes.

Identification and Insilico Analysis of Retinoblastoma Serum microRNA Profile and Gene Targets Towards Prediction of Novel Serum Biomarkers.

Retinoblastoma (RB) is a malignant tumor of the retina seen in children, and potential non invasive biomarkers are in need for rapid diagnosis and for prognosticating the therapy. This study was undertaken to identify the differentially expressed miRNAs in the serum of children with RB in comparison with the normal age matched serum, to analyze its concurrence with the existing RB tumor miRNA profile, to identify its novel gene targets specific to RB, and to study the expression of a few of the identified oncogenic miRNAs in the advanced stage primary RB patient's serum sample. MiRNA profiling was performed on 14 pooled serum from children with advanced RB and 14 normal age matched serum samples, wherein 21 miRNAs were found to be upregulated (fold change ≤ -2.0, P ≤ 0.05) and 24 to be downregulated (fold change ≥ +2.0, P ≤ 0.05). Furthermore, intersection of 59 significantly deregulated miRNAs identified from RB tumor profiles with that of miRNAs detected in serum profile revealed that 33 miRNAs had followed a similar deregulation pattern in RB serum. Later we validated a few of the miRNAs (miRNA 17-92) identified by microarray in the RB patient serum samples (n = 20) by using qRT-PCR. Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum samples exploring the potential of serum miRNAs identification as noninvasive diagnosis. Moreover, from miRNA gene target prediction, key regulatory genes of cell proliferation, apoptosis, and positive and negative regulatory networks involved in RB progression were identified in the gene expression profile of RB tumors. Therefore, these identified miRNAs and their corresponding target genes could give insights on potential biomarkers and key events involved in the RB pathway.