Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.     

Genome-wide circular RNA (circRNA) and mRNA profiling identify a circMET-miR-410-3p regulatory motif for cell growth in colorectal cancer

Circular RNA (circRNA) is a non-coding RNA molecule that lacks polyadenylated tails and is highly stable, abundant, and conserved in human cells. CircRNAs can serve as a competing endogenous RNA (ceRNA) to sponge microRNAs (miRNA) and block their effects on target mRNA expression. CircRNAs also have possible relevance to cancer and therefore may be considered as ideal biomarkers for monitoring cancer progression. Of the about 300,000 predicted human circRNAs, only a few have validated biological functions related to cancer. To better understand the ceRNA role of circRNAs in colorectal cancer (CRC), we performed genome-wide circRNA-based RNA-sequencing (RNA-Seq) on nine CRC tumor samples and their paired histologically normal adjacent tissue samples. By profiling the mRNA expression in the same patients, we further explored the expression correlation between circRNAs and mRNAs generated from the same parental gene. Focusing on the concordant differential expression between circRNAs and mRNAs, we substantially reduced the regulatory noise. In total, we identified 694 circRNA-mRNA pairs that were consistently up or downregulated between tumor and normal tissues. These 694 circRNA-mRNA pairs are from 182 protein-coding genes associated with hormone responses and chemotaxis. Of these 182 genes, 43 are downstream targets of three highly conserved miRNAs (miR-410-3p, miR-135a, and miR-30a). Interestingly, these 43 genes are highly mutated in another cohort from eight independent CRC studies, which have significant effects on patient survival time. Focusing on miR-410-3p and its target oncogene MET, we experimentally validated the ceRNA regulatory motif of circMET. Notably, circMET is substantially upregulated in CRC cell lines and could promote cell proliferation and growth. By confirming the regulatory relationship between miR-410-3p and circMET, we propose a new mechanism for the observed sustained activation of MET in CRC. In conclusion, our work identifies a novel regulatory role of circMET and provides a potential diagnostic biomarker for CRC.     

Genome-Wide Analyses of Radioresistance-Associated miRNA Expression Profile in Nasopharyngeal Carcinoma Using Next Generation Deep Sequencing

Background

Rapidly growing evidence suggests that microRNAs (miRNAs) are involved in a wide range of cancer malignant behaviours including radioresistance. Therefore, the present study was designed to investigate miRNA expression patterns associated with radioresistance in NPC.

Methods

The differential expression profiles of miRNAs and mRNAs associated with NPC radioresistance were constructed. The predicted target mRNAs of miRNAs and their enriched signaling pathways were analyzed via biological informatical algorithms. Finally, partial miRNAs and pathways-correlated target mRNAs were validated in two NPC radioreisitant cell models.

Results

50 known and 9 novel miRNAs with significant difference were identified, and their target mRNAs were narrowed down to 53 nasopharyngeal-/NPC-specific mRNAs. Subsequent KEGG analyses demonstrated that the 53 mRNAs were enriched in 37 signaling pathways. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-324-3p, miR-93-3p and miR-4501), 3 up-regulated miRNAs (miR-371a-5p, miR-34c-5p and miR-1323) and 2 novel miRNAs. Additionally, corresponding alterations of pathways-correlated target mRNAs were observed including 5 up-regulated mRNAs (ICAM1, WNT2B, MYC, HLA-F and TGF-β1) and 3 down-regulated mRNAs (CDH1, PTENP1 and HSP90AA1).

Conclusions

Our study provides an overview of miRNA expression profile and the interactions between miRNA and their target mRNAs, which will deepen our understanding of the important roles of miRNAs in NPC radioresistance.     

mRNA and microRNA selection for breast cancer molecular subtype stratification using meta-heuristic based algorithms

Cancer subtype stratification, which may help to make a better decision in treating cancerous patients, is one of the most crucial and challenging problems in cancer studies. To this end, various computational methods such as Feature selection, which enhances the accuracy of the classification and is an NP-Hard problem, have been proposed. However, the performance of the applied methods is still low and can be increased by the state-of-the-art and efficient methods. We used 11 efficient and popular meta-heuristic algorithms including WCC, LCA, GA, PSO, ACO, ICA, LA, HTS, FOA, DSOS and CUK along with SVM classifier to stratify human breast cancer molecular subtypes using mRNA and micro-RNA expression data. The applied algorithms select 186 mRNAs and 116 miRNAs out of 9692 mRNAs and 489 miRNAs, respectively. Although some of the selected mRNAs and miRNAs are common in different algorithms results, six miRNAs including miR-190b, miR-18a, miR-301a, miR-34c-5p, miR-18b, and miR-129-5p were selected by equal or more than three different algorithms. Further, six mRNAs, including HAUS6, LAMA2, TSPAN33, PLEKHM3, GFRA3, and DCBLD2, were chosen through two different algorithms. We have reported these miRNAs and mRNAs as important diagnostic biomarkers to the stratification of breast cancer subtypes. By investigating the literature, it is also observed that most of our reported mRNAs and miRNAs have been proposed and introduced as biomarkers in cancer subtypes stratification.     

Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila

Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3’UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.     

Integrative Analyses of m6A Regulators Identify that METTL3 is Associated with HPV Status and Immunosuppressive Microenvironment in HPV-related Cancers

Although m6A modifications are associated with tumor progression, and anti-tumor immune responses, the role of m6A regulators in HPV-related carcinogenesis has not been well resolved. To provide evidence for the role of m6A regulators in HPV-related carcinogenesis and identify potential therapeutic targets for HPV-related cancers, integrative analyses of m6A regulators in 1,485 head and neck squamous cell carcinoma (HNSC) patients and 507 cervical squamous cell carcinoma (CESC) patients was performed and identified that an m6A regulator, METTL3, was highly expressed in tumors and was related to the poor prognosis in HNSC and CESC. In HPV-positive tumors, METTL3 was positively associated with tumor HPV status, such as HPV integration status, E6 and unspliced-E6 expression, and p16 expression. Further analysis demonstrated that METTL3 ^high status was negatively correlated with tumor immune cell infiltrations and facilitated the expression of immunosuppressive immune checkpoint molecules (i.e., PD-L1). Cell-derived xenograft models demonstrated that METTL3 inhibitor combined with anti-PD1 therapy promoted immunotherapy of CESC in vivo. Overall, this study identified that METTL3 ^high status, is associated with poor prognosis and HPV status, and serves as a mediator of the immunosuppressive tumor microenvironment in HPV-associated cancer, which provides a promising therapeutic target for anti-cancer immunotherapy.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

Retracted: Deep integrative analysis of microRNA-mRNA regulatory networks for biomarker and target discovery in chondrosarcoma

Chondrosarcoma (CHS) is a common malignant bone sarcoma and its occurrence increases with age. microRNAs (miRNAs) are a class of noncoding RNAs that participate in various biological processes and disease pathogenesis by targeting functional messenger RNA (mRNA). However, the modulation of miRNAs in CHS remains largely unknown. In this study, we performed integrative analysis to explore the expression profiles of miRNAs and mRNAs, together with their interaction networks in human CHS tissues and cell lines by RNA-seq (miRNA and mRNA). A total of 133 and 796 differentially expressed miRNAs and mRNAs were identified (|Fold change| ≥ 2 and P-value ≤ 0.5). miRNA-mRNA regulatory interactions between 55 miRNAs and 242 mRNAs were screened by the Pearson correlation analysis and target prediction. mRNAs in the network were enriched to 145 Gene Ontology terms and 35 Kyoto Encyclopedia of Genes and Genomes pathways. Specifically, some key regulators (hub-miRNAs) in the network (miR-622, miR-4539, miR-145, miR-25, and miR-96) were suggested to play important regulatory roles in the pathogenesis of CHS. In addition, functional experiments validated that miR-622 regulated CHS cell proliferation by targeting bone morphogenetic protein 1 (BMP1).     

microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation

Studies have indicated that Nel-like molecule-1 (NELL-1) was an osteoblast-specific cytokine and some specific microRNAs (miRNAs) could serve as competing endogenous RNA (ceRNA) to partake in osteogenic differentiation of human adipose-derived stem cells (hASCs). The aim of this study was to explore the potential functional mechanisms of recombinant human NELL-1 protein (rhNELL-1) during hASCs osteogenic differentiation. rhNELL-1 was added to osteogenic medium to activate osteogenic differentiation of hASCs. High-throughput RNA sequencing (RNA-Seq) was performed and validated by real-time quantitative polymerase chain reaction. Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed to detect the functions of differentially expressed miRNAs and genes. Coding-noncoding gene co-expression network and ceRNA networks were constructed to predict the potential regulatory role of miRNAs. A total of 1010 differentially expressed miRNAs and 1762 differentially expressed messenger RNAs (mRNAs) were detected. miRNA-370-3p, bone morphogenetic protein 2 (BMP2), and parathyroid hormone like hormone (PTHLH) were differentially expressed during NELL-1-induced osteogenesis. Bioinformatic analyses demonstrated that these differentially expressed miRNAs and mRNAs enriched in Rap1 signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, Glucagon signaling pathway, and hypoxia-inducible factor-1 signaling pathway, which were important pathways related to osteogenic differentiation. In addition, miRNA-370-3p and has-miR-485-5p were predicted to interact with circ0001543, circ0002405, and ENST00000570267 in ceRNA networks. Based on the gain or loss of functional experiments by transfection, the results showed that miR-370-3p was a key regulator in osteogenic differentiation by targeting BMP2 and disturbing the expression of PTHLH, and participated in NELL-1-stimulated osteogenesis. The present study provided the primary data and evidence for further exploration on the roles of miRNAs and ceRNAs during NELL-1-induced ossification of hASCs.     

Reconstruction and analysis of the aberrant lncRNA‐miRNA‐mRNA network based on competitive endogenous RNA in CESC

A growing body of studies has demonstrated that long non‐coding RNA (lncRNA) are regarded as the primary section of the ceRNA network. This is thought to be the case owing to its regulation of protein‐coding gene expression by functioning as miRNA sponges. However, functional roles and regulatory mechanisms of lncRNA‐mediated ceRNA in cervical squamous cell carcinoma (CESC), as well as their use for potential prediction of CESC prognosis, remains unknown. The aberrant expression profiles of mRNA, lncRNA, and miRNA of 306 cervical squamous cancer tissues and three adjacent cervical tissues were obtained from the TCGA database. A lncRNA‐mRNA‐miRNA ceRNA network in CESC was constructed. Meanwhile, Gene Ontology (GO) and KEGG pathway analysis were performed using Cytoscape plug‐in BinGo and DAVID database. We identified a total of 493 lncRNA, 70 miRNA, and 1921 mRNA as differentially expressed profiles. An aberrant lncRNA‐mRNA‐miRNA ceRNA network was constructed in CESC, it was composed of 50 DElncRNA, 18 DEmiRNA, and 81 DEmRNA. According to the overall survival analysis, 3 out of 50 lncRNA, 10 out of 81 mRNA, and 1 out of 18 miRNA functioned as prognostic biomarkers for patients with CESC (P value < 0.05). We extracted the sub‐network in the ceRNA network and found that two novel lncRNA were recognized as key genes. These included lncRNA MEG3 and lncRNA ADAMTS9‐AS2. The present study provides a new insight into a better understanding of the lncRNA‐related ceRNA network in CESC, and the novel recognized ceRNA network will help us to improve our understanding of lncRNA‐mediated ceRNA regulatory mechanisms in the pathogenesis of CESC.