Exosomal MicroRNA as Biomarkers for Diagnosing or Monitoring the Progression of Ovarian Clear Cell Carcinoma: A Pilot Study

Ovarian cancer is the most common cause of gynecological malignancy-related mortality since early-stage disease is difficult to diagnose. Advanced clear cell carcinoma of the ovary (CCCO) has dismal prognosis, and its incidence has been increasing in Japan, emphasizing the need for highly sensitive diagnostic and prognostic CCCO biomarkers. Exosomal microRNAs (miRNAs) secreted by tumor cells are known to play a role in carcinogenesis; however, their involvement in ovarian cancer is unclear. In this study, we performed expression profiling of miRNAs from exosomes released by five cell lines representing different histological types of ovarian cancer. Exosomes isolated from culture media of cancer and normal cells were compared for miRNA composition using human miRNA microarray. We detected 143 exosomal miRNAs, whose expression was ≥1.5-fold higher in ovarian cancer cells than in the control. Among them, 28 miRNAs were upregulated in cells of all histological ovarian cancer types compared to control, and three were upregulated in CCCO cells compared to other types. Functional analyses indicated that miR-21 overexpressed in CCCO cells targeted tumor suppressor genes PTEN, TPM1, PDCD4, and MASP1. The identified miRNAs could represent novel candidate biomarkers to diagnose or monitor progression of ovarian cancer, particularly CCCO.     

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.     

In silico study of cancer stage-specific DNA methylation pattern in White breast cancer patients based on TCGA dataset

BackgroundBreast cancer is one of the most common types of cancer among women. As current breast cancer treatments are still ineffective, we assess the methylation pattern of White breast cancer patients across cancer stage based on The Cancer Genome Atlas (TCGA) dataset. Significant hypermethylation and hypomethylation can regulate the gene expression, thus becoming potential biomarkers in breast cancer tumorigenesis.MethodsDNA methylation data was downloaded using TCGA Assembler 2 based on race-specific metadata of TCGA - Breast Invasive Carcinoma (TCGA-BRCA) project from Genomic Data Commons (GDC) Data Portal. After the data was divided into each cancer stage, duplicated data of each patient was removed using OMICSBind, while differentially-expressed probes were identified using edgeR. The resulting probes were validated based on correlation and regression analysis with the gene expression, ANOVA between cancer stages, ROC curve per stage, as well as databases.ResultsBased on the White dataset, we found 66 significant hypermethylated genes with logFC > 1.8 between Stage I-III. From this number, three epigenetic-regulated, stage-specific genes are proposed to be the detection biomarkers of breast cancer due to significant aberrant gene expression and/or low mutation ratio among breast cancer patients: ABCC9 (Stage III), SHISA3 (Stage II), and POU4F1 (Stage I-II).ConclusionsOur study shows that ABCC9, SHISA3, and POU4F1 are potential stage-specific detection biomarkers of breast cancer for White individuals, whereas their roles in other races need to be studied further.     

The role of promoter methylation in Epstein-Barr virus (EBV) microRNA expression in EBV-infected B cell lines

Epstein-Barr virus (EBV) microRNAs (miRNAs) are expressed in EBV-associated tumors and cell lines, but the regulation mechanism of their expression is unclear yet. We investigated whether the expression of EBV miRNAs is epigenetically regulated in EBV-infected B cell lines. The expression of BART miRNAs was inversely related with the methylation level of the BART promoter at both steady-state and following 5-aza-2'-deoxycytidine treatment of the cells. The expression of BHRF1 miRNAs also became detectable with the demethylation of Cp/Wp in latency I EBV-infected cell lines. Furthermore, in vitro methylation of the BART and Cp promoters reduced the promoter-driven transactivation. In contrast, tricostatin A had little effect on the expression of EBV miRNA expression as well as on the BART and Cp/Wp promoters. Our results suggest that promoter methylation, but not histone acetylation, plays a role in regulation of the EBV miRNA expression in EBV-infected B cell lines.     

Analysis of the NCI-60 dataset for cancer-related microRNA and mRNA using expression profiles

BackgroundRecent studies have indicated that microRNA (miRNA) may play an oncogenic or tumor suppressor role in human cancer. To study the regulatory role of miRNAs in tumorigenesis, an integrated platform has been set up to provide a user friendly interface for query. The main advantage of the present platform is that all the miRNA target genes’ information and disease records are drawn from experimentally verified or high confidence records.ResultsMiRNA target gene results are annotated with reference to the disease gene as well as the pathway database. The correlation strength between miRNA and target gene expression profile is quantified by computing the correlation coefficient using the NCI-60 expression profiling data. Comprehensive analysis of the NCI-60 data found that the cumulative percentage of negative correlation coefficients for cleavage regulation is slightly higher than its positive counterpart; which indicated that the mRNA degradation mechanism is slightly dominant. In addition, the RNAHybrid and TargetScans scores are computed which potentially served as quantitative estimators for miRNA–mRNA binding events.Three scores are defined for each miRNA–mRNA pair, which are based on the disease gene and pathway information. These three scores allow user to sort out high confidence cancer-related miRNA–mRNA pairs.Statistical tests were applied to investigate the relations of three chromosomal features, i.e., CpG island, fragile site, and miRNA cluster, with cancer-related miRNAs. A web-based interface has been set up for query, which can be accessed at: http://ppi.bioinfo.asia.edu.tw/mirna_target/ConclusionsThe main advantage of the present platform on miRNA–mRNA targeting information is that all the target genes’ information and disease records are experimentally verified. Although this may limit the number of miRNA–mRNA relationships, the results provided here are more solid and have fewer false positive events. Certain novel cancer-related miRNA–mRNA pairs are identified and confirmed in the literature. Fisher's exact test suggests that CpG island and fragile site associated miRNAs tend to associate with cancer formation. In summary, the present platform provides an easy means of investigating cancer-related miRNAs.     

Expression of miRNAs Targeting <Emphasis Type="Italic">mTOR</Emphasis> and <Emphasis Type="Italic">S6K1</Emphasis> Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3′-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.     

The regulation of microRNA in each of cancer stage from two different ethnicities as potential biomarker for breast cancer

miRNA has recently emerged as a potential biomarker for breast cancer. Even though many studies have identified ethnic variation affecting miRNA regulation, the effect of cancer stage within specific ethnicities on miRNA epigenetic remains unclear. The present study is designed to investigate miRNA regulation from two distinct ethnicities in specific cancer stages (non-Hispanic white and non-Hispanic black) using the TCGA dataset. Differentially expressed miRNAs were calculated by using the edgeR package. miRNAs with the highest or lowest log fold Change from each cancer stage were selected as a potential biomarker. miRNA-gene interaction was analyzed by using spearman correlation analysis, CLUEGO, and DIANA-mirpath. The association of biomarker candidates with diagnostic and prognostic performance was assessed using ROC and Kaplan-Meier survival analysis. miRNA-gene interaction analysis revealed the involvement of selected miRNAs in cancer progression. From eleven selected aberrant miRNAs, four of the miRNAs (hsa-mir-495, hsa-mir-592, hsa-mir-6501, and hsa-mir-937) are significantly detrimental to breast cancer diagnosis and prognosis. Hence, our result provides valuable information to explore miRNA’s role in each cancer stage between non-Hispanic white and non-Hispanic black.     

Suitable reference genes for relative quantification of miRNA expression in prostate cancer

Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa- miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa- miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.     

Identifying the target mRNAs of microRNAs in colorectal cancer

MicroRNAs (miRNAs) play an important role in gene regulatory networks by inhibiting the expression of target mRNAs. There is a growing interest in identifying the relationship between miRNAs and their target mRNAs. Various experimental studies have been carried out to discover miRNAs involved in cancer and to identify their target genes. At the same time, a large volume of miRNA and mRNA expression profiles have become available owing to the development of high-throughput measurement technologies. So, there is now a pressing need to develop a computational method by which we can identify the target mRNAs of given miRNAs from such massive expression data sets. In this respect, we propose an effective linear model based identification method to unravel the relationship between miRNAs and their target mRNAs in colorectal cancer by using microarray expression profiles and sequence data.     

Computational identification of novel microRNA homologs in the chimpanzee genome

MicroRNAs are important negative regulators of gene expression in higher eukaryotes. The miRNA repertoire of the closest human animal relative, the chimpanzee (Pan troglodytes), is largely unknown. In this study, we focused on computational search of novel miRNA homologs in chimpanzee. We have searched and analyzed the chimp homologs of the human pre-miRNA and mature miRNA sequences. Based on a homology search of the chimpanzee genome with human miRNA precursor sequences as queries, we identified 639 chimp miRNA genes, including 529 novel chimp miRNAs. 91.8% of chimp mature miRNAs and 60.3% of precursors are 100% identical to their human orthologs. The pre-miRNA secondary structures, miRNA families, and clusters are also highly conserved. We also found certain sequence differences in pre-miRNAs and even mature miRNAs that occurred after the divergence of the two species. Some of these differences (especially in mature miRNAs) could have caused species-specific changes in the expression levels of their target genes which in turn could have resulted in phenotypic variation between human and chimp.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.     

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.Subject terms: Prognostic markers, Methylation analysis