miRNome landscape analysis reveals a 30 miRNA core in retinoblastoma

Abstract

BackgroundmiRNAs 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.MethodsTwelve 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.ResultsWe 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.ConclusionThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3421-3) contains supplementary material, which is available to authorized users.

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

“…In a group of those plasma samples, we tested whether miRNA profiles are able to discriminate Rb cases from healthy controls. This work is an extension of a previous effort to define the mirnomic view in primary retinoblastoma tissue [20]. We now define the mirnomic view of the corresponding plasmas, further comparing them with healthy controls.…”

“…The corresponding list of these miRNAs including the 19 exclusive miRNAs in plasma from Rb cases is found in S3 Lists. We then explored the corresponding relationships of the 19 miRNAs found exclusively in plasma from Rb cases with the central core of 142 miRNAs previously reported in the primary tumors [20]. Results are shown in Fig 2D. There is an intersection between 14 out of 19 miRNAs detected in plasma from the Rb cases that are also detected in retinoblastoma tumor tissue, indicating that 5 miRNAs in circulation are not present in the tumor.…”

“…These calibrators allowed the development of robust methods for detection calls [19], which are basically ignored by most analysis. Broad maps of what is detected and what is not can be generated with the detection score using clustering tools thus facilitating data interpretation [20]. This approach allows answering relevant questions, such as´how many´and´which´miRNAs can be detected in plasma or EVs?…”

“…Total RNA was isolated from 0.5 ml of each plasma sample, and from 100 μl of purified EVs using TRIzol reagent (Invitrogen 15596026), solubilized in nuclease free water and quantified using a Nanodrop. RNA 150 ng was poly-A tailed and biotin incorporated into tails with Flash Tag biotin for miRNA microarrays (Affymetrix 901911), including an Enzyme Linked Oligosorbent Assay (ELOSA) to confirm biotin labeling according to manufacturer instructions and as previously described [20].…”

“…To test if plasma and EVs from Rb cases share miRNA elements with the primary tumors, we generated the corresponding plasma and EV datasets and compared these with a publicly available mirnomic dataset of the corresponding tumor tissues that we had previously reported (GEO GSE 84747 and Array Express E-MTAB-4977) [20]. With unsupervised hierarchical clustering analysis (clustering both samples and miRNAs), we generated a miRNA detection two-color heatmap of these samples shown in Fig 1. This map shows a panoramic mirnomic view of detected and not detected miRNAs in all samples derived from Rb cases.…”