Supplementary Table 4 from Molecular Profiling Uncovers a p53-Associated Role for MicroRNA-31 in Inhibiting the Proliferation of Serous Ovarian Carcinomas and Other Cancers
<div>Abstract<p>MicroRNAs (miRNA) regulate complex patterns of gene expression, and the relevance of altered miRNA expression to ovarian cancer remains to be elucidated. By comprehensively profiling expression of miRNAs and mRNAs in serous ovarian tumors and cell lines and normal ovarian surface epithelium, we identified hundreds of potential miRNA-mRNA targeting associations underlying cancer. Functional overexpression of miR-31, the most underexpressed miRNA in serous ovarian cancer, repressed predicted miR-31 gene targets including the cell cycle regulator <i>E2F2. MIR31</i> and <i>CDKN2A</i>, which encode p14<sup>ARF </sup>and p16<sup>INK4A</sup>, are located at 9p21.3, a genomic region commonly deleted in ovarian and other cancers. p14<sup>ARF</sup> promotes p53 activity, and <i>E2F2</i> overexpression in p53 wild-type cells normally leads via p14<sup>ARF</sup> to an induction of p53-dependent apoptosis. In a number of serous cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, and SKOV3), miR-31 overexpression inhibited proliferation and induced apoptosis; however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Additionally, the osteosarcoma cell line U2OS and the prostate cancer cell line PC3 (p14<sup>ARF</sup>-deficient and p53-deficient, respectively) were also sensitive to miR-31. Furthermore, miR-31 overexpression induced a global gene expression pattern in OVCAR8 associated with better prognosis in tumors from patients with advanced stage serous ovarian cancer, potentially affecting many genes underlying disease progression. Our findings reveal that loss of miR-31 is associated with defects in the p53 pathway and functions in serous ovarian cancer and other cancers, suggesting that patients with cancers deficient in p53 activity might benefit from therapeutic delivery of miR-31. Cancer Res; 70(5); 1906–15</p></div>
MicroRNAs (miRNAs) are small non-coding RNAs that mediate post-transcriptional gene silencing. Over 700 human miRNAs have currently been identified, many of which are mutated or de-regulated in diseases. Here we report the identification of novel miRNAs through deep sequencing the small RNAome (<30 nt) of over 100 tissues or cell lines derived from human female reproductive organs in both normal and disease states. These specimens include ovarian epithelium and ovarian cancer, endometrium and endometriomas, and uterine myometrium and uterine smooth muscle tumors. Sequence reads not aligning with known miRNAs were each mapped to the genome to extract flanking sequences. These extended sequence regions were folded in silico to identify RNA hairpins. Sequences demonstrating the ability to form a stem loop structure with low minimum free energy (<−25 kcal) and predicted Drosha and Dicer cut sites yielding a mature miRNA sequence matching the actual sequence were considered putative novel miRNAs. Additional confidence was achieved when putative novel hairpins assembled a collection of sequences highly similar to the putative mature miRNA but with heterogeneous 3′-ends. A confirmed novel miRNA fulfilled these criteria and had its "star" sequence in our collection. We found 7 distinct confirmed novel miRNAs, and 51 additional novel miRNAs that represented highly confident predictions but without detectable star sequences. Our novel miRNAs were detectable in multiple samples, but expressed at low levels and not specific to any one tissue or cell type. To date, this study represents the largest set of samples analyzed together to identify novel miRNAs.
Abstract Retinoic acid induced 1 (RAI1) encodes a dosage-sensitive gene that when haploinsufficient results in Smith–Magenis syndrome (SMS) and when overexpressed results in Potocki–Lupski syndrome (PTLS). Phenotypic and molecular evidence illustrates that haploinsufficiency of RAI1 disrupts circadian rhythm through the dysregulation of the master circadian regulator, circadian locomotor output cycles kaput (CLOCK), and other core circadian components, contributing to prominent sleep disturbances in SMS. However, the phenotypic and molecular characterization of sleep features in PTLS has not been elucidated. Using the Pittsburgh Sleep Quality Index (PSQI), caregivers of 15 school-aged children with PTLS reported difficulties in initiating sleep. Indeed, more than 70% of individuals manifested moderate to severe sleep latency, as defined by the PSQI. Moreover, these individuals manifested difficulties in sleep maintenance, with middle of the night and early morning awakenings. When assessing daytime sleepiness through the Epworth Sleepiness Scale, approximately 21% of the individuals manifested excessive daytime somnolence. This indicates that mild dyssomnia characterizes the majority of the sleep phenotype, with occasionally problematic daytime somnolence, a phenotype different than that expressed by individuals with SMS, where daytime sleepiness is a chronic problem. Gene expression analysis of the core circadian machinery in the hypothalamus of the PTLS mouse model (Rai1-Tg) found significant dysregulation of the transcriptional activators, Clock and Arntl, and the transcriptional repressors, Per1–3 and Cry1/2, during both light and dark phases. These findings suggest a partial loss of circadian entrainment typically evoked by environmental photic cues. Examination of circadian clock gene expression in the Rai1-Tg mouse heart, liver, and kidney found unchanged expression of Clock and most of its downstream targets during both light and dark phases, suggesting an asynchronized circadian rhythm. Furthermore, examination of circadian gene expression in synchronized PTLS lymphoblasts revealed reduced transcripts of the Period (PER1–3) family and normal expression of CRY1/2. The finding that central circadian gene expression was altered while many peripheral circadian components were intact suggests a tissue-specific circadian uncoupling of the circadian machinery due to Rai1 overexpression. Overall, our results demonstrate that overexpression of RAI1 results in sleep deficiencies in individuals with PTLS due to a lack of properly regulated circadian machinery gene expression and highlight the importance of evaluating sleep concerns in individuals with PTLS.
Supplementary Table 1 from Molecular Profiling Uncovers a p53-Associated Role for MicroRNA-31 in Inhibiting the Proliferation of Serous Ovarian Carcinomas and Other Cancers
MicroRNAs (miRNAs) are short non-coding RNAs that could have large-scale biological effects by directing gene regulation through translational repression and degradation of multiple complementary target mRNAs. Like other regulatory molecules, altered miRNA expression has been implicated in the formation of cancers. To identify putative tumor suppressor microRNAs in ovarian cancer, we used Illumina next generation sequencing technology to comprehensively profile microRNAs in human ovarian cancers, cancer cell lines, and normal ovarian surface epithelium (NOSE). We found that miR-31 was the most down-regulated miRNA, decreased an average of 35-fold (range: 12-460-fold) in human serous ovarian cancers (n=8) compared to NOSE (n=4). Likewise, miR-31 levels were decreased 34-fold (range: 14-590-fold) in ovarian cancer cell lines compared to NOSE. We next profiled the mRNA expression levels in the cancers, cell lines, and NOSE cells. Using an established bioinformatic algorithms, we identified hundreds of potential miR-31:mRNA associations potentially underlying cancer. We followed up these findings by validating in vitro the interrelationship of the predicted upregulated target genes with the downregulation of the miR-31 microRNA. Overexpression of miR-31 in the serous ovarian cancer cell line, OVCAR8, resulted in inhibition of proliferation and induction of apoptosis. Similar responses to miR-31 were observed in a number of ovarian cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, SKOV3 and TOV112D); however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Moreover, the osteosarcoma cell lines U2OS and SAOS2 (p14ARF-deficient and p53-deficient, respectively) were also sensitive to miR-31. Studies on the colon cancer cell lines HCT116 (p53+/+) and HCT116 (p53-/-) further indicated that miR-31 function is associated with defects in the p53 pathway. Molecular analysis using QPCR showed that predicted miR-31 targets including E2F2 were downregulated in OVCAR8 cells overexpressing miR-31, while a pro-apoptotic gene BAK1 was upregulated. Interestingly, in HEY cells, E2F2 and BAK1 were not influenced by introducing miR-31, indicating that miR-31 may function through a direct or indirect regulation on E2F2 and BAK1. Our results reveal a tumor suppressive effect of miR-31 in ovarian cancer. Further in vivo studies to explore the impact of altered miR-31 expression are highly warranted and are being performed. (platform)
Supplementary Table Legends 1-4 from Molecular Profiling Uncovers a p53-Associated Role for MicroRNA-31 in Inhibiting the Proliferation of Serous Ovarian Carcinomas and Other Cancers
