Long noncoding RNA Glis2 regulates podocyte mitochondrial dysfunction and apoptosis in diabetic nephropathy via sponging miR-328-5p.

Podocyte apoptosis exerts a crucial role in the pathogenesis of DN. Recently, long noncoding RNAs (lncRNAs) have been gradually identified to be functional in a variety of different mechanisms associated with podocyte apoptosis. This study aimed to investigate whether lncRNA Glis2 could regulate podocyte apoptosis in DN and uncover the underlying mechanism. The apoptosis rate was detected by flow cytometry. Mitochondrial membrane potential (ΔΨM) was measured using JC-1 staining. Mitochondrial morphology was detected by MitoTracker Deep Red staining. Then, the histopathological and ultrastructure changes of renal tissues in diabetic mice were observed using periodic acid-Schiff (PAS) staining and transmission electron microscopy. We found that lncRNA Glis2 was significantly downregulated in high-glucose cultured podocytes and renal tissues of db/db mice. LncRNA Glis2 overexpression was found to alleviate podocyte mitochondrial dysfunction and apoptosis. The direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay. Furthermore, lncRNA Glis2 overexpression alleviated podocyte apoptosis in diabetic mice. Taken together, this study demonstrated that lncRNA Glis2, acting as a competing endogenous RNA (ceRNA) of miRNA-328-5p, regulated Sirt1-mediated mitochondrial dysfunction and podocyte apoptosis in DN.

Uncovering the ceRNA Network Related to the Prognosis of Stomach Adenocarcinoma Among 898 Patient Samples.

Stomach adenocarcinoma (STAD) patients are often associated with significantly high mortality rates and poor prognoses worldwide. Among STAD patients, competing endogenous RNAs (ceRNAs) play key roles in regulating one another at the post-transcriptional stage by competing for shared miRNAs. In this study, we aimed to elucidate the roles of lncRNAs in the ceRNA network of STAD, uncovering the molecular biomarkers for target therapy and prognosis. Specifically, a multitude of differentially expressed lncRNAs, miRNAs, and mRNAs (i.e., 898 samples in total) was collected and processed from TCGA. Cytoplasmic lncRNAs were kept for evaluating overall survival (OS) time and constructing the ceRNA network. Differentially expressed mRNAs in the ceRNA network were also investigated for functional and pathological insights. Interestingly, we identified one ceRNA network including 13 lncRNAs, 25 miRNAs, and 9 mRNAs. Among them, 13 RNAs were found related to the patient survival time; their individual risk score can be adopted for prognosis inference. Finally, we constructed a comprehensive ceRNA regulatory network for STAD and developed our own risk-scoring system that can predict the OS time of STAD patients by taking into account the above.

GKLOMLI: a link prediction model for inferring miRNA-lncRNA interactions by using Gaussian kernel-based method on network profile and linear optimization algorithm.

BACKGROUND: The limited knowledge of miRNA-lncRNA interactions is considered as an obstruction of revealing the regulatory mechanism. Accumulating evidence on Human diseases indicates that the modulation of gene expression has a great relationship with the interactions between miRNAs and lncRNAs. However, such interaction validation via crosslinking-immunoprecipitation and high-throughput sequencing (CLIP-seq) experiments that inevitably costs too much money and time but with unsatisfactory results. Therefore, more and more computational prediction tools have been developed to offer many reliable candidates for a better design of further bio-experiments. METHODS: In this work, we proposed a novel link prediction model based on Gaussian kernel-based method and linear optimization algorithm for inferring miRNA-lncRNA interactions (GKLOMLI). Given an observed miRNA-lncRNA interaction network, the Gaussian kernel-based method was employed to output two similarity matrixes of miRNAs and lncRNAs. Based on the integrated matrix combined with similarity matrixes and the observed interaction network, a linear optimization-based link prediction model was trained for inferring miRNA-lncRNA interactions. RESULTS: To evaluate the performance of our proposed method, k-fold cross-validation (CV) and leave-one-out CV were implemented, in which each CV experiment was carried out 100 times on a training set generated randomly. The high area under the curves (AUCs) at 0.8623 ± 0.0027 (2-fold CV), 0.9053 ± 0.0017 (5-fold CV), 0.9151 ± 0.0013 (10-fold CV), and 0.9236 (LOO-CV), illustrated the precision and reliability of our proposed method. CONCLUSION: GKLOMLI with high performance is anticipated to be used to reveal underlying interactions between miRNA and their target lncRNAs, and deciphers the potential mechanisms of the complex diseases.

Gender-specific lncRNA-miRNA-mRNA regulatory network to reveal potential genes for primary open-angle glaucoma.

BACKGROUND: Investigation of biomarkers may facilitate understanding the mechanisms of primary open-angle glaucoma (POAG) and developing therapeutic targets. This study aimed to identify potential genes based on competing endogenous RNA (ceRNA) network for POAG. METHODS: Based on long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) from the Gene Expression Omnibus (GEO) database, we identified differential expressed lncRNAs (DELs), differential expressed miRNAs (DEMis) and differential expressed mRNAs (DEMs) and then constructed a ceRNA network. Through weighted gene co-expression network analysis (WGCNA), we identified gender-specific genes for gender-associated ceRNA network construction, followed by the protein-protein interaction (PPI) network and functional enrichment analysis to screen hub genes and reveal their functions. The expression levels of hub genes were measured in steroid-induced ocular hypertension (SIOH) mice. RESULTS: A total of 175 DELs, 727 DEMs and 45 DEMis were screened between control and POAG samples. Seven modules were identified through WGCNA and one module was associated with gender of POAG patients. We discovered 41 gender-specific genes for gender-associated ceRNA construction and then identified 8 genes (NAV3, C1QB, RXRB, P2RY4, ADAM15, VAV3, ZNF207 and TOP1), which were enriched in cell cycle-related pathways and immune-related pathways. C1QB, RXRB, Top1 and ZNF207 were highly interacted with other proteins. The expression levels of NAV3 and C1QB were downregulated in SIOH, while the levels of RXRB, P2RY4, ADAM15, VAV3, ZNF207 and TOP1 were upregulated in SIOH. CONCLUSION: This study identifies hub genes associated with the pathogenesis of gender-specific POAG and provides potential biomarkers for POAG.

High eukaryotic initiation factor 5A2 expression predicts poor prognosis and may participate in the SNHG16/miR-10b-5p/EIF5A2 regulatory axis in head and neck squamous cell carcinoma.

BACKGROUND: This study attempted to investigate the significance of eukaryotic initiation factor 5A2 (EIF5A2) in the prognosis and regulatory network of head and neck squamous cell carcinoma (HNSCC). METHODS: EIF5A2 expression, prognostic information, and methylation levels of HNSCC were collected from the Cancer Genome Atlas (TCGA) database. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine EIF5A2 levels in HNSCC and normal tissue samples. R software was employed for expression analysis and prognosis assessment of EIF5A2 in HNSCC. A competing endogenous RNA (ceRNA) network was generated with the starBase database. Gene set enrichment analysis (GSEA) was used to determine the enriched physiological functions and network related to high expression of EIF5A2 in HNSCC. Immune infiltration-related outcomes were acquired from the CIBERSORT and Tumor Immune Estimation Resource (TIMER) database. RESULTS: EIF5A2 overexpression was observed in HNSCC and linked to poor progression-free survival and overall survival time. Cox regression analyses showed that EIF5A2 level was a stand-alone indicator of HNSCC patients' prognosis. A ceRNA network analysis highlighted the SNHG16/miR-10b-5p/EIF5A2 axis in EIF5A2 regulation. The GSEA results indicated that EIF5A2 was involved in complex signaling pathways. The CIBERSORT and TIMER databases revealed significant associations between EIF5A2 expression and immune cell infiltration. CONCLUSION: EIF5A2 overexpression may be a risk factor for prognosis in HNSCC and may be regulated by the SNHG16/miR-10b-5p/EIF5A2 axis.

Comprehensive ceRNA network for MACF1 regulates osteoblast proliferation.

BACKGROUND: Previous studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC­E1 pre­osteoblast cells. RESULTS: In total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes. CONCLUSION: This study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC­E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.

Identification and analysis of mitochondria-related key genes of heart failure.

Mitochondria-induced cell death is a vital mechanism of heart failure (HF). Thus, identification of mitochondria-related genes (Mito-RGs) based on transcriptome sequencing data of HF might provide novel diagnostic markers and therapeutic targets for HF. First, bioinformatics analysis was conducted on the GSE57338, GSE76701, GSE136547, and GSE77399 datasets in the Gene Expression Omnibus. Next, we analyzed HF-Mito differentially expressed genes (DEGs) using the protein-protein interaction (PPI) network for obtaining critical genes and exploring their functions. Subsequently, immune cell scores of the HF and normal groups were compared. The potential alteration mechanisms of the key genes were investigated by constructing a competing endogenous RNA network. Finally, we predicted potential therapeutic agents and validated the expression levels of the key genes. Twenty-three HF-Mito DEGs were acquired in the GSE57338 dataset, and the PPI network obtained four key genes, including IFIT3, XAF1, RSAD2, and MX1. According to gene set enrichment analysis, the key genes showed high enrichment in myogenesis and hypoxia. Immune cell analysis demonstrated that aDCs, B cells, and 20 other immune cell types varied between the HF and normal groups. Moreover, we observed that H19 might affect the expression of IFIT3, AXF1, and RSAD2. PCGEM1 might regulate RSAD2 expression. A total of 515 potential therapeutic drugs targeting the key genes, such as tretinoin, silicon dioxide, and bisphenol A, were acquired. Finally, IFIT3, RSAD2, and MX1 expression increased in HF samples compared with normal samples in the GSE76701 dataset, conforming to the GSE57338 dataset analysis. This work screened four key genes, namely, IFIT3, XAF1, RSAD2, and MX1, which can be further explored in subsequent studies for their specific molecular mechanisms in HF.

Comprehensive analysis to identify pseudogenes/lncRNAs-hsa-miR-200b-3p-COL5A2 network as a prognostic biomarker in gastric cancer.

OBJECTIVE: Gastric cancer is one of the most common and deadly types of cancer. The molecular mechanism of gastric cancer progression remains unclear. MATERIALS AND METHODS: Four hub genes were identified through GEO and TCGA database screening and analysis. Prognostic analysis revealed that COL5A2 was the most likely to affect the prognosis of gastric cancer among the four hub genes. The relationships between COL5A2 and clinical variables and immune cell infiltration were analyzed. Then, COL5A2 was analyzed for single-gene differences and related functional enrichment. Using the starBase database for prediction and analysis, miRNAs and pseudogenes/lncRNAs that might combine with COL5A2 were identified; thus, the ceRNA network was constructed. Finally, the network was verified by Cox analysis and qPCR, and a nomogram was constructed. RESULTS: First, we found that COL5A2, COL12A1, BGN and THBS2 were highly expressed in gastric cancer. COL5A2 had statistical significance in overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) analysis. Immune infiltration analysis suggested that COL5A2 might influence the changes in the tumor immune microenvironment. The StarBase database was used to predict that 3 pseudogenes and 7 lncRNAs might inhibit the hsa-miR-200b-3p-COL5A2 axis in gastric cancer. The pseudogenes/lncRNA-hsa-miR-200b-3p-COL5A2 ceRNA network was identified and verified using Cox regression analysis and PCR. Finally, we constructed a nomogram. CONCLUSIONS: We elucidated the regulatory role of the pseudogenes/lncRNA-hsa-miR-200b-3p-COL5A2 network in gastric cancer progression and constructed a nomogram. These studies may provide effective treatments and potential prognostic biomarkers for gastric cancer.

Critical Roles of Circular RNA in Tumor Metastasis via Acting as a Sponge of miRNA/isomiR.

Circular RNAs (circRNAs), a class of new endogenous non-coding RNAs (ncRNAs), are closely related to the carcinogenic process and play a critical role in tumor metastasis. CircRNAs can lay the foundation for tumor metastasis via promoting tumor angiogenesis, make tumor cells gain the ability of migration and invasion by regulating epithelial-mesenchymal transition (EMT), interact with immune cells, cytokines, chemokines, and other non-cellular components in the tumor microenvironment, damage the normal immune function or escape the immunosuppressive network, and further promote cell survival and metastasis. Herein, based on the characteristics and biological functions of circRNA, we elaborated on the effect of circRNA via circRNA-associated competing endogenous RNA (ceRNA) network by acting as miRNA/isomiR sponges on tumor angiogenesis, cancer cell migration and invasion, and interaction with the tumor microenvironment (TME), then explored the potential interactions across different RNAs, and finally discussed the potential clinical value and application as a promising biomarker. These results provide a theoretical basis for the further application of metastasis-related circRNAs in cancer treatment. In summary, we briefly summarize the diverse roles of a circRNA-associated ceRNA network in cancer metastasis and the potential clinical application, especially the interaction of circRNA and miRNA/isomiR, which may complicate the RNA regulatory network and which will contribute to a novel insight into circRNA in the future.

The long noncoding RNA Arrl1 inhibits neurite outgrowth by functioning as a competing endogenous RNA during neuronal regeneration in rats.

The intrinsic regeneration ability of neurons is a pivotal factor in the repair of peripheral nerve injury. Therefore, identifying the key modulators of nerve regeneration may help improve axon regeneration and functional recovery after injury. Unlike for classical transcription factors and regeneration-associated genes, the function of long noncoding RNAs (lncRNAs) in the regulation of neuronal regeneration remains mostly unknown. In this study, we used RNA-Seq-based transcriptome profiling to analyze the expression patterns of lncRNAs and mRNAs in rat dorsal root ganglion (DRG) following sciatic nerve injury. Analyses using the lncRNA-mRNA co-expression network, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway databases indicated that the lncRNA Arrl1 decreases neurite outgrowth after neuronal injury. shRNA-mediated Arrl1 silencing increased axon regeneration both in vitro and in vivo and improved functional recovery of the sciatic nerve. Moreover, inhibiting an identified target gene of Arrl1, cyclin-dependent kinase inhibitor 2B (Cdkn2b), markedly promoted neurite outgrowth of DRG neurons. We also found that Arrl1 acts as a competing endogenous RNA that sponges a Cdkn2b repressor, microRNA-761 (miR-761), and thereby up-regulates Cdkn2b expression during neuron regeneration. We conclude that the lncRNA Arrl1 affects the intrinsic regeneration of DRG neurons by derepressing Cdkn2b expression. Our findings indicate a role for an lncRNA-microRNA-kinase pathway in the regulation of axon regeneration and functional recovery following peripheral nerve injury in rats.

Genome-wide identification and characterization of long non-coding RNAs involved in flag leaf senescence of rice.

KEY MESSAGE: This study showed the systematic identification of long non-coding RNAs (lncRNAs) involving in flag leaf senescence of rice, providing the possible lncRNA-mRNA regulatory relationships and lncRNA-miRNA-mRNA ceRNA networks during leaf senescence. LncRNAs have been reported to play crucial roles in diverse biological processes. However, no systematic identification of lncRNAs associated with leaf senescence in plants has been studied. In this study, a genome-wide high throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. A total of 3953 lncRNAs and 38757 mRNAs were identified, of which 343 lncRNAs and 9412 mRNAs were differentially expressed. Through weighted gene co-expression network analysis (WGCNA), 22 continuously down-expressed lncRNAs targeting 812 co-expressed mRNAs and 48 continuously up-expressed lncRNAs targeting 1209 co-expressed mRNAs were considered to be significantly associated with flag leaf senescence. Gene Ontology results suggested that the senescence-associated lncRNAs targeted mRNAs involving in many biological processes, including transcription, hormone response, oxidation-reduction process and substance metabolism. Additionally, 43 senescence-associated lncRNAs were predicted to target 111 co-expressed transcription factors. Interestingly, 8 down-expressed lncRNAs and 29 up-expressed lncRNAs were found to separately target 12 and 20 well-studied senescence-associated genes (SAGs). Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 6 down-expressed lncRNAs possibly regulated 51 co-expressed mRNAs through 15 miRNAs, and 14 up-expressed lncRNAs possibly regulated 117 co-expressed mRNAs through 21 miRNAs. Importantly, by expression validation, a conserved miR164-NAC regulatory pathway was found to be possibly involved in leaf senescence, where lncRNA MSTRG.62092.1 may serve as a ceRNA binding with miR164a and miR164e to regulate three transcription factors. And two key lncRNAs MSTRG.31014.21 and MSTRG.31014.36 also could regulate the abscisic-acid biosynthetic gene BGIOSGA025169 (OsNCED4) and BGIOSGA016313 (NAC family) through osa-miR5809. The possible regulation networks of lncRNAs involving in leaf senescence were discussed, and several candidate lncRNAs were recommended for prior transgenic analysis. These findings will extend the understanding on the regulatory roles of lncRNAs in leaf senescence, and lay a foundation for functional research on candidate lncRNAs.

Benzo[a]pyrene diol epoxide-induced transformed cells identify the significance of hsa_circ_0051488, a ERCC1-derived circular RNA in pulmonary squamous cell carcinoma.

ERCC1 is a gene for repairing DNA damage whose function is related to carcinogenic-induced tumorigenesis and the effectiveness of platinum therapies. Circular RNAs (circRNAs) are products of posttranscriptional regulation with pleiotropic effects on the pathogenesis of lung cancer. We aim to identify that specific circRNAs derived from ERCC1 can regulate key biological processes involved in the development of lung cancer. We performed bioinformatics analysis, in vitro experiments, and analyzed clinical samples, to determine the biological features of a certain ERCC1-derived circRNA termed as hsa_circ_0051488 in benzo[a]pyrene diol epoxide-induced malignant transformed cell and lung cancer cell. The well-established model of transformed cells provided an ideal platform for analyzing the molecular characteristics of this circRNA in the malignant transformation of lung epithelial cell, which supports that hsa_circ_0051488 functions in the onset and growth of lung squamous cell carcinoma (LUSC). Further analysis indicates that the absence of hsa_circ_0051488 promoted the proliferation of cells with the malignant phenotype. Extensive experiments confirm that hsa_circ_0051488 is present in the cytoplasm and functioned as a competing endogenous RNA. In particular, hsa_circ_0051488 binds to mir-6717-5p, thereby modulating the expression of SATB2 gene, a lung cancer suppressor. Furthermore, our in silico experiments indicate that SATB2 can inhibit multiple tumor pathways and its expression positively correlated with the tumor suppressor gene CRMP1. These findings suggest a possible regulatory mechanism of hsa_circ_0051488 in LUSC, and that the newly discovered hsa_circ_0051488/miR-6717-5p/SATB2 axis may be a potential route for therapeutic intervention of LUSC.

Systematic identification and characterization of circular RNAs involved in flag leaf senescence of rice.

MAIN CONCLUSION: Circular RNAs (circRNAs) identification, expression profiles, and construction of circRNA-parental gene relationships and circRNA-miRNA-mRNA ceRNA networks indicate that circRNAs are involved in flag leaf senescence of rice. Circular RNAs (circRNAs) are a class of 3'-5' head-to-tail covalently closed non-coding RNAs which have been proved to play important roles in various biological processes. However, no systematic identification of circRNAs associated with leaf senescence in rice has been studied. In this study, a genome-wide high-throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. Here, a total of 6612 circRNAs were identified, among which, 113 circRNAs were differentially expressed (DE) during the leaf senescence process. Moreover, 4601 (69.59%) circRNAs were derived from the exons or introns of their parental genes, while 2110 (71%) of the parental genes produced only one circRNA. The sequence alignment analysis showed that hundreds of rice circRNAs were conserved among different plant species. Gene Ontology (GO) enrichment analysis revealed that parental genes of DE circRNAs were enriched in many biological processes closely related to leaf senescence. Through weighted gene co-expression network analysis (WGCNA), six continuously down-expressed circRNAs, 18 continuously up-expressed circRNAs and 15 turn-point high-expressed circRNAs were considered to be highly associated with leaf senescence. Additionally, a total of 17 senescence-associated circRNAs were predicted to have parental genes, in which, regulations of three circRNAs to their parental genes were validated by qRT-PCR. The competing endogenous RNA (ceRNA) networks were also constructed. And a total of 11 senescence-associated circRNAs were predicted to act as miRNA sponges to regulate mRNAs, in which, regulation of two circRNAs to eight mRNAs was validated by qRT-PCR. It is discussed that senescence-associated circRNAs were involved in flag leaf senescence probably through mediating their parental genes and ceRNA networks, to participate in several well-studied senescence-associated processes, mainly including the processes of transcription, translation, and posttranslational modification (especially protein glycosylation), oxidation-reduction process, involvement of senescence-associated genes, hormone signaling pathway, proteolysis, and DNA damage repair. This study not only showed the systematic identification of circRNAs involved in leaf senescence of rice, but also laid a foundation for functional research on candidate circRNAs.

A circRNA-miRNA-mRNA regulatory network associated with the treatment response to tuberculosis.

OBJECTIVES: The high morbidity and mortality of tuberculosis (TB) have severe socio-economic consequences, and there is an urgent need to explore the mechanisms driving TB development and progression. The aim of this study was to analyze the regulatory RNAs and target genes involved in TB, in order to identify key genetic biomarkers for diagnosing and treating TB. METHODS: Circular RNAs (circRNAs), microRNAs (miRNAs) and messenger RNA (mRNAs) expression profiles of TB patients and healthy controls were downloaded from the GEO database. A circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was constructed using the differentially expressed circRNAs (DEcircRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs). The DEmRNAs in this network were functionally annotated using GO and KEGG analyses, and ordinal regression analysis was used to identify the genes correlated to the treatment response in TB patients. RESULTS: We identified 133 DEmRNAs, 37 DEcircRNAs and 173 DEmiRNAs between the TB and healthy controls, from which 30 DECircRNAs, 27 DEmiRNAs and 35 DEmRNAs were used to construct the ceRNA network. CACNA1I, IGF2BP3, LPCAT2, SPOCK2 and IRF2 were significantly correlated with the anti-TB therapeutic response (P < 0.05). CONCLUSION: A TB-associated DEcircRNA-miRNA-mRNA ceRNA network was constructed, of which some DEmRNAs potentially influence the treatment response.

Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes.

BACKGROUND: miRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes. METHODS: Small and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8. RESULTS: We revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs - 43 miRNAs - 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16. CONCLUSIONS: The multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.

Long noncoding RNAs sustain high expression levels of exogenous octamer-binding protein 4 by sponging regulatory microRNAs during cellular reprogramming.

Long noncoding RNAs (lncRNAs) modulate gene expression as competing endogenous RNAs (ceRNAs) that sponge regulatory microRNAs (miRNAs). During cellular reprogramming, genes associated with pluripotency establishment need to be up-regulated, and developmental genes need to be silenced. However, how ceRNAs control cellular reprogramming still awaits full elucidation. Here, we used doxycycline-inducible expression of the four transcription factors octamer-binding protein 4 (OCT4), SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc (c-Myc) to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Using RNA-Seq and bioinformatics approaches, we found that the expression levels of miRNAs from MEFs remain high from day 0 to 6 after the doxycycline induction. Many genes targeted by these miRNAs were up-regulated, and long intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs), which have complementary binding sites to these miRNAs, were highly expressed, indicating lincRNAs and circRNAs may function as ceRNAs. Intriguingly, knockdown of the linc/circRNAs that sponge the miRNAs, which target OCT4 down-regulated exogenous OCT4, decreased reprogramming efficiency, and resulted in low-grade iPSCs. Our results suggest that the ceRNA network plays an important role in cellular reprogramming.

Comprehensive analysis of key genes associated with ceRNA networks in nasopharyngeal carcinoma based on bioinformatics analysis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with high morbidity rates in the east and southeast Asia. The molecular mechanisms of NPC remain largely unknown. We explored the pathogenesis, potential biomarkers, and prognostic indicators of NPC. METHODS: We analyzed mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) in the whole transcriptome sequencing dataset of our hospital (five normal tissues vs. five NPC tissues) and six microarray datasets (62 normal tissues vs. 334 NPC tissues) downloaded from the Gene Expression Omnibus (GSE12452, GSE13597, GSE95166, GSE126683, and GSE70970, GSE43039). Differential expression analyses, gene ontology (GO) enrichment, kyoto encyclopedia of genes and genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted. The lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed using the miRanda and TargetScan database, and a protein-protein interaction (PPI) network of differentially expressed genes (DEGs) was built using Search Tool for the Retrieval of Interacting Genes (STRING) software. Hub genes were identified using Molecular Complex Detection (MCODE), NetworkAnalyzer, and CytoHubba. RESULTS: We identified 61 mRNAs, 14miRNAs, and 10 lncRNAs as shared DEGs related to NPC in seven datasets. Changes in NPC were enriched in the chromosomal region, sister chromatid segregation, and nuclear chromosome segregation. GSEA indicated that the mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol-3 OH kinase/protein kinase B (PI3K-Akt) pathway, apoptotic pathway, and tumor necrosis factor (TNF) were involved in the initiation and development of NPC. Finally, 20 hub genes were screened out via the PPI network. CONCLUSIONS: Several DEGs and their biological processes, pathways, and interrelations were found in our current study by bioinformatics analyses. Our findings may offer insights into the biological mechanisms underlying NPC and identify potential therapeutic targets for NPC.

Comprehensive analysis of TPX2-related ceRNA network as prognostic biomarkers in lung adenocarcinoma.

Background and aim: Competing endogenous RNA (ceRNA) is believed to play vital roles in tumorigenesis. The goal of this study was to screen prognostic biomarkers in lung adenocarcinoma (LUAD). Methods: Common differentially expressed genes (DEGs) were collected from Gene Expression Omnibus (GEO) databases and The Cancer Genome Atlas databases (TCGA) using GEO2R and "limma" package in R, respectively. Overlapping DEGs were conducted using enrichment of functions and protein-protein interaction (PPI) network to discover significant candidate genes. By using a comprehensive analysis, we constructed an mRNA mediated ceRNA network. Survival rates were used Kaplan-Meier analysis. Statistical analysis was used to further identify the prognosis of studied genes. Results: Integrated analysis of GSE32863 and TCGA databases, a total of 886 overlapping DEGs, including 279 up-regulated and 607 down-regulated genes were identified. Considering the highest term of candidate genes in PPI, we identified TPX2, which was enriched in cell division signaling pathway. Besides, 35 differentially expressed miRNAs (DEmiRNAs) were predicted to target TPX2 and only 7 DEmiRNAs were identified to be prognostic biomarkers in LUAD. Then, 30 differentially expressed lncRNAs (DElncRNAs) were predicted to bind these 7 DEmiRNAs. Finally, we found that 7 DElncRNAs were correlated with the overall survival (all p <0.05). Furthermore, we identified elevated TPX2 was strongly correlated with the worse survival rate among 458 samples. Univariate and multivariate cox analysis showed TPX2 may act as an independent factor for prognosis in LUAD (p <0.05). Then pathway enrichment results suggested that TPX2 may facilitate tumorigenesis by participating in several cancer-related signaling pathways in LUAD, especially in Notch signal pathway. Conclusions: TPX2-related lncRNAs and miRNAs are related to the survival of LUAD. 7 lncRNAs, 7 miRNAs and TPX2 may serve as prognostic biomarkers in LUAD.

MIR205HG acts as a ceRNA to expedite cell proliferation and progression in lung squamous cell carcinoma via targeting miR-299-3p/MAP3K2 axis.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been associated with many types of cancers, but their molecular mechanisms in lung squamous cell carcinoma (LUSC) have not been fully studied. Therefore, the current study investigated the regulation role of microRNA-205 host gene (MIR205HG) in LUSC and recognized the target genes managed by this lncRNA. METHODS: MIR205HG expression was assessed by the quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The effects of silenced MIR205HG on cell biological behaviors were detected by colony formation assay, transwell assay, flow cytometry analysis and western blot analysis. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to proof the binding relationship between miR-299-3p and MIR205HG/mitogen-activated protein kinase kinase kinase 2 (MAP 3 K2). RESULTS: The expression levels of MIR205HG in LUSC tissues and cell lines were obviously up-regulated. Down-regulation of MIR205HG expression remarkably reduced cell proliferation, migration and epithelial-to-mesenchymal transition (EMT) progression, whereas promoted cell apoptosis. MIR205HG could bind with miR-299-3p and down-regulation of MIR205HG elevated miR-299-3p expression. MAP 3 K2 acted as the target gene of miR-299-3p and was up-regulated by MIR205HG overexpression. Overexpressing MAP 3 K2 could counteract the effects of down-regulating MIR205HG on LUSC progression to some degree. CONCLUSION: MIR205HG acts as a competing endogenous RNA (ceRNA) to expedite cell proliferation and progression via targeting miR-299-3p in LUSC.

Microarray expression profiles analysis revealed lncRNA OXCT1-AS1 promoted bladder cancer cell aggressiveness via miR-455-5p/JAK1 signaling.

Bladder cancer (BCa) is one of the most prevalent cancers of the urinary system worldwide. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) perform a vital function in the pathogenesis and progression of BCa. In the current study, we identified a novel lncRNA OXCT1-AS1 and investigated its role and potential mechanisms in BCa. The microarray results showed the expression of lncRNAs, microRNAs, and messenger RNAs between BCa primary tumor tissues and metastatic lymph nodes were significantly different. The quantitative polymerase chain reaction verification was performed to ensure the reliability of the screening results. The Cell Counting Kit 8 and transwell assay were used to assess the tumor cell proliferation and invasion abilities in vitro, respectively. The dual-luciferase activity assay was performed to investigate the potential mechanism of competing endogenous RNA network. lncRNA OXCT1-AS1, which elevated in metastasis lymph node, was significantly upregulated in BCa cell lines compared with SVHUC-1. We demonstrated OXCT1-AS1 inhibited miR-455-5p to decrease its binding to the JAK1 3'-untranslated region, which could upregulate the expression of JAK1 at the protein level, thus promoting BCa proliferation and invasion. Therefore, lncRNA OXCT1-AS1 could act as a potential biomarker and therapeutic target for patients with BCa.