Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High-risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. The need for biomarkers that can select patients not responding well to therapy in an early phase is therefore needed. In this study, we used next generation sequencing technology to determine the expression profiles in high-risk neuroblastoma cell lines established before and after therapy. Using partial least squares-discriminant analysis (PLS-DA) with least absolute shrinkage and selection operator (LASSO) and leave-one-out cross-validation, we identified a panel of 55 messenger RNAs and 17 long non-coding RNAs (lncRNAs) which were significantly altered in the expression between cell lines isolated from primary and recurrent tumors. From a neuroblastoma patient cohort, we found 20 of the 55 protein-coding genes to be differentially expressed in patients with unfavorable compared with favorable outcome. We further found a twofold increase or decrease in hazard ratios in these genes when comparing patients with unfavorable and favorable outcome. Gene set enrichment analysis (GSEA) revealed that these genes were involved in proliferation, differentiation and regulated by Polycomb group (PcG) proteins. Of the 17 lncRNAs, 3 upregulated ( NEAT1, SH3BP5-AS1, NORAD) and 3 downregulated lncRNAs ( DUBR, MEG3, DHRS4-AS1) were also found to be differentially expressed in favorable compared with unfavorable outcome. Moreover, using expression profiles on both miRNAs and mRNAs in the same cohort of cell lines, we found 13 downregulated and 18 upregulated experimentally observed miRNA target genes targeted by miR-21, -424 and -30e, -29b, -138, -494, - 181a, -34a, -29b, respectively. The advantage of analyzing biomarkers in a clinically relevant neuroblastoma model system enables further studies on the effect of individual genes upon gene perturbation. In summary, this study identified several genes, which may aid in the prediction of response to therapy and tumor recurrence.

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Differentially Expressed Long Noncoding RNAs Involved in FUBP1 Promoting Hepatocellular Carcinoma Cells Proliferation

BioMed Research International ◽

10.1155/2021/6664519 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Xianpeng Li ◽

Huaixi Yu ◽

Feng Xu ◽

Yifeng Wu ◽

Jifang Sheng

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Noncoding Rna ◽

Target Genes ◽

Expression Profiles ◽

Differentially Expressed ◽

Cell Counting ◽

Rt Pcr ◽

Upstream Element ◽

Hcc Cells

Background. Far upstream element-binding protein 1 (FUBP1) is reported to be involved in cancer development by regulating the transcription of c-myc gene through binding to far upstream element. Highly expressed FUBP1 was negatively correlated with survival rate of patients with hepatocellular carcinoma (HCC) and could promote the proliferation of HCC cells. However, the downstream mechanism of FUBP1 has not yet been clearly explained. This study is aimed at identifying the expression profiles of long noncoding RNA (lncRNA) in HCC cells in response to FUBP1 overexpression and at investigating the possible lncRNAs that participated in cell proliferation process regulated by FUBP1. Methods. The overexpression of FUBP1 was mediated by lentiviral infection on 3 different types of HCC cell lines (MHCC97-H, MHCC97-L, and Huh-7). The expression of target genes was detected by quantitative reverse transcription-PCR (RT-PCR) and western blotting assays. Microarray and quantitative RT-PCR were applied to screen the differentially expressed lncRNAs in HCC cells after FUBP1 overexpression. The Cell Counting Kit-8 assay was used to confirm the growth vitality of HCC cells. Results. The growth vitality of HCC cells was significantly increased after lentivirus infection. A total of 12 lncRNAs had the same expression trend in the 3 HCC cell lines in response to FUBP1 overexpression, including 3 upregulated lncRNAs and 9 downregulated lncRNAs. Coexpression analysis of dysregulated lncRNAs-mRNAs network showed that lnc-LYZ-2 was the lncRNA most relevant to FUBP1. Inhibition of lnc-LYZ-2 could significantly relieve the proproliferation effect of FUBP1 on HCC cells, suggesting that lnc-LYZ-2 was partially involved in proproliferation regulation of FUBP1. Conclusions. Our results indicated that FUBP1 induced the abnormal expression of lncRNAs and the FUBP1-lncRNAs coexpression network in HCC cells, which could provide theoretical and experimental basis for FUBP1-lncRNAs network involved in HCC development.

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Bioinformatics Analysis of Circular RNA Expression Profiles in HBx-Transfected HepG2 Cells by Transcriptional Sequencing

10.21203/rs.3.rs-435183/v1 ◽

2021 ◽

Author(s):

Luzheng Liu ◽

Jiacheng Chen ◽

Liang Chen ◽

Cheng Chen ◽

Dafeng Xu ◽

...

Keyword(s):

Cell Lines ◽

Hepg2 Cells ◽

Target Genes ◽

Expression Profiles ◽

Circular Rna ◽

Differentially Expressed ◽

Circular Rnas ◽

Bioinformatics Analyses ◽

Go Analysis ◽

Micro Rnas

Abstract BACKGROUND Circular RNA (CircRNA) and HBx genes separately play essential roles in the occurrence and development of hepatitis B (HBV)-related hepatocellular carcinoma (HCC). However, whether HBx expression in HCC is co-related to differential circRNA patterns remains unknown. METHODS HCC cell lines with HBx overexpression (HepG2 H6679) and empty vector control (HepG2 H5298) were successfully constructed. The high-throughput second-generation transcriptome sequencing technology (RNA-seq) was employed to sequence the two cell lines, and the selected circRNAs were verified by qPCR (quantitative real-time PCR). The differentially expressed circRNAs were analyzed. Bioinformatics analyses, including clustering, differential expression, GO analysis, and KEGG pathway, were performed. Target Scan and Miranda software were employed to predict miRNAs corresponding with circRNAs. RESULTS We identified 1120 circRNAs upregulated and 1447 circRNAs downregulated in HepG2 cell lines with HBx overexpression compared to its control. We selected 36 circRNAs with significant differences (also consistent with log2fold change absolute value ≥ 1.0 or P ≤ 0.05) displayed by cluster analysis and then performed qPCR validation. Among them, 15 circRNAs (hsa_circ_0005603, hsa_circ_0004448, hsa_circ_0006845, hsa_circ_0064654, hsa_circ_0006460, hsa_circ_0045350, hsa_circ_0000824, hsa_circ_0005227, hsa_circ_0067991, hsa_circ_0064656, hsa_circ_0005224, circRNA11716, circRNA759, circRNA14848 and circRNA13751) are consistent with sequencing results. Hsa_circ_0005603 and hsa_circ_0006845 showed significant differences and were chosen for further study. GO analysis shows that many target genes are involved in biological processes, cellular components, and molecular functions. Nearly 193 target genes were enriched on KEGG pathways analysis. Actin cytoskeleton regulation, tight junction, and FoxO signaling pathway are among the top three pathways involved in most genes. We predicted that hsa_circ_0005603 might interact with micro-RNAs, including miR-182-5p, hsa-miR-27a-3p, hsa-miR-98-5p, and hsa-miR-198, that might thereby regulate downstream genes involved in tumor progression. Similarly, hsa_circ_0006845 was predicted to be referred to HBV-related HCC by acting as a sponge for hsa-miR-106a-3p and hsa-miR-198. Furthermore, we discovered two novel circular RNAs (circRNA11716 and circRNA13751) which might be involved in HCC occurrence. CONCLUSION In this study, we comprehensively explored the differentially expressed circRNAs in HepG2 cells with different HBx expression, and our results indicate that hsa_circ_0005603, hsa_circ_0006845, and novel circular RNAs (circRNA11716 and circRNA13751) might play an important role in HBV-related HCC, deserving further research.

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Genome-wide transcriptome profiling uncovers differential miRNAs and lncRNAs in ovaries of Hu sheep at different developmental stages

Scientific Reports ◽

10.1038/s41598-021-85245-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samina Shabbir ◽

Prerona Boruah ◽

Lingli Xie ◽

Muhammad Fakhar-e-Alam Kulyar ◽

Mohsin Nawaz ◽

...

Keyword(s):

Target Genes ◽

Expression Profiles ◽

Ovarian Follicle ◽

Follicular Development ◽

Differentially Expressed ◽

Ovary Development ◽

Estrogen Metabolism ◽

Genome Wide ◽

Micro Rnas ◽

Hu Sheep

AbstractOvary development is an important determinant of the procreative capacity of female animals. Here, we performed genome-wide sequencing of long non-coding RNAs (lncRNAs) and mRNAs on ovaries of 1, 3 and 8 months old Hu sheep to assess their expression profiles and roles in ovarian development. We identified 37,309 lncRNAs, 45,404 messenger RNAs (mRNAs) and 330 novel micro RNAs (miRNAs) from the transcriptomic analysis. Six thousand, seven hundred and sixteen (6716) mRNAs and 1972 lncRNAs were significantly and differentially expressed in ovaries of 1 month and 3 months old Hu sheep (H1 vs H3). These mRNAs and target genes of lncRNAs were primarily enriched in the TGF-β and PI3K-Akt signalling pathways which are closely associated with ovarian follicular development and steroid hormone biosynthesis regulation. We identified MSTRG.162061.1, MSTRG.222844.7, MSTRG.335777.1, MSTRG.334059.16, MSTRG.188947.6 and MSTRG.24344.3 as vital genes in ovary development by regulating CTNNB1, CCNA2, CDK2, CDC20, CDK1 and EGFR expressions. A total of 2903 mRNAs and 636 lncRNAs were differentially expressed in 3 and 8 months old ovaries of Hu sheep (H3 vs H8); and were predominantly enriched in PI3K-Akt, progesterone-mediated oocyte maturation, estrogen metabolism, ovulation from the ovarian follicle and oogenesis pathways. These lncRNAs were also found to regulate FGF7, PRLR, PTK2, AMH and INHBA expressions during follicular development. Our result indicates the identified genes participate in the development of the final stages of follicles and ovary development in Hu sheep.

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Robust Metabolic Transcriptional Components in 34,494 Patient-Derived Samples and Cell Lines

10.21203/rs.3.rs-659596/v1 ◽

2021 ◽

Author(s):

Vincent Christiaan Leeuwenburgh ◽

Carlos G. Urzúa-Traslaviña ◽

Arkajyoti Bhattacharya ◽

Marthe T.C. Walvoort ◽

Mathilde Jalving ◽

...

Keyword(s):

Cell Lines ◽

Expression Profiles ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Metabolic Processes ◽

Gene Sets ◽

Transcriptional Changes ◽

Cancer Tissues ◽

Tumor Biopsies ◽

Transcriptional Landscape

Abstract Background: Patient-derived bulk expression profiles of cancers can provide insight into transcriptional changes that underlie reprogrammed metabolism in cancer. These profiles represent the average expression pattern of all heterogeneous tumor and non-tumor cells present in biopsies of tumor lesions. Hence, subtle transcriptional footprints of metabolic processes can be concealed by other biological processes and experimental artifacts. However, consensus Independent Component Analyses (c-ICA) can capture statistically independent transcriptional footprints, of both subtle and more pronounced metabolic processes. Methods: We performed c-ICA with 34,494 bulk expression profiles of patient-derived tumor biopsies, non-cancer tissues, and cell lines. Gene set enrichment analysis with 608 gene sets that describe metabolic processes was performed to identify transcriptional components enriched for metabolic processes (mTCs). The activity of these mTCs were determined in all samples to create a metabolic transcriptional landscape. Results: A set of 555 mTCs were identified of which many were robust across different datasets, platforms, and patient-derived tissues and cell lines. We demonstrate how the metabolic transcriptional landscape defined by the activity of these mTCs in samples can be used to explore associations between the metabolic transcriptome and drug sensitivities, patient outcomes, and the composition of the immune tumor microenvironment. Conclusions: To facilitate the use of our transcriptional metabolic landscape, we have provided access to all data via a web portal ( www.themetaboliclandscapeofcancer.com ). We believe this resource will contribute to the formulation of new hypotheses on how to metabolically engage the tumor or its (immune) microenvironment.

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Identification of prognostic gene signature associated with microenvironment of lung adenocarcinoma

PeerJ ◽

10.7717/peerj.8128 ◽

2019 ◽

Vol 7 ◽

pp. e8128 ◽

Cited By ~ 12

Author(s):

Cheng Yue ◽

Hongtao Ma ◽

Yubai Zhou

Keyword(s):

High Risk ◽

Lung Adenocarcinoma ◽

Prognostic Model ◽

Cox Regression ◽

Risk Group ◽

Expression Profiles ◽

Gene Signature ◽

Low Risk ◽

Differentially Expressed ◽

Lasso Regression

Background Lung cancer has the highest morbidity and mortality worldwide, and lung adenocarcinoma (LADC) is the most common pathological subtype. Accumulating evidence suggests the tumor microenvironment (TME) is correlated with the tumor progress and the patient’s outcome. As the major components of TME, the tumor-infiltrated immune cells and stromal cells have attracted more and more attention. In this study, differentially expressed immune and stromal signature genes were used to construct a TME-related prognostic model for predicting the outcomes of LADC patients. Methods The expression profiles of LADC samples with clinical information were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) related to the TME of LADC were identified using TCGA dataset by Wilcoxon rank sum test. The prognostic effects of TME-related DEGs were analyzed using univariate Cox regression. Then, the least absolute shrinkage and selection operator (LASSO) regression was performed to reduce the overfit and the number of genes for further analysis. Next, the prognostic model was constructed by step multivariate Cox regression and risk score of each sample was calculated. Then, survival and Receiver Operating Characteristic (ROC) analyses were conducted to validate the model using TCGA and GEO datasets, respectively. The Kyoto Encyclopedia of Genes and Genomes analysis of gene signature was performed using Gene Set Enrichment Analysis (GSEA). Finally, the overall immune status, tumor purity and the expression profiles of HLA genes of high- and low-risk samples was further analyzed to reveal the potential mechanisms of prognostic effects of the model. Results A total of 93 TME-related DEGs were identified, of which 23 DEGs were up-regulated and 70 DEGs were down-regulated. The univariate cox analysis indicated that 23 DEGs has the prognostic effects, the hazard ratio ranged from 0.65 to 1.25 (p < 0.05). Then, seven genes were screened out from the 23 DEGs by LASSO regression method and were further analyzed by step multivariate Cox regression. Finally, a three-gene (ADAM12, Bruton Tyrosine Kinase (BTK), ERG) signature was constructed, and ADAM12, BTK can be used as independent prognostic factors. The three-gene signature well stratified the LADC patients in both training (TCGA) and testing (GEO) datasets as high-risk and low-risk groups, the 3-year area under curve (AUC) of ROC curves of three GEO sets were 0.718 (GSE3141), 0.646 (GSE30219) and 0.643 (GSE50081). The GSEA analysis indicated that highly expressed ADAM12, BTK, ERG mainly correlated with the activation of pathways involving in focal adhesion, immune regulation. The immune analysis indicated that the low-risk group has more immune activities and higher expression of HLA genes than that of the high-risk group. In sum, we identified and constructed a three TME-related DEGs signature, which could be used to predict the prognosis of LADC patients.

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Proteomic Study of HPV-Positive Head and Neck Cancers: Preliminary Results

BioMed Research International ◽

10.1155/2014/430906 ◽

2014 ◽

Vol 2014 ◽

pp. 1-16 ◽

Cited By ~ 3

Author(s):

Géraldine Descamps ◽

Ruddy Wattiez ◽

Sven Saussez

Keyword(s):

Head And Neck ◽

Cell Lines ◽

Expression Profiles ◽

Elongation Factor ◽

Head And Neck Carcinoma ◽

Hpv Infection ◽

Differentially Expressed ◽

Hpv Status ◽

Clinical Series ◽

Protein Expression Profiles

Human papillomavirus (HPV) was recently recognized as a new risk factor for head and neck squamous cell carcinoma. For oropharyngeal cancers, an HPV+ status is associated with better prognosis in a subgroup of nonsmokers and nondrinkers. However, HPV infection is also involved in the biology of head and neck carcinoma (HNC) in patients with a history of tobacco use and/or alcohol consumption. Thus, the involvement of HPV infection in HN carcinogenesis remains unclear, and further studies are needed to identify and analyze HPV-specific pathways that are involved in this process. Using a quantitative proteomics-based approach, we compared the protein expression profiles of two HPV+ HNC cell lines and one HPV− HNC cell line. We identified 155 proteins that are differentially expressed (P<0.01) in these three lines. Among the identified proteins, prostate stem cell antigen (PSCA) was upregulated and eukaryotic elongation factor 1 alpha (EEF1α) was downregulated in the HPV+ cell lines. Immunofluorescence and western blotting analyses confirmed these results. Moreover, PSCA and EEF1αwere differentially expressed in two clinical series of 50 HPV+ and 50 HPV− oral cavity carcinomas. Thus, our study reveals for the first time that PSCA and EEF1αare associated with the HPV-status, suggesting that these proteins could be involved in HPV-associated carcinogenesis.

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Identification of Exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under Drought Stress by High-Throughput Sequencing

Genes ◽

10.3390/genes11010030 ◽

2019 ◽

Vol 11 (1) ◽

pp. 30

Author(s):

Yaodong Zhao ◽

Wenjing Ma ◽

Xiaohong Wei ◽

Yu Long ◽

Ying Zhao ◽

...

Keyword(s):

Nitric Oxide ◽

Drought Stress ◽

Medicago Sativa ◽

High Throughput ◽

Drought Resistance ◽

High Throughput Sequencing ◽

Target Genes ◽

Expression Profiles ◽

Differentially Expressed ◽

Medicago Sativa L

Alfalfa (Medicago sativa L.) is a high quality leguminous forage. Drought stress is one of the main factors that restrict the development of the alfalfa industry. High-throughput sequencing was used to analyze the microRNA (miRNA) profiles of alfalfa plants treated with CK (normal water), PEG (polyethylene glycol-6000; drought stress), and PEG + SNP (sodium nitroprusside; nitric oxide (NO) sprayed externally under drought stress). We identified 90 known miRNAs belonging to 46 families and predicted 177 new miRNAs. Real-time quantitative fluorescent PCR (qRT-PCR) was used to validate high-throughput expression analysis data. A total of 32 (14 known miRNAs and 18 new miRNAs) and 55 (24 known miRNAs and 31 new miRNAs) differentially expressed miRNAs were identified in PEG and PEG + SNP samples. This suggested that exogenous NO can induce more new miRNAs. The differentially expressed miRNA maturation sequences in the two treatment groups were targeted by 86 and 157 potential target genes, separately. The function of target genes was annotated by gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) analysis. The expression profiles of nine selected miRNAs and their target genes verified that their expression patterns were opposite. This study has documented that analysis of miRNA under PEG and PEG + SNP conditions provides important insights into the improvement of drought resistance of alfalfa by exogenous NO at the molecular level. This has important scientific value and practical significance for the improvement of plant drought resistance by exogenous NO.

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Integrated Transcriptomic Analysis of Necrosis-related Gene in Diffuse Gliomas

Journal of Neurological Surgery Part A Central European Neurosurgery ◽

10.1055/s-0039-1683448 ◽

2019 ◽

Vol 80 (04) ◽

pp. 240-249

Author(s):

Jiajia Wang ◽

Jie Ma

Keyword(s):

Gene Expression ◽

High Risk ◽

Differentially Expressed Genes ◽

Expression Profiles ◽

Prognostic Index ◽

Gene Expression Profiles ◽

Risk Groups ◽

Low Risk ◽

Differentially Expressed ◽

Low Grade

Glioblastoma multiforme (GBM), an aggressive brain tumor, is characterized histologically by the presence of a necrotic center surrounded by so-called pseudopalisading cells. Pseudopalisading necrosis has long been used as a prognostic feature. However, the underlying molecular mechanism regulating the progression of GBMs remains unclear. We hypothesized that the gene expression profiles of individual cancers, specifically necrosis-related genes, would provide objective information that would allow for the creation of a prognostic index. Gene expression profiles of necrotic and nonnecrotic areas were obtained from the Ivy Glioblastoma Atlas Project (IVY GAP) database to explore the differentially expressed genes.A robust signature of seven genes was identified as a predictor for glioblastoma and low-grade glioma (GBM/LGG) in patients from The Cancer Genome Atlas (TCGA) cohort. This set of genes was able to stratify GBM/LGG and GBM patients into high-risk and low-risk groups in the training set as well as the validation set. The TCGA, Repository for Molecular Brain Neoplasia Data (Rembrandt), and GSE16011 databases were then used to validate the expression level of these seven genes in GBMs and LGGs. Finally, the differentially expressed genes (DEGs) in the high-risk and low-risk groups were subjected to gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathway, and gene set enrichment analyses, and they revealed that these DEGs were associated with immune and inflammatory responses. In conclusion, our study identified a novel seven-gene signature that may guide the prognostic prediction and development of therapeutic applications.

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RNA-Seq Reveals the Expression Profiles of Long Non-Coding RNAs in Lactating Mammary Gland from Two Sheep Breeds with Divergent Milk Phenotype

Animals ◽

10.3390/ani10091565 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1565

Author(s):

Zhiyun Hao ◽

Yuzhu Luo ◽

Jiqing Wang ◽

Jiang Hu ◽

Xiu Liu ◽

...

Keyword(s):

Mammary Gland ◽

Signaling Pathway ◽

Target Genes ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Wnt Signaling Pathway ◽

Mammary Epithelial ◽

Differentially Expressed ◽

Rna Seq ◽

Non Coding Rnas

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA with >200 nucleotides in length. Some lncRNAs have been proven to have clear regulatory functions in many biological processes of mammals. However, there have been no reports on the roles of lncRNAs in ovine mammary gland tissues. In the study, the expression profiles of lncRNAs were studied using RNA-Seq in mammary gland tissues from lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes with different milk yield and ingredients. A total of 1894 lncRNAs were found to be expressed. Compared with the GAM ewes, the expression levels of 31 lncRNAs were significantly up-regulated in the mammary gland tissues of STH ewes, while 37 lncRNAs were remarkably down-regulated. Gene Ontogeny (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the target genes of differentially expressed lncRNAs were enriched in the development and proliferation of mammary epithelial cells, morphogenesis of mammary gland, ErbB signaling pathway, and Wnt signaling pathway. Some miRNA sponges of differentially expressed lncRNAs, reported to be associated with lactation and mammary gland morphogenesis, were found in a lncRNA-miRNA network. This study reveals comprehensive lncRNAs expression profiles in ovine mammary gland tissues, thereby providing a further understanding of the functions of lncRNAs in the lactation and mammary gland development of sheep.

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Analysis of the miRNA transcriptome during testicular development and spermatogenesis of the Mongolian horse

Reproduction Fertility and Development ◽

10.1071/rd19133 ◽

2020 ◽

Vol 32 (6) ◽

pp. 582

Author(s):

Bei Li ◽

Xiaolong He ◽

Yiping Zhao ◽

Dongyi Bai ◽

Dandan Li ◽

...

Keyword(s):

Target Genes ◽

Expression Profiles ◽

Mammalian Species ◽

Mature Mirnas ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

P Value ◽

Testicular Development ◽

Interaction Sites ◽

Mongolian Horse

Numerous studies have shown that microRNAs (miRNAs) are essential for testicular development and spermatogenesis. In order to further characterise these physiological processes, three immature and three mature testes of the Mongolian horse were collected and six libraries were established. Using small RNA sequencing technology, 531 mature miRNAs were identified, including 46 novel miRNAs without previously ascribed functions. Among the 531 miRNAs, 421 were expressed in both immature and mature libraries, 65 miRNAs were found solely in immature testis libraries and 45 miRNAs were found solely in mature testis libraries. Furthermore, among the miRNAs that were identified in both immature and mature libraries, 107 were significantly differentially expressed (corrected P value (padj)<0.05). Among the miRNAs that were only expressed in immature testes, two miRNAs were differentially expressed, whereas among the miRNAs that were only expressed in mature testes, nine miRNAs were differentially expressed. Comprehensive analysis of miRNA and mRNA expression profiles predicted 107 miRNA–mRNA interaction sites. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted target genes suggested roles of the differentially expressed miRNAs in testicular development and spermatogenesis. These findings identify miRNAs as key factors in the development of the testes and spermatogenesis in the Mongolian horse, which may also help us to understand the mechanisms of fertility in related mammalian species.

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