miRNome landscape analysis reveals a 30 miRNA core in retinoblastoma.

BACKGROUND: miRNAs exert their effect through a negative regulatory mechanism silencing expression upon hybridizing to their target mRNA, and have a prominent position in the control of many cellular processes including carcinogenesis. Previous miRNA studies on retinoblastoma (Rb) have been limited to specific miRNAs reported in other tumors or to medium density arrays. Here we report expression analysis of the whole miRNome on 12 retinoblastoma tumor samples using a high throughput microarray platform including 2578 mature miRNAs. METHODS: Twelve retinoblastoma tumor samples were analyzed using an Affymetrix platform including 2578 mature miRNAs. We applied RMA analysis to normalize raw data, obtained categorical data from detection call values, and also used signal intensity derived expression data. We used Diana-Tools-microT-CDS to find miRNA targets and ChromDraw to map miRNAs in chromosomes. RESULTS: We discovered a core-cluster of 30 miRNAs that were highly expressed in all the cases and a cluster of 993 miRNAs that were uniformly absent in all cases. Another 1022 miRNA were variably present in the samples reflecting heterogeneity between tumors. We explored mRNA targets, pathways and biological processes affected by some of these miRNAs. We propose that the core-cluster of 30 miRs represent miRNA machinery common to all Rb, and affecting most pathways considered hallmarks of cancer. In this core, we identified miR-3613 as a potential and critical down regulatory hub, because it is highly expressed in all the samples and its potential mRNA targets include at least 36 tumor suppressor genes, including RB1. In the variably expressed miRNA, 36 were differentially expressed between males and females. Some of the potential pathways targeted by these 36 miRNAs were associated with hormonal production. CONCLUSION: These findings indicate that Rb tumor samples share a common miRNA expression profile regardless of tumor heterogeneity, and shed light on potential novel therapeutic targets such as mir-3613 This is the first work to delineate the miRNA landscape in retinoblastoma tumor samples using an unbiased approach.

[Level of expression of gene CTSL and its correlation with natural killer T-Cells in mexican pediatric patients with recent-onset type 1 diabetes]. / Asociación de células NKT con la expresión del gen CTSL en población pediátrica mexicana con diabetes tipo 1 de reciente diagnóstico.

OBJECTIVE: To compare the level of expression of the gene CTSL and its correlation with NKT cells in patients with recent-onset type 1 diabetes (T1D), their siblings, and healthy controls. METHODS: Analytical cross-sectional design. Patients with T1D < 3 months evolution, their siblings, and healthy controls were included. Percentages and absolute numbers of NKT cells were measured with expression of the CTSL gene. RESULTS: 124 subjects: with T1D (n = 48), siblings (n = 44) and controls (n = 32) were included. HbA1c was greater and C-peptide lower in T1D than the other groups and sibling age was higher (p < 0.001). There were no differences in NKT cells between T1D (0.176 ± 0.202) and controls (0.118 ± 0.133), but the percentage was higher in siblings (0.246 ± 0.188; p = 0.002). Lower level of expression of the CTSL gene associated with both absolute number (r: 0.4607; 95% CI: -0.08425 to -0.7935; p = 0.043) and percentage of NKT cells (r: 0.4540; 95% CI: -0.0927 to -0.7903; p = 0.045) in the T1D group. CONCLUSIONS: Patients with T1D have lower percentage and absolute number of NKT cells compared to their siblings. NKT cells absolute numbers are correlated with the expression of CTSL in T1D patients.

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Circulating miRNome detection analysis reveals 537 miRNAS in plasma, 625 in extracellular vesicles and a discriminant plasma signature of 19 miRNAs in children with retinoblastoma from which 14 are also detected in corresponding primary tumors.

miRNAs regulate post-transcriptional gene expression in metazoans, and thus are involved in many fundamental cellular biological processes. Extracellular miRNAs are also found in most human biofluids including plasma. These circulating miRNAs constitute a long distance inter cellular communication system and are potentially useful biomarkers. High throughput technologies like microarrays are able to scan a complete miRNome providing useful detection scores that are underexplored. We proposed to answer how many and which miRNAs are detectable in plasma or extracellular vesicles as these questions have not yet been answered. We set out to address this knowledge gap by analyzing the mirRNome in plasma and corresponding extracellular vesicles (EVs) from 12 children affected by retinoblastoma (Rb) a childhood intraocular malignant tumor, as well as from 12 healthy similarly aged controls. We calculated an average of 537 detectable miRNAs in plasma and 625 in EVs. The most miRNA enriched compartment were EVs from Rb cases with an average of 656 detectable elements. Using hierarchical clustering with the detection scores, we generated broad detection mirnome maps and identified a plasma signature of 19 miRNAs present in all Rb cases that is able to discriminate cases from controls. An additional 9 miRNAs were detected in all the samples; within this group, miRNA-5787 and miRNA-6732-5p were highly abundant and displayed very low variance across all the samples, suggesting both are good candidates to serve as plasma references or normalizers. Further exploration considering participant's sex, allowed discovering 5 miRNAs which corresponded only to females and 4 miRNAs corresponding only to males. Target and pathway analysis of these miRNAs revealed hormonal function including estrogen, thyroid signaling pathways and testosterone biosynthesis. This approach allows a comprehensive unbiased survey of a circulating miRNome landscape, creating the possibility to define normality in mirnomic profiles, and to locate where in these miRNome profiles promising and potentially useful circulating miRNA signatures can be found.