ceRNA Regulatory Network Research Articles

Dysfunction of the ST7-AS1/miR-301b-3p/BTG1 ceRNA network promotes immune escape of triple-negative breast cancer

Functional interactions in ceRNA regulatory networks coordinate a wide array of biologic processes and, contribute to cancer pathogenesis when perturbed. The current study was performed to explore the role of a newly constructed lncRNA-miRNA-mRNA ceRNA network in immune escape in triple-negative breast cancer (TNBC). We constructed an orthotopic tumor model of TNBC in nude mice, where tumor tissue was collected for whole transcriptome sequencing. The ceRNA regulatory network was constructed according to the data from TNBC-related whole transcriptome sequencing and differential analysis of TCGA database. Accordingly, a ceRNA regulatory network ST7-AS1/miR-301b-3p/BTG1 related to the prognosis of TNBC patients was identified. The in vivo experiments further confirmed that the expression of ST7-AS1 and BTG1 was down-regulated, but miR-301b-3p expression was up-regulated in orthotopic transplanted tumor tissues of mice. Furthermore, ST7-AS1 might upregulate BTG1 expression by sequestering miR-301b-3p, which promoted the activity of CD8 + T cells, thus inhibiting the development of TNBC. Taken together, our study emphasized that ST7-AS1 might promote BTG1 expression by competitively binding to miR-301b-3p, thereby restricting immune escape in TNBC.

Identification of potential immune-related ceRNA Regulatory Network in UVB-irradiated human skin

ABSTRACT For a better understanding the molecular biomarkers in UVB-induced skin damage, and its potential mechanism, we downloaded two microarray data sets on skin UVB damage from the Gene Expression Omnibus (GEO): GSE21429, GSE56754. By using the Limma package to analyze differential gene expression and co-expression network analysis to screen module genes, 16 common genes were identified (16 up-regulated). Gene Ontology analysis to explore the functional roles of these genes indicated that the common genes were associated mainly with melanin biosynthetic process and metabolic process. Gene Set Enrichment Analysis provided evidence that the most gene sets enriched in immune and inflammation-related signaling pathways in the UVB-treated subjects, as compared with the untreated subjects. The PPI network genes were ranked according to the degree of connectivity, the top three ranked genes: ”MLANA”, ”GPR143” and ”SFTPC” were identified as potential biomarkers using the area under the receiver operating characteristic curve. The relative proportion of 22 immune cell types was then calculated by using the CIBERSORT algorithm. A higher follicular helper T cell ratio in UVB-treated samples compared to untreated samples was observed. Moreover, three hub genes have also been shown to be associated with immune cells. Finally, through multiple online miRNA databases, we propose MLANA-miR-573-MALAT1/NEAT1, GPR143-miR-138-5p-MALAT1/ KCNQ1OT1 might be potential RNA regulatory pathways that control disease progression in UVB-induced skin damage. In summary, the present results provide novel insights into the UVB-radiation related biological process changes, and further offer a new clinical application for prognosis and diagnostic prediction of UVB radiation-mediated skin damage.

Identification and validation of hub genes and potential drugs involved in osteoarthritis through bioinformatics analysis.

Purpose: Osteoarthritis (OA) is a common degenerative disease, which still lacks specific therapeutic drugs. Synovitis is one of the most important pathological process in OA. Therefore, we aim to identify and analyze the hub genes and their related networks of OA synovium with bioinformatics tools to provide theoretical basis for potential drugs. Materials and methods: Two datasets were obtained from GEO. DEGs and hub genes of OA synovial tissue were screened through Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment as well as protein-protein interaction (PPI) network analysis. Subsequently, the correlation between expression of hub genes and ferroptosis or pyroptosis was analyzed. CeRNA regulatory network was constructed after predicting the upstream miRNAs and lncRNAs. The validation of hub genes was undertook through RT-qPCR and ELISA. Finally, potential drugs targeting pathways and hub genes were identified, followed by the validation of the effect of two potential drugs on OA. Results: A total of 161 commom DEGs were obtained, of which 8 genes were finally identified as hub genes through GO and KEGG enrichment analysis as well as PPI network analysis. Eight genes related to ferroptosis and pyroptosis respectively were significantly correlated to the expression of hub genes. 24 miRNAs and 69 lncRNAs were identified to construct the ceRNA regulatory network. The validation of EGR1, JUN, MYC, FOSL1, and FOSL2 met the trend of bioinformatics analysis. Etanercept and Iguratimod reduced the secretion of MMP-13 and ADAMTS5 of fibroblast-like synoviocyte. Conclusion: EGR1, JUN, MYC, FOSL1, and FOSL2 were identified as hub genes in the development of OA after series of bioinformatics analysis and validation. Etanercept and Iguratimod seemed to have opportunities to be novel drugs for OA.

FZD1/KLF10-hsa-miR-4762-5p/miR-224-3p-circular RNAs axis as prognostic biomarkers and therapeutic targets for glioblastoma: a comprehensive report

BackgroundThe circular RNA (circRNA) plays a vital role in the pathogenesis of tumors as a competitive endogenous RNA (ceRNA). Given the high aggressiveness and fatality rate of glioblastoma (GBM) as well as poor prognosis, it is necessary to construct a circRNA-related ceRNA network for further studies on the mechanism of GBM and identify possible biomarkers as well as therapeutic drugs.MethodsThree datasets from the gene expression omnibus (GEO) database were downloaded to distinguish differential circRNAs, microRNAs, and messenger RNAs respectively in GBM. With the help of GEPIA2, circBank, CSCD, TargetScan, miRDB, and miRTarBase databases, we established a circRNAs-related ceRNA network in GBM. Functional enrichments were employed to profile the most relevant mRNAs to indirectly clarify the mechanisms of the ceRNA network. Based on the expression profile data and survival information of GBM patients from the GEO and the cancer genome atlas (TCGA) databases, we performed survival analysis to select prognostic mRNAs and constructed a novel circRNA-miRNA-mRNA central regulatory subnetwork. The DGIdb database was used to find potential drug–gene interactions.ResultsThe datasets obtained from the GEO and TCGA databases were analyzed, and 504 differentially expressed mRNAs (DEmRNAs), 71 differentially expressed microRNAs (DEmiRNAs), and 270 differentially expressed circRNAs (DEcircRNAs) were screened out. The novel ceRNA regulatory network included 22 circRNAs, 11 miRNAs, and 15 mRNAs. FZD1 and KLF10 were significantly correlated with the overall survival rate of patients with GBM (P < 0.05). The final survival subnetwork contained six circRNAs, two miRNAs, and two mRNAs. Two small-molecule compounds and one antibody could be used as therapeutic drugs for GBM. Interestingly, the Wnt signaling pathway appeared in both KEGG and GO functional terms.ConclusionsResults of this study demonstrate that FZD1 and KLF10 may exert regulatory functions in GBM, and the ceRNA-mediated network could be a therapeutic strategy for GBM.

HELLPAR/RRM2 axis related to HMMR as novel prognostic biomarker in gliomas

BackgroundGliomas are the most frequent type of central nervous system tumor, accounting for more than 70% of all malignant CNS tumors. Recent research suggests that the hyaluronan-mediated motility receptor (HMMR) could be a novel potential tumor prognostic marker. Furthermore, mounting data has highlighted the important role of ceRNA regulatory networks in a variety of human malignancies. The complexity and behavioural characteristics of HMMR and the ceRNA network in gliomas, on the other hand, remained unknown.MethodsTranscriptomic expression data were collected from TCGA, GTEx, GEO, and CGGA database.The relationship between clinical variables and HMMR was analyzed with the univariate and multivariate Cox regression. Kaplan–Meier method was used to assess OS. TCGA data are analyzed and processed, and the correlation results obtained were used to perform GO, GSEA, and ssGSEA. Potentially interacting miRNAs and lncRNAs were predicted by miRWalk and StarBase.ResultsHMMR was substantially expressed in gliomas tissues compared to normal tissues. Multivariate analysis revealed that high HMMR expression was an independent predictive predictor of OS in TCGA and CGGA. Functional enrichment analysis found that HMMR expression was associated with nuclear division and cell cycle. Base on ssGSEA analysis, The levels of HMMR expression in various types of immune cells differed significantly. Bioinformatics investigation revealed the HEELPAR-hsa-let-7i-5p-RRM2 ceRNA network, which was linked to gliomas prognosis. And through multiple analysis, the good predictive performance of HELLPAR/RRM2 axis for gliomas patients was confirmed.ConclusionThis study provides multi-layered and multifaceted evidence for the importance of HMMR and establishes a HMMR-related ceRNA (HEELPAR-hsa-let-7i-5p-RRM2) overexpressed network related to the prognosis of gliomas.

LncRNA CASC19 Enhances the Radioresistance of Nasopharyngeal Carcinoma by Regulating the miR-340-3p/FKBP5 Axis.

Radioresistance remains a serious obstacle encountered in the radiotherapy of nasopharyngeal carcinoma (NPC). Both mRNAs and non-coding RNAs (ncRNAs), including long ncRNA (lncRNA) and microRNA (miRNA), play essential roles in radiosensitivity. However, the comprehensive expression profiles and competing endogenous RNA (ceRNA) regulatory networks among lncRNAs, miRNAs, and mRNAs in NPC radioresistance are still bewildering. In this study, we performed an RNA-sequencing (RNA-seq) assay in the radioresistant NPC cells CNE2R and its parental cells CNE2 to identify the differentially expressed lncRNAs, miRNAs, and mRNAs. The ceRNA networks containing lncRNAs, miRNAs, and mRNAs were predicted on the basis of the Pearson correlation coefficients and authoritative miRanda databases. In accordance with bioinformatic analysis of the data of the tandem mass tag (TMT) assay of CNE2R and CNE2 cells and the gene chip assay of radioresistant NPC samples in pre- and post-radiotherapy, the radioresistance-related signaling network of lncRNA CASC19, miR-340-3p, and FKBP5 was screened and further verified using an RT-qPCR assay. CASC19 was positively associated with FKBP5 expression while negatively correlated with miR-340-3p, and the target binding sites of CASC19/miR-340-3p and miR-340-3p/FKBP5 were confirmed using a dual-luciferase reporter assay. Moreover, using an mRFP-GFP-LC3 maker, it was found that autophagy contributed to the radioresistance of NPC. MiR-340-3p inhibition or FKBP5 overexpression could rescue the suppression of autophagy and radioresistance induced by CASC19 knockdown in CNE2R cells. In conclusion, the CASC19/miR-340-3p/FKBP5 network may be instrumental in regulating NPC radioresistance by enhancing autophagy, which provides potential new therapeutic targets for NPC.

Identification and verification of m7G-Related genes as biomarkers for prognosis of sarcoma.

Background: Increasing evidence indicates a crucial role for N7-methylguanosine (m7G) methylation modification in human disease development, particularly cancer, and aberrant m7G levels are closely associated with tumorigenesis and progression via regulation of the expression of multiple oncogenes and tumor suppressor genes. However, the role of m7G in sarcomas (SARC) has not been adequately evaluated. Materials and methods: Transcriptome and clinical data were gathered from the TCGA database for this study. Normal and SARC groups were compared for the expression of m7G-related genes (m7GRGs). The expression of m7GRGs was verified using real-time quantitative PCR (RT-qPCR) in SARC cell lines. Then, differentially expressed genes (DEGs) were identified between high and low m7GRGs expression groups in SARC samples, and GO enrichment and KEGG pathways were evaluated. Next, prognostic values of m7GRGs were evaluated by Cox regression analysis. Subsequently, a prognostic model was constructed using m7GRGs with good prognostic values by Lasso regression analysis. Besides, the relationships between prognostic m7GRGs and immune infiltration, clinical features, cuproptosis-related genes, and antitumor drugs were investigated in patients with SARC. Finally, a ceRNA regulatory network based on m7GRGs was constructed. Results: The expression of ten m7GRGs was higher in the SARC group than in the control group. DEGs across groups with high and low m7GRGs expression were enriched for adhesion sites and cGMP-PKG. Besides, we constructed a prognostic model that consists of EIF4A1, EIF4G3, NCBP1, and WDR4 m7GRGs for predicting the survival likelihood of sarcoma patients. And the elevated expression of these four prognostic m7GRGs was substantially associated with poor prognosis and elevated expression in SARC cell lines. Moreover, we discovered that these four m7GRGs expressions were negatively correlated with CD4+ T cell levels, dendritic cell level and tumor purity, and positively correlated with tumor mutational burden, microsatellite instability, drug sensitivity and cuproptosis-related genes in patients with sarcomas. Then, a triple regulatory network of mRNA, miRNA, and lncRNA was established. Conclusion: The current study identified EIF4A1, EIF4G3, NCBP1, and WDR4 as prognostic genes for SARC that are associated with m7G.These findings extend our knowledge of m7G methylation in SARC and may guide the development of innovative treatment options.

Identification of non-coding RNAs and their functional network associated with optic nerve invasion in retinoblastoma

Optic nerve invasion (ONI) is an important high-risk feature and prognostic indicator of retinoblastoma (RB). Emerging evidence has revealed that non-coding RNAs (ncRNAs) play important roles in tumor perineural invasion (PNI). Nevertheless, the regulatory role of ncRNAs in the ONI of RB is poorly understood. In the current study, whole-transcriptome sequencing was performed to assess the expression profiles of ncRNAs and mRNAs in RB tissues, with or without ONI. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, we predicted the biological functions of differentially expressed (DE) mRNAs. We then constructed competing endogenous RNA (ceRNA) regulatory networks based on bioinformatics analysis. The hsa_circ_0015965/lncRNA MEG3-hsa-miR-378a-5p-NOTCH1 pathway was selected and validated by real-time qPCR, western blotting, and dual luciferase reporter assays. Moreover, we demonstrated that NOTCH1 promotes the malignant progression of RB. Taken together, our results provide novel insights into the mechanism underlying optic nerve invasion in RB.

Regulation of oxidized LDL-induced proliferation and migration in human vascular smooth muscle cells by a novel circ_0007478/miR-638/ROCK2 ceRNA network.

Circular RNAs (circRNAs) have been implicated in the pathogenesis of atherosclerosis (AS) and the migration and proliferation of vascular smooth muscle cells (VSMCs) under oxidized low-density lipoprotein (ox-LDL). Here, we defined the exact action of human circ_0007478 in VSMC migration and proliferation induced by ox-LDL. Human VSMCs (HVSMCs) were exposed to ox-LDL. Circ_0007478, microRNA (miR)-638, and rho-associated protein kinase 2 (ROCK2) levels were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell viability and proliferation were assessed by MTT and EdU assays, respectively. Transwell assays were used to detect cell migration and invasion. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-638 and circ_0007478 or ROCK2. Our data indicated that circ_0007478 expression was augmented in AS serum samples and ox-LDL-treated HVSMCs. Depletion of circ_0007478 attenuated HVSMC proliferation, migration, and invasion induced by ox-LDL. Mechanistically, circ_0007478 targeted miR-638 by directly pairing to miR-638. Reduction of miR-638 reversed the effects of circ_0007478 depletion on ox-LDL-evoked proliferation, migration, and invasion in HVSMCs. ROCK2 was a direct miR-638 target and miR-638-mediated inhibition of ROCK2 relieved ox-LDL-evoked HVSMC proliferation, migration, and invasion. Furthermore, circ_0007478 was identified as a competing endogenous RNA (ceRNA) for miR-638 to modulate ROCK2 expression. Our present study establishes an undescribed ceRNA regulatory network, in which circ_0007478 targets miR-638 to upregulate ROCK2, thereby contributing to ox-LDL-induced proliferation and migration in HVSMCs.

Circ_0001274 Competitively Binds miR-143-3p to Upregulate VWF Expression to Improve Acute Traumatic Coagulopathy.

Accumulating evidence has noted the circRNA-microRNA- (circRNA-miRNA-) mRNA competing endogenous RNA (ceRNA) regulatory network in disease development and progression. The current study explored the ceRNA network in acute traumatic coagulopathy (ATC). Potential ATC-related genes were screened, and upstream miRNAs and circRNAs of VWF (the candidate target) were assayed through database searching and high-throughput sequencing technology. circ_0001274/miR-143-3p/VWF ceRNA regulatory network was constructed and validated. The expression of circ_0001274/miR-143-3p/VWF was determined in the peripheral blood samples from ATC patients and ATC mouse models. Online database and circRNA sequencing analysis results identified VWF as a key gene in ATC as supported by assays and that VWF was lowly expressed in ATC patients and mice. Further experiments demonstrated that miR-143-3p could target and inhibit VWF, and circ_0001274 could competitively sponge miR-143-3p. Functionally, circ_0001274 could competitively sequester miR-143-3p to upregulate VWF expression, potentially improving ATC. Our study highlights the critical role of circ_0001274/miR-143-3p/VWF axis in improving ATC.

Circular RNA ame_circ_000115 regulates expression of genes in larval gusts of Apis mellifera ligustica stressed by Ascosphaera apis

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.

Differential Circular RNA Expression Profiles in Induced Sputum of Patients with Asthma

Introduction: circRNA played a role in a variety of diseases. This paper aimed to explore the differentially expressed circRNA in induced sputum cells of asthma patients, so as to provide new potential biomarkers and new ideas for the study of asthma. Methods: All subjects were from the First Affiliated Hospital of Sun Yat-sen University. Differentially expressed circRNAs of asthma patients were screened by high-throughput sequencing. qRT-PCR was used to verify the expression of differential circRNAs. The association between circRNA and asthma was explored by analyzing the correlation between circRNA and clinical data and some cytokines of asthma patients. The possible ceRNA network was analyzed and predicted by online software, and the expression of each molecule in the network was preliminary verified by qRT-PCR in induced sputum cells and 16HBE cell. Results: We screened a total of 49 circRNAs differentially expressed in asthma patients (including 12 circRNAs with elevated expression and 37 circRNAs with decreased expression), among which has_circSORT1 and has_circSERPINB1 were significantly elevated. Correlation analysis showed that has_circSORT1 was correlated with FeNO, EOS%, IL-17A, IFN-γ, and PC20, and has_circSERPINB1 was correlated with IL-6, IL-17A, IFN-γ, FEV1%, and FVC%. The possible existence of has_circSORT1/has-miR-185-3p/ZNFX1, a ceRNA regulatory network, in induced sputum cells of asthma patients was hypothesized by online software prediction and qRT-PCR in sputum cells and 16HBE. Conclusion: Differentially expressed circRNAs existed in induced sputum cells of asthma patients, among which has_circSORT1 and has_circSERPINB1 were significantly upregulated and may be involved in the process of asthma disease, which could be expected to be a potential biomarker for asthma diagnosis. In addition, a ceRNA regulatory network, has_circSORT1/has-miR-185-3p/ZNFX1, may exist in asthma.

Bioinformatic analysis of differentially expressed profiles of lncRNAs and miRNAs with their related ceRNA network in endometrial cancer.

Increasing evidence suggests that long non-coding riboneucleic acids (lncRNAs), as competing endogenous RNA (ceRNA), play a key role in the initiation, invasion, and metastasis of cancer. As a new hypothesis, the lncRNA-micro RNA (miRNA)-messenger RNA (mRNA), ceRNA regulatory network has been successfully constructed in a variety of cancers. However, lncRNA, which plays a ceRNA function in endometrial cancer (EC), is still poorly understood. In this study, we downloaded EC expression profiling from The Cancer Genome Atlas database and used the R software "edgeR" package to analyze the differentially expressed genes between EC and normal endometrium samples. Then, differentially expressed (DE) lncRNAs, miRNAs and mRNAs were selected to construct a lncRNA-miRNA-mRNA prognosis-related regulatory network based on interaction information. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed on the genes in the network to predict the potential underlying mechanisms and functions of lncRNAs in EC. Kaplan-Meier method and the log-rank test were used for survival analysis. Based on the "ceRNA hypothesis," we constructed a co-expression network of mRNA and lncRNA genes mediated by miRNA in the process of tumor genesis. Furthermore, we successfully constructed a dysregulated lncRNA-associated ceRNA network containing 96 DElncRNAs, 27 DEmiRNAs, and 74 DEmRNAs. Through Kaplan-Meier curve analysis, we found that 9 lncRNAs, 3 miRNAs, and 12 mRNAs were significantly correlated with the overall survival rate of patients among all lncRNAs, miRNAs, and mRNAs involved in ceRNA (P < .05). Our research provides a new perspective for the interaction among lncRNAs, miRNAs, and mRNA and lays the foundation for further research on the mechanism of lncRNAs in the occurrence of EC.

RNA modification writers pattern in relation to tumor microenvironment and prognosis in prostate cancer.

Background: RNA modifications are important in the study of epigenetic regulatory mechanisms in immune responses and tumorigenesis. When RNA writers are mutated or disrupted in expression, the genes associated with the pathways they modify are also disrupted and can activate or repress related pathways, affecting tumorigenesis and progression. However, the potential role of RNA writers in prostate cancer is unclear. Methods: Based on data from three datasets, we describe 26 RNA writers that mediate gene expression and genetic mutation in prostate cancer and assess their expression patterns in 948 prostate cancer samples. Using principal component analysis algorithms, the RM Score was developed to quantify the RNA modification patterns of specific tumors. Results: Two different categories were determined by unsupervised clustering methods, and survival analysis showed significant differences in OS prognosis between these two categories. Differentially expressed genes between the different categories were detected and the RNA writers-mediated scoring model RM_Score were constructed based on this. Also, the RM_Score was analyzed in relation to clinical characteristics, immune infiltration level, drug response, and efficacy of chemotherapy and immunotherapy. Those results confirm that multilayer alterations in epitope-modified RNA writers are associated with patient prognosis and with immune cell infiltration characteristics. Finally, we examined differentially expressed mRNA, lncRNA and miRNA between high and low RM_Score groups, based on which a ceRNA regulatory network was constructed. Conclusion: This work is a comprehensive analysis of modified writers in prostate cancer and identified them to have a role in chemotherapy and immunotherapy.

Evaluation of Circ_0000977-Mediated Regulatory Network in Breast Cancer: A Potential Discriminative Biomarker for Triple-Negative Tumors.

Previous investigations have revealed that circular RNAs (circRNAs) play pivotal roles in cancer development and progression by participating in several biological procedures, such as competing endogenous RNA (ceRNA) networks. Recently, circRNAs have been proposed as non-invasive, stable, and affordable cell-free biomarkers for cancer screening and test monitoring. Although, their clinical usefulness vastly remains to be evaluated in breast cancer (BC). Triple-negative breast cancer (TNBC), as the most challenging BC subtype, is an urgent requirement of identifying specific biomarkers and discovering the molecular mechanisms that lead to aggressive behaviors of tumor cells. The therapeutic strategies for TN patients have remained limited due to the impracticality of endocrine therapies and a remarkable portion of patients with TNBC experience recurrence, chemoresistance, and metastasis. TNBC Microarray expression profile analysis found that circ_0000977 is one of the most dysregulated circRNA in TNBC in comparison with non-TNBC. It could be a clue referring to the potential clinical utility of circ_0000977 in TNBC. The current study aims to assess the clinical implications and potential ceRNA regulatory network of circ_0000977 in TNBC. We confirmed circ_0000977 down-regulation in TNBC cell lines and tumors versus non-TNBC samples by real-time PCR. Subsequently, an assessment of the diagnostic value of circ_0000977 in plasma samples from triple-negative patients revealed a potential diagnostic cell-free biomarker in triple-negative BC. Finally, our integrative approach uncovered potential circ-0000977/miR-135b-5p/mRNAs regulatory network in TNBC. The inhibitory effect of miR-135b-5p on its downstream mRNAs was assessed by knocking down it in MDA-MB-231 cells. Functional and correlation analyses revealed APC and GATA3 could be regulated by circ_0000977/miR-135b-5p ceRNA axis, which presents valuable insight into circ-0000977-mediated gene silencing involved in the ceRNA network of TNBC. This study uncovered the potential clinical implication of circ_0000977 for the diagnosis and treatment of TNBC patients.

Identification and validation of immune cells and hub genes alterations in recurrent implantation failure: A GEO data mining study.

Introduction: Recurrent implantation failure (RIF) is a distressing problem in assisted reproductive technology (ART). Immunity plays a vital role in recurrent implantation failure (RIF) occurrence and development, but its underlying mechanism still needs to be fully elucidated. Through bioinformatics analysis, this study aims to identify the RIF-associated immune cell types and immune-related genes. Methods: The differentially expressed genes (DEGs) were screened based on RIF-associated Gene Expression Omnibus (GEO) datasets. Then, the enrichment analysis and protein-protein interaction (PPI) analysis were conducted with the DEGs. The RIF-associated immune cell types were clarified by combining single sample gene set enrichment analysis (ssGSEA) and CIBERSORT. Differentially expressed immune cell types-related modules were identified by weighted gene co-expression network analysis (WGCNA) and local maximal quasi-clique merger (lmQCM) analysis. The overlapping genes between DEGs and genes contained by modules mentioned above were delineated as candidate hub genes and validated in another two external datasets. Finally, the microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) that interacted with hub genes were predicted, and the competing endogenous RNA (ceRNA) regulatory network was structured. Results: In the present study, we collected 324 DEGs between RIF and the control group, which functions were mainly enriched in immune-related signaling pathways. Regarding differential cell types, the RIF group had a higher proportion of activated memory CD4 T cells and a lower proportion of γδ T cells in the endometrial tissue. Finally, three immune-related hub genes (ALOX5AP, SLC7A7, and PTGS2) were identified and verified to effectively discriminate RIF from control individuals with a specificity rate of 90.8% and a sensitivity rate of 90.8%. In addition, we constructed a key ceRNA network that is expected to mediate molecular mechanisms in RIF. Conclusion: Our study identified the intricate correlation between immune cell types and RIF and provided new immune-related hub genes that offer promising diagnostic and therapeutic targets for RIF.

LncRNA UCA1 inhibits mitochondrial dysfunction of skeletal muscle in type 2 diabetes mellitus by sequestering miR-143-3p to release FGF21.

Increasing evidence suggests that insulin resistance in type 2 diabetes mellitus (T2DM) is associated with mitochondrial dysfunction in skeletal muscle, while the underlying molecular mechanisms remain elusive. This study aims to construct a ceRNA regulatory network that is involved in mitochondrial dysfunction of skeletal muscle in T2DM. Based on GEO database analysis, differentially expressed lncRNA and mRNA profiles were identified in skeletal muscle tissues of T2DM. Next, LASSO regression analysis was conducted to predict the key lncRNAs related to T2DM, which was validated by receiver operating characteristic (ROC) analysis. Moreover, the miRNAs related to skeletal muscle in T2DM were identified by WGCNA, followed by construction of gene-gene interaction network and GO and KEGG enrichment analyses. It was found that 12 lncRNAs and 6 miRNAs were related to skeletal muscle in T2DM. Moreover, the lncRNA-miRNA-mRNA ceRNA network involving UCA1, miR-143-3p, and FGF21 was constructed. UCA1, and FGF21 were downregulated, while miR-143-3p was upregulated in skeletal muscle cells (SkMCs) exposed to palmitic acid. Additionally, ectopic expression experiments were performed in SkMCs to confirm the effects of UCA1/miR-143-3p/FGF21 on mitochondrial dysfunction by determining mitochondrial ROS, oxygen consumption rate (OCR), membrane potential, and ATP level. Overexpression of miR-143-3p increased ROS accumulation and reduced the OCR, fluorescence ratio of JC-1, and ATP level, which were reversed by upregulation of UCA1 or FGF21. Collectively, lncRNA UCA1 inhibited mitochondrial dysfunction of skeletal muscle in T2DM by sequestering miR-143-3p away from FGF21, therefore providing a potential therapeutic target for alleviating mitochondrial dysfunction of skeletal muscle in T2DM.

Excavation and characterization of key circRNAs for milk fat percentage in Holstein cattle.

Milk fat percentage is one of the significant indicators governing the price and quality of milk and is regulated by a variety of non-coding RNAs. We used RNA sequencing (RNA-seq) techniques and bioinformatics approaches to explore potential candidate circular RNAs (circRNAs) regulating milk fat metabolism. After analysis, compared with low milk fat percentage (LMF) cows, 309 circRNAs were significantly differentially expressed in high milk fat percentage (HMF) cows. Functional enrichment and pathway analysis revealed that the main functions of the parental genes of differentially expressed circRNAs (DE-circRNAs) were related to lipid metabolism. We selected four circRNAs (Novel_circ_0000856, Novel_circ_0011157, novel_circ_0011944, and Novel_circ_0018279) derived from parental genes related to lipid metabolism as key candidate DE-circRNAs. Their head-to-tail splicing was demonstrated by linear RNase R digestion experiments and Sanger sequencing. However, the tissue expression profiles showed that only Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 were expressed with high abundance in breast tissue. Based on the subcellular localization found that Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 mainly function as competitive endogenous RNAs (ceRNAs) in the cytoplasm. Therefore, we constructed their ceRNA regulatory networks, and the five hub target genes (CSF1, TET2, VDR, CD34, and MECP2) in ceRNAs were obtained by CytoHubba and MCODE plugins in Cytoscape, as well as tissue expression profiles analysis of target genes. These genes play a key role as important target genes in lipid metabolism, energy metabolism, and cellular autophagy. The Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 regulate the expression of hub target genes through interaction with miRNAs and constitute key regulatory networks that may be involved in milk fat metabolism. The circRNAs obtained in this study may act as miRNA sponges and thus influence mammary gland development and lipid metabolism in cows, which improves our understanding of the role of circRNAs in cow lactation.

Whole transcriptome sequencing reveals core genes related to spermatogenesis in Bactrian camels.

Bactrian camels survive and reproduce better in extreme climatic conditions than other domestic animals can. However, the reproductive efficiency of camels under their natural pastoral conditions is low. Several factors affect mammalian reproductive performance, including testicular development, semen quality, libido, and mating ability. Testis is a main reproductive organ of the male and is responsible for producing spermatozoa and hormones. However, our understanding of the expression patterns of the genes in camel testis is minimal. Thus, we performed total RNA-sequencing to investigate the gene expression pattern. As a result, 1,538 differential expressed mRNAs (DEmRNAs), 702 differential expressed long non-coding RNAs (DElncRNAs), and 61 differential expressed microRNAs (DEmiRNAs) were identified between pubertal and adult Bactrian camel testes. Then the genomic features, length distribution, and other characteristics of the lncRNAs and mRNAs in the Bactrian camel testis were investigated. Target genes of the DEmiRNAs and DEmRNAs were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Genes, such as AMHR2, FGF1, ACTL7A, GATA4, WNT4, ID2, LAMA1, IGF1, INHBB, and TLR2, were mainly involved in the TGF-β, PI3K-AKT, Wnt, GnRH, and Hippo signaling pathways which relate to spermatogenesis. Some of the DEmiRNAs were predicted to be associated with numerous DElncRNAs and DEmRNAs through competing endogenous RNA (ceRNA) regulatory network. At last, the candidate genes were validated by RT-qPCR, dual fluorescent reporter gene, and a fluorescence in situ hybridization (FISH) assay. This research provides high-throughput RNA sequencing data of the testes of Bactrian camels across different developmental stages. It lays the foundation for further investigations on lncRNAs, miRNAs, and mRNAs that involved in Bactrian camel spermatogenesis.

Screening of Biomarkers Related to Myocardial Infarction Based on the Construction of a ceRNA Regulation Network.

This study aimed to identify the biomarkers related to myocardial infarction based on building lncRNA-miRNA-mRNA ceRNA regulation network. The expression profile data were obtained from the Gene Expression Omnibus database. The differentially expressed RNAs (DElncRNAs, DEmiRNAs, and DEmRNAs) were analyzed using the limma package of R. In addition, the differential myocardial infarction-related genes were obtained and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway on the differential myocardial infarction-related genes were analyzed. The co-expression network of lncRNA-mRNA was constructed, and the Kyoto Encyclopedia of Genes and Genomes pathway of such a co-expression network was analyzed. Moreover, the lncRNA-miRNA-mRNA ceRNA network associated with myocardial infarction was built, and the key biomarkers of the ceRNA network were verified using the GSE141512 and GSE66752 datasets. In total, we acquired 51 DElncRNAs, 276 DEmiRNAs, and 1200 DEmRNAs as well as 291 differential myocardial infarction-related genes. Moreover, the co-expression network of lncRNA-mRNA was built, and mRNAs were found to be associated with 109 Kyoto Encyclopedia of Genes and Genomes pathways (the target gene of RP3-394A18.1 was significantly related to lipid and atherosclerosis). In addition, the ceRNA network related to myocardial infarction was constructed, and the dataset analyses of the RNAs were validated between the myocardial infarction and normal groups. It was also found that the expression levels of hsa-miR-1291 and Retinoid acid receptor-related orphan receptor α (RORA) in the myocardial infarction group were noticeably lower than those in the normal group, whereas the expression levels of ENTPD1, QSOX1, and TIMP2 in the myocardial infarction group were noticeably higher than those in the normal group. In this study, we built a ceRNA regulation network of myocardial infarction. This study could help identify the biomarkers related to myocardial infarction.