Protein-coding mRNAs Research Articles

Abstract 3492: Exosome-based plasma RNA biomarkers for high-risk prostate cancer

Abstract Introduction: There is an unmet need for liquid biopsy tests to support the management of patients with intermediate and high-risk prostate cancer (PCa). In treatment naïve patients, risk stratification by standard pathology and tissue based genomic testing can be influenced by sampling errors inherent to PCa biopsy. Exosome-based liquid biopsies are emerging as a clinically effective platform for minimally invasive and highly sensitive diagnostics for diverse types of cancer. In this project we undertake the development of a plasma exosome-based liquid biopsy to stratify patients with clinically significant PCa that may circumvent the sampling challenges associated with tissue-based genomic tests. Exosomes are small double-lipid membrane vesicles that cells actively shed into various biofluids and that provide stable packages for RNA, DNA and protein molecules. Here we take advantage of a well-developed exosomal biomarker analysis platform to profile plasma exosomal RNA markers that differentiate treatment-naïve men diagnosed with NCCN low risk vs high risk PCa as the first step in the development of a plasma exosomal liquid biopsy to support the management of patients with intermediate and high-risk disease. Methods: We compared plasma exosomal RNA profiles from 11 high-risk and 9 low-risk, treatment-naïve, biopsy-confirmed patients together with 4 healthy controls. Exosomes were isolated from 1 ml plasma samples and exosomal RNA prepared using the ExosomeDx ExoLution platform. A hybrid-capture RNA Next Generation Sequencing analysis was performed to enrich for transcripts of exons, 3’ and 5’ untranslated RNA and long non-coding RNA (lncRNA) to identify differentially expressed RNAs. Results: We detected over 10,000 protein coding genes and ~400 lncRNAs in each plasma sample using exosomal RNAseq. 273 genes were differentially expressed between high-risk vs control but not in low-risk vs control. We identified four potential plasma exosomal biomarkers of high-risk PCa. These include three protein coding mRNA transcripts that showed greater than 20-fold lower expression in plasma from high-risk in comparison to low-risk patients; TCGA data show that two of these mRNA markers are also downregulated in PCa tissue in patients with worse survival. In addition, we identified one lncRNA that showed greater than 20-fold higher expression in plasma from high-risk in comparison to low-risk patients. We also detected multiple alternate androgen receptor transcripts in plasma exosomal RNA from the high-risk patients. Together, these represent early data for potentially novel liquid biopsy RNA biomarkers for high-risk PCa. Conclusions: Here we report results of a liquid biopsy biomarker discovery study to develop an exosome-based long RNA test for to support the management of patients with intermediate and high-risk PCa. Plasma exosomal RNA provides a rich reservoir of currently untapped biomarkers for high-risk PCa. Citation Format: Sandra M. Gaston, Johan K. Skog, Benjamin Spieler, Alan Dal Pra, Radka Stoyanova, Christian Fischer, Yevgenia Khodor, Grannum Sant, Seth Yu, Vasisht Tadigotla, Sudipto K. Chakrabortty, Sanoj Punnen, Alan Pollack. Exosome-based plasma RNA biomarkers for high-risk prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3492.

Impact of Diminished Expression of circRNA on Multiple Sclerosis Pathomechanisms.

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing–remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated (P < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa_circRNA_101145 and hsa_circRNA_001896. Their expression was significantly decreased during remission in RRMS (P = 0.0000332, FC = 0.385 and P = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa_circRNA_101145, P = 0.0695; hsa_circRNA_001896, P = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood–brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS.

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.

Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5' splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.

HTG transcriptome panel (HTP): An accurate and robust tool for transcriptome-wide gene expression profiling.

e15063 Background: Analysis of transcriptome-wide gene expression has the potential to provide insights into biological pathways and molecular mechanisms that regulate disease progression and treatment response. RNA-Seq is considered the gold standard for transcriptome analysis; however, it may not perform well with older, archival formalin-fixed paraffin embedded (FFPE) tissues or low-quality/low-quantity RNA. To help researchers overcome these challenges, HTG Molecular Diagnostics developed a Research Use Only, extraction-free, targeted human transcriptome panel with an automated, proprietary web-based data analysis pipeline that provides fast, accurate, and repeatable quantitative gene expression data for 19,398 protein-coding mRNA targets from lysed FFPE tissue samples or extracted RNA (eRNA). Methods: Panel precision was evaluated by Lin’s Concordance Correlation Coefficient (Lc) using FFPE tissue lysates and eRNA from FFPE and fresh frozen tissues, including FFPE blocks that were older than ten years. The accuracy of the HTP to perform differential expression analysis was assessed by comparing it to RNA-Seq using FFPE samples from different cancer indications. The agreement of the log fold changes between the two platforms was measured by Pearson Correlation Coefficient (Pc). Additionally, a cohort of 83 breast cancer FFPE samples, that had also been evaluated for estrogen (ER) and progesterone (PR) receptor expression by immunohistochemistry (IHC), were used to determine the correlation between HTP gene expression and orthogonal IHC data. Results: The precision of the panel measured by Lc, ranged from 0.90-0.94 across multiple operators, instruments, lots, and processing days, demonstrating that the HTP is a precise and robust panel. In addition, HTP is a competitive alternate to RNA-seq (estimated log-fold changes, Pc = 0.83 between the platforms). Archival samples demonstrated high repeatability (Pc &gt; 0.9) with sample pass rates of 100% for HTP, which further confirms panel’s robust performance. Furthermore, the HTP gene expression results correlated with the IHC markers for ER (Pc = 0.92) and PR (Pc = 0.88) receptor status in a cohort of 83 breast cancer samples, which highlights the potential of the panel to replace IHC staining while providing expression data on thousands of additional genes. Conclusions: The results demonstrate the HTP is a powerful tool for comprehensive gene expression analysis that addresses several key limitations of RNA-Seq. The HTP is precise, accurate, user-friendly (avoids extraction-bias, fewer steps, automated data analysis), and efficient (quicker, requires lower amounts of sample, works well with samples of low quality and archival samples). Collectively, the data demonstrate HTP has the potential to be used for biomarker discovery and the development of clinical solutions.

The LINC01929/miR-6875-5p/ADAMTS12 Axis in the ceRNA Network Regulates the Development of Advanced Bladder Cancer.

Considering its speedy development and extremely low 5-year overall survival rate worldwide, bladder cancer (BCa) is one of the most common and highly malignant tumors. Increasing evidence suggests that protein-coding mRNAs and non-coding RNAs, including long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs), play an essential role in regulating the biological processes of cancer. To investigate the molecular regulation associated with poor prognosis during advanced BCa development, we constructed a competitive endogenous RNA (ceRNA) network. Using transcriptome profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases, we performed differential expression (DE) analysis, weighted gene co-expression network analysis, functional enrichment analysis, survival analysis, prediction of miRNA targeting, and Pearson correlation analysis. Through layers of selection, 8 lncRNAs-28 mRNAs and 8 miRNAs-28 mRNAs pairs shared similar expression patterns, constituting a core ceRNA regulatory network related to the invasion, progression, and metastasis of advanced clinical stage (ACS) BCa. Subsequently, we conducted real time qPCR, western blotting, and immunohistochemistry to validate expression trend bioinformatics analysis on 3, 2, and 3 differentially expressed mRNAs, lncRNAs, and miRNAs, respectively. The most significantly differentially expressed LINC01929, miR-6875-5p and ADAMTS12 were selected for in vitro experiments to assess the functional role of the LINC01929/miR-6875-5p/ADAMTS12 axis. RNA pull-down, luciferase assays, and rescue assays were performed to examine the binding of LINC01929 and miR-6875-5p. Increasing trends in COL6A1, CDH11, ADAMTS12, LINC01705, and LINC01929 expression variation were verified as consistent with previous DE analysis results in ACS-BCa, compared with low clinical stage (LCS) BCa. Expression trends in parts of these RNAs, such as hsa-miR-6875-5p, hsa-miR-6784-5p, COL6A1, and CDH11, were measured in accordance with DE analysis in LCS-BCa, compared with normal bladder urothelium. Through experimental validation, the cancer-promoting molecule ADAMST12 was found to play a key role in the development of advanced BCa. Functionally, ADAMTS12 knockdown inhibited the progression of bladder cancer. Overexpression of LINC01929 promoted bladder cancer development, while overexpression of miR-6785-5p inhibited bladder cancer development. Mechanistically, LINC01929 acted as a sponge for miR-6785-5p and partially reversed the role of miR-6785-5p. Our findings provide an elucidation of the molecular mechanism by which advanced bladder cancer highly expressed LINC01929 upregulates ADAMTS12 expression through competitive adsorption of miR-6875-5p. It provides a new target for the prognosis and diagnosis of advanced bladder cancer.

MP27-12 DEVELOPMENT OF EXOSOME-BASED PLASMA RNA BIOMARKERS FOR HIGH-RISK PROSTATE CANCER

You have accessJournal of UrologyCME1 May 2022MP27-12 DEVELOPMENT OF EXOSOME-BASED PLASMA RNA BIOMARKERS FOR HIGH-RISK PROSTATE CANCER Sandra Gaston, Benjamin Spieler, Alan Dal Pra, Radka Stoyanova, Yevgenia Khodor, Sudipto Chakrabortty, Vasisht Tadigotla, Seth Yu, Grannum Sant, Christian Fischer, Sanoj Punnen, Johan Skog, and Alan Pollack Sandra GastonSandra Gaston More articles by this author , Benjamin SpielerBenjamin Spieler More articles by this author , Alan Dal PraAlan Dal Pra More articles by this author , Radka StoyanovaRadka Stoyanova More articles by this author , Yevgenia KhodorYevgenia Khodor More articles by this author , Sudipto ChakraborttySudipto Chakrabortty More articles by this author , Vasisht TadigotlaVasisht Tadigotla More articles by this author , Seth YuSeth Yu More articles by this author , Grannum SantGrannum Sant More articles by this author , Christian FischerChristian Fischer More articles by this author , Sanoj PunnenSanoj Punnen More articles by this author , Johan SkogJohan Skog More articles by this author , and Alan PollackAlan Pollack More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002570.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: There is an unmet need for liquid biopsy tests to support the management of patients with intermediate and high-risk prostate cancer (PCa). In treatment naïve patients, risk stratification by standard pathology and tissue based genomic testing can be influenced by sampling errors inherent to PCa biopsy. Exosome-based liquid biopsies are emerging as a clinically effective platform for minimally invasive and highly sensitive diagnostics for diverse types of cancer. Exosomes are small double-lipid membrane vesicles that cells shed into various biofluids and that provide packages for RNA, DNA and protein molecules. In this project we undertake the development of a plasma exosome-based liquid biopsy to stratify patients with clinically significant PCa that may circumvent the sampling challenges associated with tissue-based genomic tests. METHODS: Our pilot study included plasma samples from 11 NCCN high-risk and 9 NCCN low-risk, treatment-naïve, biopsy-confirmed PCa patients together with 4 healthy controls. Exosomes were isolated from 1 ml plasma using the ExosomeDx ExoLution platform. A hybrid-capture RNA Next Generation Sequencing analysis was performed to enrich for transcripts of exons, 3’ and 5’ untranslated RNA and long non-coding RNA (lncRNA). Data analysis included identification of differentially expressed RNAs in high-risk vs low-risk vs control samples. RESULTS: In each plasma exosome sample we detected transcripts of over 10,000 protein coding genes and ∼400 lncRNAs using exosomal RNAseq. 273 genes were differentially expressed between high-risk vs control but not between low-risk vs control. We identified four potential plasma exosomal biomarkers of high-risk PCa. These include three protein coding mRNA transcripts that showed > 20-fold lower expression in plasma from high-risk compared to low-risk patients; TCGA data show that two of these transcripts are also downregulated in PCa tissue in patients with worse survival. In addition, we identified one lncRNA that showed > 20-fold higher expression in plasma from high-risk compared to low-risk patients. We also detected multiple alternate androgen receptor transcripts in plasma exosomal RNA from high-risk patients. Together, these represent early data for potentially novel liquid biopsy RNA biomarkers for high-risk PCa. CONCLUSIONS: Here we report results of a liquid biopsy biomarker discovery study to develop an exosome-based RNA test to support the management of patients with intermediate and high-risk PCa. Plasma exosomal RNA provides a rich reservoir of currently untapped biomarkers for high-risk PCa. Source of Funding: NIH Grants R01CA189295, U01CA239141 and P30CA240139 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e454 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sandra Gaston More articles by this author Benjamin Spieler More articles by this author Alan Dal Pra More articles by this author Radka Stoyanova More articles by this author Yevgenia Khodor More articles by this author Sudipto Chakrabortty More articles by this author Vasisht Tadigotla More articles by this author Seth Yu More articles by this author Grannum Sant More articles by this author Christian Fischer More articles by this author Sanoj Punnen More articles by this author Johan Skog More articles by this author Alan Pollack More articles by this author Expand All Advertisement PDF DownloadLoading ...

Inhibition of Nonsense-mediated Decay Causes Hyperalgesic Priming in Mice

RNA stability permeates neurobiology. Yet, the contribution of a dominant pathway of RNA metabolism termed nonsense-mediated decay (NMD) to pain is unclear. NMD safeguards against translation of mRNAs that harbor premature termination codons. It also controls the stability of roughly 10% of typical protein-coding mRNAs. NMD hinges on the activity of a conserved kinase designated SMG-1. We sought to determine the role of SMG-1 and NMD in pain. We found that SMG-1 is expressed in sensory neurons throughout the DRG. We identified SMG-1 targets in DRG neurons using high-throughput sequencing. We analyzed multiple features present in mRNA to establish the characteristics of NMD substrates in DRG neurons. The most dominant feature was structural content in the 3’UTR. This implies that factors that bind to structured motifs likely recruit NMD machinery to specific mRNAs. We identify numerous NMD targets linked to pain including components of the integrated stress response such as ATF-4. We found that inhibition of SMG-1 with a small molecule results in hyperalgesic priming in mice. Given that ATF-4 is targeted by NMD, we next asked if priming was due to activation of the integrated stress response. Indeed, a small molecule inhibitor of the integrated stress response called ISRIB prevented priming triggered by inhibition of NMD. Collectively, our results indicate that (i) inhibition of NMD is pro-nociceptive, (ii) priming induced by NMD blockade requires the integrated stress response, (iii) and structure is a defining feature of NMD targets. This work establishes a clear link between NMD and pain-associated behaviors in mice. RNA stability permeates neurobiology. Yet, the contribution of a dominant pathway of RNA metabolism termed nonsense-mediated decay (NMD) to pain is unclear. NMD safeguards against translation of mRNAs that harbor premature termination codons. It also controls the stability of roughly 10% of typical protein-coding mRNAs. NMD hinges on the activity of a conserved kinase designated SMG-1. We sought to determine the role of SMG-1 and NMD in pain. We found that SMG-1 is expressed in sensory neurons throughout the DRG. We identified SMG-1 targets in DRG neurons using high-throughput sequencing. We analyzed multiple features present in mRNA to establish the characteristics of NMD substrates in DRG neurons. The most dominant feature was structural content in the 3’UTR. This implies that factors that bind to structured motifs likely recruit NMD machinery to specific mRNAs. We identify numerous NMD targets linked to pain including components of the integrated stress response such as ATF-4. We found that inhibition of SMG-1 with a small molecule results in hyperalgesic priming in mice. Given that ATF-4 is targeted by NMD, we next asked if priming was due to activation of the integrated stress response. Indeed, a small molecule inhibitor of the integrated stress response called ISRIB prevented priming triggered by inhibition of NMD. Collectively, our results indicate that (i) inhibition of NMD is pro-nociceptive, (ii) priming induced by NMD blockade requires the integrated stress response, (iii) and structure is a defining feature of NMD targets. This work establishes a clear link between NMD and pain-associated behaviors in mice.

Circular RNA Signatures of Human Healing and Nonhealing Wounds

Venous ulcers (VUs) have complex and obscure pathogenicity, and effective VU therapies are still lacking. Circular RNAs (circRNAs) have emerged as powerful gene regulators with important roles in health and disease. In this study, we used paired total RNA and small RNA sequencing to profile circRNAs, protein-coding mRNAs, and microRNAs expression in a unique collection of clinical samples: healthy skin and acute wounds at inflammatory and proliferative phases and wound-edge VU biopsies. We unravel a dynamically changed expression pattern of circRNAs during human skin repair and their abnormal expression signature in VU, which are presented as a searchable web resource (www.xulandenlab.com/humanwounds-circrna). We analyzed the coexpression relationship between the circRNAs and mRNAs with weighted correlation network analysis and constructed circRNA‒mRNA‒microRNA networks. This allowed us to expose the regulatory networks specific to the inflammatory and proliferative phases of wound repair and VU, the biological processes the circRNAs may regulate, and the circRNAs that could sponge microRNAs in human wounds. Importantly, we found that hsa-CHST15_0003 and hsa-TNFRSF21_0001, two circRNAs upregulated in VU, hampered epidermal keratinocyte migration while promoting proliferation by modulating gene networks underpinning these cellular processes. This study paves the way to decipher the functional significance of circRNAs in tissue repair.

Identification of a Prognostic Signature Composed of GPI, IL22RA1, CCT6A and SPOCK1 for Lung Adenocarcinoma Based on Bioinformatic Analysis of lncRNA-Mediated ceRNA Network and Sample Validation.

Lung adenocarcinoma (LUAD) is one of the most common malignant tumors with high morbidity and mortality in China and worldwide. Long non-coding RNAs (lncRNAs) as the competing endogenous RNA (ceRNA) play an essential role in the occurrence and development of LUAD. However, identifying lncRNA-related biomarkers to improve the accuracy of LUAD prognosis remains to be determined. This study downloaded RNA sequence data from The Cancer Genome Atlas (TCGA) database and identified the differential RNAs by bioinformatics. A total of 214 lncRNA, 198 miRNA and 2989 mRNA were differentially identified between LUAD and adjacent nontumor samples. According to the ceRNA hypothesis, we constructed a lncRNA-miRNA-mRNA network including 95 protein-coding mRNAs, 7 lncRNAs and 15 miRNAs, and found 24 node genes in this network were significantly associated with the overall survival of LUAD patients. Subsequently, through LASSO regression and multivariate Cox regression analyses, a four-gene prognostic signature composed of GPI, IL22RA1, CCT6A and SPOCK1 was developed based on the node genes of the lncRNA-mediated ceRNA network, demonstrating high performance in predicting the survival and chemotherapeutic responses of low- and high-risk LUAD patients. Finally, independent prognostic factors were further analyzed and combined into a well-executed nomogram that showed strong potential for clinical applications. In summary, the data from the current study suggested that the four-gene signature obtained from analysis of lncRNA-mediated ceRNA could serve as a reliable biomarker for LUAD prognosis and evaluation of chemotherapeutic response.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B

IntroductionHemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. Patient and methodsThe patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. ResultsA 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. ConclusionA novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

The intricate balance between microRNA-induced mRNA decay and translational repression.

Post-transcriptional regulation of messenger RNAs (mRNAs) (i.e., mechanisms that control translation, stability and localization) is a critical focal point in spatiotemporal regulation of gene expression in response to changes in environmental conditions. The human genome encodes ~ 2000 microRNAs (miRNAs), each of which could control the expression of hundreds of protein-coding mRNAs by inducing translational repression and/or promoting mRNA decay. While mRNA degradation is a terminal event, translational repression is reversible and can be employed for rapid response to internal or external cues. Recent years have seen significant progress in our understanding of how miRNAs induce degradation or translational repression of the target mRNAs. Here, we review the recent findings that illustrate the cellular machinery that contributes to miRNA-induced silencing, with a focus on the factors that could influence translational repression vs. decay.

Comparative transcriptome analysis identifies important maternal molecules and associated biological pathways for pig and human mature oocytes.

Recent researches reveal that during oocyte maturation, species-specific molecular profile exists and has important functional roles. However, molecular differences between pig (a larger animal model for human reproduction) and human mature oocytes remain unknown. Here, by comparative transcriptome analyses of single-cell RNA-seq data, we aimed to identify the common and unique maternal factors and associated biological processes between in vivo and in vitro matured pig oocytes, and between in vitro matured human and pig oocytes. Annotated protein-coding mRNAs were identified in pig in vivo (11,147) and in vitro (11,997), and human in vitro (14,491) MII oocytes, respectively. For in vivo and in vitro derived pig MII oocytes, 10,551 annotated maternal mRNAs were common, mainly enriched in signalling pathways such as cell cycle, oocyte meiosis, microtubule cytoskeleton, MAPK, RNA processing/binding. Besides, in vivo (596) and in vitro (1446) pig MII-specific mRNAs and their involved signalling pathways (in vivo: Bmp, calcium-mediated signalling, PI3K-Akt; in vitro: growth factor activity, JAK-STAT, cytokine-cytokine receptor interaction, calcium signalling pathway) were also found. As for in vitro derived human and pig MII oocytes, 10,285 annotated mRNAs were common, enriched in a variety of signalling pathways (cell cycle, oocyte meiosis, microtubule, AMPK, RNA splicing, protein serine/threonine kinase activity, etc). In vitro MII-specific mRNAs were found for humans (4206) and pigs (1712), which were also enriched in species-specific signalling pathways (humans: golgi-related terms, transcription repressor and hormone activity; pigs: ATP biosynthetic process, G protein-coupled peptide receptor activity, animoacyl-tRNA biosynthesis), respectively. These findings improve our understanding on oocyte maturation, and also the limitations of pig model for human oocyte maturation and fertilization.

Roles of LINC01473 and CD74 in osteoblasts in multiple myeloma bone disease

The suppression of osteoblast (OB) activity is partially responsible for multiple myeloma (MM) bone disease. Long non-coding RNAs (lncRNAs) play a vital role in bone formation and resorption. However, their...

Insights from Dysregulated mRNA Expression Profile of β-Cells in Response to Proinflammatory Cytokines.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that is characterized by autoimmunity and its mediated β-cell damage. Chronic exposure of β-cells to proinflammatory cytokines is known to regulate the expression of many genes, subsequently resulting in the impairment of some signaling pathways involved with insulin production and secretion and/or β-cell apoptosis. In our study, RNA sequencing technology was applied to identify differentially expressed mRNAs in MIN6 cells treated with a mix of cytokines, including IL-1β, TNF-α, and IFN-γ. The results showed 809 upregulated and 946 downregulated protein-coding mRNAs in MIN6 cells upon the stimulation of cytokines. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses were performed to predict the functions of dysregulated genes. The networks of circRNA-mRNA were constructed between differentially mRNAs and dysregulated expressed circRNAs in our previous study. In addition, we selected 8 dysregulated mRNAs for further validation by quantitative real-time PCR. The RNA sequencing data showed 809 upregulated and 946 downregulated protein-coding mRNAs. GO analysis showed that the top 10 significant “biological processes,” “cellular components,” and “molecular functions” for upregulated mRNAs include “immune system process,” “inflammatory response,” and “innate immune response” and the top 10 for downregulated mRNAs include “cell cycle,” “mitotic cytokinesis,” and “cytoplasm.” KEGG analysis showed that these differentially expressed genes were involved with “antigen processing and presentation,” “TNF signaling pathway” and “type 1 diabetes,” “cell cycle,” “necroptosis,” and “Rap1 signaling pathway.” We also constructed the networks of differentially expressed circRNAs and mRNAs. We observed that upregulated circRNA 006029 and downregulated circRNA 000286 and 017277 were associated with the vast majority of selected dysregulated mRNAs, while circRNA 013053 was only related to the protein-coding gene, Slc7a2. To the summary, these data indicated that differentially expressed mRNAs may play key or partial roles in cytokine-mediated β-cell dysfunction and gave us the hint that circRNAs might regulate mRNAs, thereby contributing to the development of T1DM. The current study provided a systematic perspective on the potential functions and possible regulatory mechanisms of mRNAs in proinflammatory cytokine-induced β-cell destruction.

Research advances on the role of competing endogenous RNAs in wound healing

Wound healing, as one of the important public health issues, has been a worldwide problem. Due to the unique biological wound environment, wound healing is a very complex process with current treatments requiring long cycles, being poorly effective, and bringing high economic burden to patients. An increasing number of studies have shown that non-coding RNAs (ncRNAs) play important roles in wound healing process. The competing endogenous RNAs (ceRNAs) hypothesis in recent years is a new proposal on the inter-regulation of RNAs, which suggests a "mode of communication" between different RNAs. ceRNA regulatory network (ceRNET) combines the functions of protein-coding mRNA with ncRNA (e.g., microRNA, long non-coding RNA, pseudogenes, and circular RNA). Recent studies have shown that ceRNAs play important roles in wound healing, which may provide new effective therapeutic targets for wound healing. This paper starting with ceRNET systematically reviewed the research progress on the effects of various ceRNAs in wound healing and the future research challenges, with the aim to deeply explore the molecular mechanisms and clinical significance of ceRNAs in the process of wound healing.

Identification of Differentially Expressed and Prognostic lncRNAs for the Construction of ceRNA Networks in Lung Adenocarcinoma.

Background Long noncoding RNAs (lncRNAs) could function as competitive endogenous RNAs (ceRNAs) to competitively adsorb microRNAs (miRNAs), thereby regulating the expression of their target protein-coding mRNAs. In this study, we aim to identify more effective diagnostic and prognostic markers for lung adenocarcinoma (LUAD). Methods We obtained differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) for LUAD by using The Cancer Genomes Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) was performed to unveil core gene modules associated with LUAD. The Cox proportional hazards model was performed to determine the prognostic significance of DElncRNAs. The diagnostic and prognostic significance of DElncRNAs was further verified based on the receiver operating characteristic curve (ROC). Cytoscape was used to construct the ceRNA networks comprising the lncRNAs-miRNAs-mRNAs axis based on the correlation obtained from the miRcode, miRDB, and TargetScan. Results Compared with normal lung tissues, 2355 DElncRNAs, 820 DEmiRNAs, and 17289 DEmRNAs were identified in LUAD tissues. We generated 8 WGCNA core modules in the lncRNAs coexpression network, 5 modules in the miRNAs, and 12 modules in the mRNAs coexpression network, respectively. One lncRNA module (blue) consisting of 441 lncRNAs, two miRNA modules (blue and turquoise) containing 563 miRNAs, and one mRNA module (turquoise), which consisted of 15162 mRNAs, were mostly significantly related to LUAD status. Furthermore, 67 DEmRNAs were found to be tumor-associated as well as the target genes of the DElncRNAs-DEmiRNAs axis. Survival analyses showed that 6 lncRNAs (LINC01447, WWC2-AS2, OGFRP1, LINC00942, LINC01168, and AC005863.1) were significantly correlated with the prognosis of LUAD patients. Ultimately, the potential ceRNA networks including 6 DElncRNAs, 4 DEmiRNAs, and 22 DEmRNAs were constructed. Conclusion Our study indicated that 6 DElncRNAs had the possibilities as diagnostic and prognostic biomarkers for LUAD. The lncRNA-mediated ceRNA networks might provide novel insights into the molecular mechanisms of LUAD progression.

Generation of ENSEMBL-based proteogenomics databases boosts the identification of non-canonical peptides.

SummaryWe have implemented the pypgatk package and the pgdb workflow to create proteogenomics databases based on ENSEMBL resources. The tools allow the generation of protein sequences from novel protein-coding transcripts by performing a three-frame translation of pseudogenes, lncRNAs and other non-canonical transcripts, such as those produced by alternative splicing events. It also includes exonic out-of-frame translation from otherwise canonical protein-coding mRNAs. Moreover, the tool enables the generation of variant protein sequences from multiple sources of genomic variants including COSMIC, cBioportal, gnomAD and mutations detected from sequencing of patient samples. pypgatk and pgdb provide multiple functionalities for database handling including optimized target/decoy generation by the algorithm DecoyPyrat. Finally, we have reanalyzed six public datasets in PRIDE by generating cell-type specific databases for 65 cell lines using the pypgatk and pgdb workflow, revealing a wealth of non-canonical or cryptic peptides amounting to >5% of the total number of peptides identified.Availability and implementationThe software is freely available. pypgatk: https://github.com/bigbio/py-pgatk/ and pgdb: https://nf-co.re/pgdb.Supplementary information Supplementary data are available at Bioinformatics online.

The Aflibercept-Induced MicroRNA Profile in the Vitreous of Proliferative Diabetic Retinopathy Patients Detected by Next-Generation Sequencing.

Purpose: Vascular endothelial growth factor-A (VEGF-A) is an important pathogenic factor in proliferative diabetic retinopathy (PDR), and aflibercept (Eylea) is one of the widely used anti-VEGF agents. This study investigated the microRNA (miRNA) profiles in the vitreous of 5 idiopathic macular hole patients (non-diabetic controls), 5 untreated PDR patients (no-treatment group), and 5 PDR patients treated with intravitreal aflibercept injection (treatment group). Methods: Next-generation sequencing was performed to determine the miRNA profiles. Deregulated miRNAs were validated with quantitative real-time PCR (qRT-PCR) in another cohort. The mRNA profile data (GSE160310) of PDR patients were retrieved from the Gene Expression Omnibus (GEO) database. The function of differentially expressed miRNAs and mRNAs was annotated by bioinformatic analysis and literature study. Results: Twenty-nine miRNAs were significantly dysregulated in the three groups, of which 19,984 target mRNAs were predicted. Hsa-miR-3184-3p, hsa-miR-24-3p, and hsa-miR-197-3p were validated to be remarkably upregulated in no-treatment group versus controls, and significantly downregulated in treatment group versus no-treatment group. In the GSE160310 profile, 204 deregulated protein-coding mRNAs were identified, and finally 179 overlapped mRNAs between the 19,984 target mRNAs and 204 deregulated mRNAs were included for further analysis. Function analysis provided several roles of aflibercept-induced miRNAs, promoting the alternation of drug sensitivity or resistance-related mRNAs, and regulating critical mRNAs involved in angiogenesis and retinal fibrosis. Conclusion: Hsa-miR-3184-3p, hsa-miR-24-3p, and hsa-miR-197-3p were highly expressed in PDR patients, and intravitreal aflibercept injection could reverse this alteration. Intravitreal aflibercept injection may involve in regulating cell sensitivity or resistance to drug, angiogenesis, and retinal fibrosis.

RNA G-quadruplex structures control ribosomal protein production

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.

MiR-146a modulates TLR1/2 and 4 induced inflammation and links it with proliferation and lipid production via the indirect regulation of GNG7 in human SZ95 sebocytes

Activation of Toll-like receptors (TLR) 1/2 and 4 are central in inducing inflammation in sebocytes by regulating the expression of protein coding mRNAs, however the microRNA (miRNA) profile in response to TLR activation and thus the possible role of miRNAs in modulating sebocyte functions has not been elucidated. In this work we identified miR-146a to have the highest induction in the TLR1/2 and 4 activated SZ95 sebocytes and found that its increased levels led to the down-regulation of IL-8 secretion, decreased the chemoattractant potential and stimulated the proliferation of sebocytes. Assessing the gene expression profile of SZ95 sebocytes treated with a miR-146a inhibitor, the induction of GNG7 was one of the highest, while when cells were treated with a miR-146a mimic, the expression of GNG7 was down-regulated. These findings correlated with our in situ hybridization results, that compared with control, miR-146a showed an increased, while GNG7 a decreased expression in sebaceous glands of acne samples. Further studies revealed, that when inhibiting the levels of GNG7 in SZ95 sebocytes, cells increased their lipid content and decreased their proliferation. Our findings suggest, that miR-146a could be a potential player in acne pathogenesis by regulating inflammation, inducing proliferation and, through the indirect down-regulation of GNG7, promoting the lipid production of sebocytes.