Search

Additional file 1: Figure S1. Validation of circRNA expression. Figure S2. Validation of circRNA exon retention. Figure S3. Pathway enrichment among differentially expressed circRNAs. Figure S4. Pathway enrichment among differentially expressed linear RNAs. Figure S5. Log2 fold-change values of top increased circRNAs and their corresponding linear host RNAs. Figure S6. Variant analysis in circRNA exons. Table S5. Source studies for compilation de novo variants identified in individuals with Schizophrenia or Schizoaffective disorder. Figure S7. SFPQ and TIAL1 target detection and siRNA knockdown. Figure S8. Extended expression profile of SFPQ and identified circRNA targets in NES differentiation. Figure S9. In Situ detection of circRNAs during NES differentiation. Figure S10. Analysis of introns from circRNA host genes. Table S8. Primer sequences.

(a, b) Luciferase assay. The HK-2 and the HCT116 cells were transfected with Negative Control miRNA Mimic, miR-17-5p, miR-106b-5p, anti-miR-17-5p or anti-miR-106b-5p, along with pMIR luciferase reporter construct containing p21 3’UTR. Cells were lysed and luciferase activity was determined as described in the Material and Methods section. Transfection efficacy was normalized by Renilla Luciferase activity. Data represent the averages of at least three independent experiments with their standard deviations. ** p-value

(a, b) Expression levels of miR-17-5p and miR-106b-5p were measured by RT-qPCR in HCT116 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. (c-e) Expression levels of TRIM8, p21, miR-34a and miR-17-5p were measured by RT-qPCR in the RCC-Shaw cells transfected with Negative Control miRNA Mimic or anti-miR-17-5p plus control short hairpin-RNA (control shRNA) or specific short hairpin against TRIM8 (shRNA-TRIM8). After transfection the cells were treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a and miR-17-5p. * p-value

(a-f) Expression levels of miR-17-5p and miR-106b-5p were measured by RT-qPCR in HK-2, RCC-Shaw and UOK-257 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. ** p-value

(a) miR-106a-5p expression in ccRCC samples (T) and their paired non-tumour tissues (NT). The analysis was performed considering the Fuhrman grading of the tumour samples. Data are represented in box-and-whisker plots showing median and 10th, 25th, 75th and 90th percentiles for each category of sample. Expression data were measured respect to one normal sample chosen arbitrarily as calibrator and then normalized by the expression levels of U6 snRNA. The bars represent the Standard Error of the Mean. It is also reported the sequence alignment between the miR-106a-5p “seed” sequence and the TRIM8 3’UTR. (b) miR-106a-5p, miR-17-5p and miR-106b-5p expression in 4 oncocytoma samples (T) and their paired non-tumour tissues (NT). Data are represented in box-and-whisker plots showing median and 10th, 25th, 75th and 90th percentiles for each category of sample. Expression data were measured respect to one normal sample chosen arbitrarily as calibrator and then normalized by the expression levels of U6 snRNA. The bars represent the Standard Error of the Mean. (PDF 79 kb)

(a-d) Expression levels of TRIM8, p21, miR-34a and miR-17-5p were measured by RT-qPCR in the RCC-Shaw cells transfected with Negative Control miRNA Mimic or anti-miR-17-5p plus control short hairpin-RNA (control shRNA) or specific short hairpin against TRIM8 (shRNA-TRIM8). After transfection the cells were treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a and miR-17-5p. ** p-value

(a, b) Expression levels of miR-17-5p and miR-106b-5p were measured by qPCR in HK-2, RCC-Shaw, UOK-257 and HCT116, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p. Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. The bars represent the Standard deviation of the Mean. (c) Colony suppression assays. Cell growth was measured in HK-2, RCC-Shaw, UOK-257 and HCT116 cells transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p. (PDF 1350 kb)

(a-b) Densitometry analysis of p53, Trim8, N-MYC and p21 proteins in HK-2, UOK-257, RCC-Shaw and HCT116 cells transfected with negative control miRNA Mimic, anti-miR-17-5p (a) or anti-miR-106b-5p (b). The relative amounts of proteins, reported in the table below the western blotting, have been calculated normalizing to actin and calibrating to the respective control. (PDF 925 kb)

(a-i) Expression levels of TRIM8, p21 and miR-34a were measured by RT-qPCR in HK-2, RCC-Shaw and UOK-257 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a. * p-value

Adenine to Inosine RNA editing is a widespread co- and post-transcriptional mechanism mediated by ADAR enzymes acting on double stranded RNA. It has a plethora of biological effects, appears to be particularly pervasive in humans with respect to other mammals, and is implicated in a number of diverse human pathologies. Here we present the first human inosinome atlas comprising 3,041,422 A-to-I events identified in six tissues from three healthy individuals. Matched directional total-RNA-Seq and whole genome sequence datasets were generated and analysed within a dedicated computational framework, also capable of detecting hyper-edited reads. Inosinome profiles are tissue specific and edited gene sets consistently show enrichment of genes involved in neurological disorders and cancer. Overall frequency of editing also varies, but is strongly correlated with ADAR expression levels. The inosinome database is available at: http://srv00.ibbe.cnr.it/editing/.

The PGC1 family (Peroxisome proliferator-activated receptor γ (PPAR γ) coactivators) of transcriptional coactivators are considered master regulators of mitochondrial biogenesis and function. The PGC1α isoform is expressed especially in metabolically active tissues, such as the liver, kidneys and brain, and responds to energy-demanding situations. Given the altered and highly adaptable metabolism of tumor cells, it is of interest to investigate PGC1α in cancer. Both high and low levels of PGC1α expression have been reported to be associated with cancer and worse prognosis, and PGC1α has been attributed with oncogenic as well as tumor suppressive features. Early in carcinogenesis PGC1α may be downregulated due to a protective anticancer role, and low levels likely reflect a glycolytic phenotype. We suggest mechanisms of PGC1α downregulation and how these might be connected to the increased cancer risk that obesity is now known to entail. Later in tumor progression PGC1α is often upregulated and is reported to contribute to increased lipid and fatty acid metabolism and/or a tumor cell phenotype with an overall metabolic plasticity that likely supports drug resistance as well as metastasis. We conclude that in cancer PGC1α is neither friend nor foe, but rather the obedient servant reacting to metabolic and environmental cues to benefit the tumor cell.

Background TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. Methods We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. Results We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. Conclusions In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0634-7) contains supplementary material, which is available to authorized users.