Xiaoyi Huang

Characterization of human plasma-derived exosomal RNAs by deep sequencing

BackgroundExosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).ResultsFrom the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.ConclusionsThis study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.

Exosomal miR-1290 and miR-375 as Prognostic Markers in Castration-resistant Prostate Cancer

BACKGROUND Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer. OBJECTIVE To identify and evaluate plasma exosomal miRNAs for prognosis in castration-resistant prostate cancer (CRPC). DESIGN, SETTING, AND PARTICIPANTS RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using quantitative real-time polymerase chain reaction in a follow-up cohort of 100 CRPC patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Cox regression and Kaplan-Meier survival analyses were used to evaluate survival association using candidate miRNAs along with clinical prognostic factors. RESULTS AND LIMITATIONS RNA sequencing in screening cohort generated approximately 6.80 million mappable reads per patient. Of those with normalized read counts ≥ 5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (false discover rate < 0.05). Of those, higher levels of miR-1290 and -375 were significantly associated with poor overall survival (p < 0.004) in the follow-up cohort. Incorporation of miR-1290/-375 into putative clinical prognostic factors-based models in CRPC stage significantly improved predictive performance with a time-dependent area under the curve increase from 0.66 to 0.73 (p = 6.57 × 10(-6)). CONCLUSIONS Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further evaluation of these candidate miRNAs. PATIENT SUMMARY In this study, we evaluated whether small RNAs circulating in blood could be used to predict clinical outcomes in late-stage prostate cancer patients. We identified two blood-based small RNAs whose levels showed significant association with survival. Our results warrant further investigation because the noninvasive blood-based test has great potential in the management of late-stage prostate cancer.

Extracellular microRNAs in urologic malignancies: chances and challenges.

Small noncoding RNAs that are 19–23 nucleotides long, known as microRNAs (miRNAs), are involved in almost all biological mechanisms during carcinogenesis. Recent studies show that miRNAs released from live cells are detectable in body fluids and may be taken up by other cells to confer cell-cell communication. These released miRNAs (here referred to as extracellular miRNAs) are often protected by RNA-binding proteins or embedded inside circulating microvesicles. Due to their relative stability, extracellular miRNAs are believed to be promising candidates as biomarkers for diagnosis and prognosis of disease, or even as therapeutic agents for targeted treatment. In this review, we first describe biogenesis and characteristics of these miRNAs. We then summarize recent publications involving extracellular miRNA profiling studies in three representative urologic cancers, including: prostate cancer, bladder cancer, and renal cell carcinoma. We focus on the diagnostic, prognostic, and therapeutic potential of these miRNAs in biological fluids, such as serum, plasma, and urine. Finally, we discuss advantages and challenges of these miRNAs in clinical applications.

Plasma extracellular RNA profiles in healthy and cancer patients.

Extracellular vesicles are selectively enriched in RNA that has potential as disease biomarkers. To systemically characterize circulating extracellular RNA (exRNA) profiles, we performed RNA sequencing analysis on plasma extracellular vesicles derived from 50 healthy individuals and 142 cancer patients. Of ~12.6 million raw reads for each individual, the number of mappable reads aligned to RNA references was ~5.4 million including miRNAs (~40.4%), piwiRNAs (~40.0%), pseudo-genes (~3.7%), lncRNAs (~2.4%), tRNAs (~2.1%), and mRNAs (~2.1%). By expression stability testing, we identified a set of miRNAs showing relatively consistent expression, which may serve as reference control for exRNA quantification. By performing multivariate analysis of covariance, we identified significant associations of these exRNAs with age, sex and different types of cancers. In particular, down-regulation of miR-125a-5p and miR-1343-3p showed an association with all cancer types tested (false discovery rate <0.05). We developed multivariate statistical models to predict cancer status with an area under the curve from 0.68 to 0.92 depending cancer type and staging. This is the largest RNA-seq study to date for profiling exRNA species, which has not only provided a baseline reference profile for circulating exRNA, but also revealed a set of RNA candidates for reference controls and disease biomarkers.

Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

Circulating exosomal miR-125a-3p as a novel biomarker for early-stage colon cancer

Circulating exosome holds great potentials as biomarker for diagnosis and prognosis of human cancers. Previously, we have applied small RNA sequencing to identify aberrantly expressed exosomal miRNAs as candidates for diagnostic markers in colon cancer patients. In this validation cohort, plasma derived exosomal miRNA was isolated from 50 early-stage colon cancer patients and 50 matched healthy volunteers. Real-time qRT-PCR revealed that miR-125a-3p, miR-320c were significantly up-regulated in plasma exosomes of the patients with early stage colon cancer. ROC curve showed that miR-125a-3p abundant level may predict colon cancer with an area of under the curve (AUC) of 68.5%, in comparison to that of CEA at 83.6%. Combination of miR-125a-3P and CEA improved the AUC to 85.5%. In addition, plasma exosome level of miR-125a-3p and miR-320c showed significant correlation with nerve infiltration (P < 0.01), but not with tumor size, infiltration depth, and differentiation degree (P > 0.05). On the contrary, plasma CEA level is correlated with tumor size, infiltration depth, and differentiation degree (P < 0.05, r = 0.3009–0.7270), but not with nerve infiltration (P = 0.744). In conclusion, this follow-up study demonstrated circulating plasma exosomal miR-125a-3p is readily accessible as diagnosis biomarker for early-stage colon cancer. When combined with conventional diagnostic markers, miR-125a-3p can improve the diagnostic power.

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing

BackgroundRNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers.ResultsWe developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification” includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module “mRNA identification” includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module “Target screening” provides expression profiling analyses and graphic visualization. The module “Self-testing” offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program’s functionality.ConclusionseRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

Aberrant regulation of miR-15b in human malignant tumors and its effects on the hallmarks of cancer

MicroRNAs encoded by the miR-15b/16-2 cluster act as tumor suppressors. Aberrant regulation of miR-15b in human malignant tumors is reportedly involved in cancer development, contributing to cell death, reduced proliferation, angiogenesis and metastasis resistance, metabolism reprogramming, genome instability, and tumor-associated inflammation. In this review, we summarize the mechanisms involved in regulating miR-15b expression in mammalian tumors and discuss the effects of miR-15b dysregulation on the hallmarks of cancer and highlight its role as a potentially valuable target for future cancer therapeutic strategies.

Combined evaluation of the expression of NUCKS and Ki-67 proteins as independent prognostic factors for patients with gastric adenocarcinoma

Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) has been recently documented in various malignancies. However, data regarding the expression and prognostic value of NUCKS in gastric adenocarcinoma are limited. Specimens from 159 gastric adenocarcinoma patients who underwent primary gastrectomy were collected. Immunohistochemical method was used to evaluate NUCKS and Ki-67 expression. The correlations between NUCKS and clinical significance were analyzed. Elevated NUCKS expression was significantly associated with TNM stage (P = 0.034), depth of invasion (P = 0.001), and expression of Ki-67 (P = 0.035). Kaplan–Meier analysis indicated that NUCKS overexpression alone (P = 0.045 for overall survival) or in combination with Ki-67 (P = 0.007 for disease-free survival, P = 0.002 for overall survival) was correlated with adverse prognosis of the patients. Multivariate analysis revealed that combined NUCKS and Ki-67 overexpression was an independent prognostic factor for both disease-free survival (hazard ratio = 1.623, P = 0.02) and overall survival (hazard ratio = 1.667, P = 0.016) in gastric adenocarcinoma patients. The combination of NUCKS and Ki-67 overexpression in gastric adenocarcinoma further distinguished a subgroup of patients with poor prognosis.

miR-15b-5p resensitizes colon cancer cells to 5-fluorouracil by promoting apoptosis via the NF-κB/XIAP axis

Drug resistance, which is closely correlated with an imbalance in apoptosis, endows colorectal cancer (CRC) with enhanced progression capacity irrespective of the treatment with therapeutics. We report that miR-15b-5p is a tumor suppressor whose level is globally decreased in CRC cells and tissues. Over-expression of miR-15b-5p not only promoted 5-fluorouracil (5-FU)-induced cellular apoptosis but also reversed the chemoresistance of 5-FU in vitro and in vivo. As a key mediator of inflammation-induced cancer, miR-15b-5p enhances these therapeutic effects are mainly attributed to targeting of the NF-κB signaling pathway through negative regulation of NF-κB1 and one of its kinase complexes IKK-α. miR-15b-5p mediates NF-ĸB regulation by targeting the anti-apoptosis protein XIAP in vitro. Together, these results establish an axis of miR-15b-mediated apoptosis regulation, which reverses chemoresistance and suppresses CRC progression. These findings suggest that miR-15b-5p may be a potential agent for CRC treatment, particularly for 5-FU-resistant CRC.