Potential Regulatory miRNAs Research Articles

Construction of a novel mRNA–miRNA–lncRNA/circRNA triple subnetwork associated with immunity and aging in intervertebral disc degeneration

Objective Intervertebral disk degeneration (IVDD) is one of the most common causes of low back pain. However, in the etiology of IVDD, the specific method by which nucleus pulposus (NP) cell senescence and the immune response induce disease is uncertain. Methods Gene Expression Omnibus database was used to find differentially expressed genes (DEGs), differentially expressed miRNAs (DE miRNAs), differentially expressed lncRNAs (DE lncRNAs), and differentially expressed circRNAs (DE circRNAs). Functional enrichment analysis was performed through Enrichr database. Potential regulatory miRNAs, lncRNAs and circRNAs of mRNAs were predicted by ENCORI and circBank, respectively. Results We identified 198 upregulated and 131 downregulated genes, 39 upregulated and 22 downregulated miRNAs, 2152 upregulated and 564 downregulated lncRNAs, and 352 upregulated and 279 downregulated circRNAs as DEGs, DE miRNAs, DE lncRNAs, DE circRNAs, respectively. Functional enrichment analysis revealed that they were significantly enriched in Toll-like receptor signaling route and the NF-kappa B signaling pathway. An mRNA–miRNA–lncRNA/circRNA network linked to the pathogenesis of NP cells in IVDD was constructed based on node degree and differential expression level. Eight immune-related DEGs (6 upregulated and 2 downregulated genes) and five aging-related DEGs (3 upregulated and 2 downregulated genes) were identified in the critical network. Conclusion We established a novel immune-related and aging-related triple regulatory network of mRNA–miRNA–lncRNA/circRNA ceRNA, among which all RNAs may be utilized as the pathogenesis biomarker of NP cells in IVDD.

MiR-92a-2-5p suppresses esophageal squamous cell carcinoma cell proliferation and invasion by targeting PRDX2

MicroRNAs (miRNAs) can function as negative regulators of gene expression by binding to the 3′-untranslated region (3′-UTR) of target genes. The aberrant expression of miRNAs in neoplasm is extensively associated with tumorigenesis and cancer progression, including esophageal squamous cell carcinoma (ESCC). Our previous investigation has identified the oncogenic roles of Peroxiredoxin2 (PRDX2) in ESCC progression; however, its upstream regulatory mechanism remains to be elucidated. By merging the prediction results from miRWalk2.0 and miRNA differential expression analysis results based on The Cancer Genome Atlas Esophageal Carcinoma (TCGA-ESCA) database, eight miRNA candidates were predicted to be the potential regulatory miRNAs of PRDX2, followed by further identification of miR-92a-2-5p as the putative miRNA of PRDX2. Subsequent functional studies demonstrated that miR-92a-2-5p can suppress ESCC cell proliferation and migration, as well as tumor growth in subcutaneous tumor xenograft models, which might be mediated by the suppression of AKT/mTOR and Wnt3a/β-catenin signaling pathways upon miR-92a-2-5p mimic transfection condition. These data revealed the tumor suppressive functions of miR-92a-2-5p in ESCC by targeting PRDX2.

Activating transcription factor 3 is a new biomarker correlation with renal clear cell carcinoma progression.

Background: Clear cell renal cell carcinoma (ccRCC) is the most invasive type of cancer, with a high risk of metastasis and recurrence. Therefore, there is an urgent need to identify novel prognostic predictors and therapeutic targets of ccRCC. Activating transcription factor 3 (ATF3), a tumor oncogene or repressor, has rarely been examined in ccRCC. In the present study, we comprehensively elucidate the prognostic value and potential functions of ATF3 in ccRCC.Methods: Several TCGA-based online databases were used to analyze ATF3 expression in ccRCC and determine ccRCC prognosis. The upstream-binding micro (mi) RNAs of ATF3 and long non-coding (lnc)RNAs were predicted using the StarBase database.Results: Analysis of several TCGA-based online databases showed that ATF3 expression is decreased in ccRCC, suggesting a significant association with the prognosis of patients with ccRCC. Furthermore, we found hsa-miR-221-3p to be potential regulatory miRNA of ATF3 in ccRCC. Prediction and analysis of the upstream lncRNAs indicated that PAXIP1-AS2 and OIP5-AS1 were the most potent upstream lncRNAs of the hsa-miR-221-3p/ATF3 axis in ccRCC. The results of the GO and KEGG analyses implied that ATF3 is likely involved in the regulation of apoptotic signaling in response to endoplasmic reticulum (ER) stress in ccRCC. Correlation analysis revealed a positive relationship between ATF3 expression and ER stress.Conclusions: Our in silico findings highlighted that ATF3 expression was low in ccRCC and negatively correlated with poor prognosis. Furthermore, PAXIP1-AS2 and the OIP5-AS1/hsa-miR-221-3p/ATF3 axis were identified as significant potential regulators of ER stress-mediated apoptosis in ccRCC.

Bioinformatics analysis of competing endogenous RNA network in decidual natural killer cell from unexplained recurrent spontaneous abortion.

Decidual natural killer (dNK) cell plays a pivotal role in maintaining pregnancy, especially in the first trimester. Noncoding-RNAs (ncRNAs) are critical regulators of transcription and protein expression. Dysregulation of ncRNAs may be involved in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the role of competing endogenous RNA (ceRNA) based on mRNA-miRNA-lncRNA network in regulating the incidence and progression of URSA remains elusive. The aim of the study is to identify the regulatory network of mRNA-miRNA-LncRNA ceRNA based on bioinformatics analysis in dNK from patients with URSA. Eligible studies were retrieved from PubMed, Embase, and the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs), miRNAs and LncRNAs in dNK cells of patients with URSA. Protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software. Potential regulatory miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and RNA22 and subject to bioinformatics analysis. A total of 634 DEGs were screened, including 290 upregulated and 344 downregulated DEGs. Among 207 differentially expressed lncRNAs, 110 lncRNAs were upregulated and 97 were downregulated. According to node degree, 30 hub genes were identified for subsequent research. After drawing the Venn diagram and matching to Cytoscape, an mRNA-miRNA-lncRNA network linked to the pathogenesis of URSA in dNK cells was constructed. A novel regulatory network of mRNA-miRNA-lncRNA ceRNA is established in dNK cells from patients with URSA. All RNAs might be used as the biomarkers of the pathogenesis of URSA.

Identification of altered miRNAs and their targets in placenta accreta.

Placenta accreta spectrum (PAS) is one of the major causes of maternal morbidity and mortality worldwide with increasing incidence. PAS refers to a group of pathological conditions ranging from the abnormal attachment of the placenta to the uterus wall to its perforation and, in extreme cases, invasion into surrounding organs. Among them, placenta accreta is characterized by a direct adhesion of the villi to the myometrium without invasion and remains the most common diagnosis of PAS. Here, we identify the potential regulatory miRNA and target networks contributing to placenta accreta development. Using small RNA-Seq followed by RT-PCR confirmation, altered miRNA expression, including that of members of placenta-specific miRNA clusters (e.g., C19MC and C14MC), was identified in placenta accreta samples compared to normal placental tissues. In situ hybridization (ISH) revealed expression of altered miRNAs mostly in trophoblast but also in endothelial cells and this profile was similar among all evaluated degrees of PAS. Kyoto encyclopedia of genes and genomes (KEGG) analyses showed enriched pathways dysregulated in PAS associated with cell cycle regulation, inflammation, and invasion. mRNAs of genes associated with cell cycle and inflammation were downregulated in PAS. At the protein level, NF-κB was upregulated while PTEN was downregulated in placenta accreta tissue. The identified miRNAs and their targets are associated with signaling pathways relevant to controlling trophoblast function. Therefore, this study provides miRNA:mRNA associations that could be useful for understanding PAS onset and progression.

Intra‑tumor heterogeneity of cancer stem cell‑related genes and their potential regulatory microRNAs in metastasizing colorectal carcinoma.

Intra‑tumor heterogeneity (ITH) is related to cancer progression, therapy resistance and recurrences, and is one of the challenging fields in cancerogenesis research. Cancer stem cells (CSC) are thought to be crucially involved in the pathogenesis of several cancer types, including colorectal carcinoma (CRC), and associated with ITH. In the present study, the expression gradient of four genes related to CSC (L1TD1, SLITRK6, ST6GALNAC1 and TCEA3) and their potential regulatory microRNAs (miRNAs) were investigated in the central part and invasive front of the primary tumor, as well as in lymph node and liver metastases. In total, 63 formalin‑fixed paraffin‑embedded biopsy samples of primary tumor (central part, invasive tumor front), as well as lymph node and liver metastases from 19patients with CRC, were analyzed. The expression of selected genes (L1TD1, SLITRK6, ST6GALNAC1 and TCEA3) and miRNAs (miR‑199a‑3p, miR‑425‑5p, miR‑1225‑3p, miR‑1233‑3p and miR‑1303) was evaluated using reverse transcription‑quantitative PCR. Significant differences in expression were identified for all investigated genes in lymph node metastasis, but not in the liver metastases. All investigated miRNAs were significantly differentially expressed in lymph node metastasis, and miR‑199a‑3p, miR‑425‑5p and miR‑1233‑3p in liver metastasis. Furthermore, a negative correlation between the expression of miR‑199a‑3p and expression of its potential target gene SLITRK6 was confirmed. The present results provide further evidence that expression of CSC‑related genes and their potential regulatory miRNAs contribute to ITH in CRC, lymph node and liver metastasis. The SLITRK6 gene and its regulatory miRNA miR‑199a‑3p are promising for further validation in functional studies to deepen the present understanding of the regulation of CSC‑related genes in CRC.

MicroRNA levels in hemodialysis patients following resistance training: Associations with functional performance, inflammatory profile, sestrins-2, and nitric oxide.

Investigate the effects of long-term resistance training (RT) on expression of the four selected microRNAs (miRNA or mir) and further association with biomarkers related to functional performance in older end-stage renal disease (ESRD) patients undergoing hemodialysis. Twenty-five older hemodialysis patients (glomerular filtration rate <15mL/min/1.73m2 aged 68.28±1.06) were recruited for the study. Patients were allocated to two groups (control, n=12 and RT, n=13). The RT group completed 24weeks of training, with sessions held three times per week on alternate days. Blood samples were collected pre- and post- intervention for miRNA and biochemical assays. Results were considered significant at P<0.05. RT promoted benefits in inflammatory profile, nitric oxide, sestrins-2, anthropometric data, and functional performance. Trained subjects presented a 51% decrease in miRNA-31 after intervention. In addition, miRNA-1 increased 128% after RT protocol. miRNA-1 significantly correlated with functional performance, inflammatory profile, sestrins-2, and nitric oxide (all P<0.05). These results suggest that the upregulation of miRNA-1 could be associated with physiological benefits promoted by RT in hemodialysis patients, providing novel understanding for potential regulatory miRNA effects on physiological RT response. These findings might point out to strategic direction for future studies.

MiR-375 Enriched in Bone Marrow Mesenchymal Stem Cells (BMSC) Exosomes Inhibits Prostate Cancer Cell Migration and Invasion by Down-Regulating Trefoil Factor 3 (TFF3)

To study the effect and mechanism of miR-375 enriched in BMSC exosomes on prostate cancer (PC) cells. Bioinformatics assessed the potential regulatory miRNA of TFF3 and miR-375 level in breast cancer cells and breast cancer clinical samples was detected by PCR. Dual luciferase assay validated the relationship between TFF3 and miR-375. miR-375 mimics or sh-TFF3 was transfected into PC cells, followed by measuring miR-375 and TFF3 by PCR and Western-blot. Cell proliferation, invasion, migration and apoptosis by Edu staining, transwell and flow cytometry. The BMSC exosomes were then isolated and co-cultured with PC cells to detect cell proliferation and invasion. PC cells and tissues showed the expression of miR-375 was decreased, indicated that miR-375 specifically inhibited TFF3 level. miR-375 was enriched in MSC-derived exosomes and could be transferred to PC cells. miR-375 mimics, exosome miR-375 or silenced TFF3 inhibited TFF3 level, up-regulated PCNA, MMP-2/9 expression, thereby inhibiting cell proliferation and metastasis, and promoting cell apoptosis. miR-375 is enriched in BMSC exosomes and inhibits PC cell migration and invasion by reducing TFF3.

MiR-514a-3p: a novel SHP-2 regulatory miRNA that modulates human cytotrophoblast proliferation.

Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP-2), encoded by the PTPN11 gene, forms a central component of multiple signalling pathways and is required for insulin-like growth factor (IGF)-induced placental growth. Altered expression of SHP-2 is associated with aberrant placental and fetal growth indicating that drugs modulating SHP-2 expression may improve adverse pregnancy outcome associated with altered placental growth. We have previously demonstrated that placental PTPN11/SHP-2 expression is controlled by miRNAs. SHP-2 regulatory miRNAs may have therapeutic potential; however, the individual miRNA(s) that regulate SHP-2 expression in the placenta remain to be established. We performed in silico analysis of 3’UTR target prediction databases to identify libraries of Hela cells transfected with individual miRNA mimetics, enriched in potential SHP-2 regulatory miRNAs. Analysis of PTPN11 levels by quantitative (q) PCR revealed that miR-758-3p increased, while miR-514a-3p reduced PTPN11 expression. The expression of miR-514a-3p and miR-758-3p within the human placenta was confirmed by qPCR; miR-514a-3p (but not miR-758-3p) levels inversely correlated with PTPN11 expression. To assess the interaction between these miRNAs and PTPN11/SHP-2, specific mimetics were transfected into first-trimester human placental explants and then cultured for up to 4 days. Overexpression of miR-514a-3p, but not miR-758-3p, significantly reduced PTPN11 and SHP-2 expression. microRNA-ribonucleoprotein complex (miRNP)-associated mRNA assays confirmed that this interaction was direct. miR-514a-3p overexpression attenuated IGF-I-induced trophoblast proliferation (BrdU incorporation). miR-758-3p did not alter trophoblast proliferation. These data demonstrate that by modulating SHP-2 expression, miR-514a-3p is a novel regulator of IGF signalling and proliferation in the human placenta and may have therapeutic potential in pregnancies complicated by altered placental growth.

Tumor suppressive microRNA-485-5p targets PRRX1 in human skin melanoma cells, regulating epithelial-mesenchymal transitionand apoptosis.

Melanoma is one of the most aggressive skin cancers. Existing evidence has reported the aberrant expression of microRNAs (miRNAs) in melanoma, but their putative targets and underlying downstream effects remain to be further understood. Herein, we explored the suppressive role of miR-485-5p in melanoma progression. Initial bioinformatics analyses showed that the PRRX1 gene was differentially expressed in melanoma, while miR-485-5p was predicted to be a potential regulatory miRNA binding to PRRX1 mRNA. We confirmed that PRRX1 was upregulated, while miR-485-5p was downregulated in human melanoma samples compared with adjacent normal skin tissues. We then showed that PRRX1 was a target gene of miR-485-5p by dual-luciferase reporter gene assay. Moreover, a reduction in the expression of PRRX1 and downregulation of important proteins of the transforming growth factor-beta (TGFβ) signaling pathway was observed after miR-485-5p overexpression. Furthermore, miR-485-5p overexpression or PRRX1 knockdown suppressed epithelial-mesenchymal transition, cell viability, migration, and invasion, and promoted cell apoptosis in melanoma cells. Our study demonstrates the tumor-suppressive functions of miR-485-5p in the development of human melanoma, providing a potential target for therapy.

Identification of the transcription factor, AFF4, as a new target of miR-203 in CNS

MiR-203 was identified as a hub of a potential regulatory miRNA network in central nervous system. Overexpressing of miR-203 in the frontal cortex of C57BL/6J wild type mouse induced neurodegeneration by increasing the apoptotic pathway and neuron death. AFF4, a transcription factor, was identified as a new bona fida protein target of miR-203 in CNS. The miRNA:mRNA interaction of miR-203 and AFF4 was verified using Dural-luciferase assay. Down-regulated expression of AFF4 was induced by overexpressing miR-203 both in vitro and in vivo. Open field test, Y maze and Morris water maze test were conducted for the behavioral assessment of the mice with stereotactic injection of lentiviral vector overexpressing miR-203 in the hippocampus. No anxiety-like behavior or impaired cognition was noticed in these mice. Consistent with the results of the behavioral assessment, the electron micrograph and Nissl staining revealed no significant change in the synaptic density and no neuron injuries in the hippocampus of mice overexpressing miR-203, respectively. Our results indicated that instead of promoting neurodegenerative phenotype, a more profound function should be ascribed to miR-203 in regulating neuron behavioral activities and cognition. Neuron-type specific functions of miR-203 are likely to be executed via its various downstream protein interactors.

Integrated analysis of mRNA and miRNA profiles revealed the role of miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer

BackgroundBreast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated.MethodsThe miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively.ResultsThe most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3.ConclusionsIn summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers

Sequencing of Transcriptome and Small RNA Revealed the Xenobiotic Metabolism-Related Genes and Potential Regulatory miRNA in Asian Tramp Snail.

The Asian tramp snail, Bradybaena similaris (Ferusssac), is an invasive land snail species and has been a rising agricultural pest in south of China. As a pest, it also plays a role in transmission of Angiostrongylus cantonensis. However, present studies on this species are rare and the molecular information is limited. For this purpose, we sequenced the transcriptome and small RNA of B. similaris collected from citrus orchards. In total, 89,747 unigenes with an N50 size of 1287 bp and an average length of 817 bp were generated from ∼8.9 Gb transcriptome and 31 Mb clean reads were generated from ∼36 Mb small RNA library. To demonstrate the usefulness of these two datasets, we analyzed a series of genes associated with xenobiotic metabolism and core RNAi machinery. Analysis of the transcripts resulted in annotation of 126 putative genes encoding cytochrome P450 monooxygenases (CYP, 45), carboxyl/cholinesterases (CCE, 13), glutathione-S-transferases (GST, 24), and ATP-binding cassette transporters (ABC, 44). Analysis of the small RNA detected 42 miRNAs. In addition, four genes involved in small RNA pathways (miRNA, piRNA, and siRNA) were identified, and a total of 430 genes that can be targeted by miRNAs were predicted. Moreover, we found that a few miRNAs could target certain genes involved in xenobiotic metabolism. Therefore, we believe that these two datasets and the characterization of the identified/predicted genes will facilitate the molecular study of this species as well as other land snails with agricultural importance.

NR5A2 and potential regulatory miRNAs in the bovine CL during early pregnancy.

Progesterone, which is secreted from the corpus luteum, is indispensable for the establishment and maintenance of pregnancy. The orphan nuclear receptor subfamily 5 group A member 2 (NR5A2) is a regulator of murine luteinization, but neither its regulation nor its role in the fully differentiated, mature corpus luteum (CL) have been described. Therefore, the goal of this study was to profile abundance and investigate the regulation and functions of NR5A2 in the bovine CL. Treatment of cultured luteal steroidogenic cells with a pharmacological inhibitor of NR5A2 decreased progesterone production and tended to decrease abundance of HSD3B1 mRNA. Luteal NR5A2 mRNA increased and NR5A2 protein tended to increase between days 4 and 6 of the estrous cycle, coincident with increased steroidogenic capacity of the CL. Luteal NR5A2 mRNA decreased by 8 h after prostaglandin (PG) F2A injection. During early pregnancy, luteal NR5A2 mRNA was less on days 20 and 23 compared to day 14, but protein abundance did not change. Neither 1 nor 10 ng/mL interferon tau (IFNT) altered NR5A2 abundance in cultured luteal steroidogenic cells, but 10 ng/mL PGF2A decreased NR5A2. Because of discrepancies between mRNA and protein abundance of NR5A2, regulation by miRNA that changed during early pregnancy was investigated. miR-27b-3p, miR-432-5p, and miR-369-3p mimics decreased NR5A2 protein abundance and miR-369-3p also inhibited progesterone production. Overall, the results of this study show that NR5A2 may be maintained by miRNA during early pregnancy and may be an important regulator of luteal progesterone production.

MiR‑1299/NOTCH3/TUG1 feedback loop contributes to the malignant proliferation of ovarian cancer.

Recent studies have revealed the oncogenic role of notch reporter 3 (NOTCH3) in ovarian cancer (OC). However, the possible regulators and mechanisms underlying notch receptor 3 (NOTCH3)-mediated behaviors in OC remain to be completely investigated. In the present study, we aimed to identify regulators of NOTCH3 and their interactions underlying the pathogenesis of OC. Bioinformatics analysis and luciferase reporter assay were used to identify potential regulatory miRNAs and lncRNAs of NOTCH3 in OC. Several in vivo and in vitro assays were performed to evaluate their effects on the proliferative ability mediated by NOTCH3. We identified microRNA-1299 (miR-1299) as a novel negative regulator of NOTCH3. miR-1299 was downregulated in OC and was found to be considerably correlated with tumor differentiation. Upregulation of miR-1299 inhibited cell proliferation, colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation, as well as induced cell cycle arrest in the G0G1 phase in OC cells. Overexpression of miR-1299 in xenograft mouse models suppressed tumor growth in vivo. The lncRNA taurine upregulated gene 1 (TUG1), acting as a sponge of miR-1299, was found to upregulate NOTCH3 expression and promote cell proliferation in OC through the competing endogenous RNA mechanism. In addition, TUG1 was found to be a potential downstream target of NOTCH3, forming a miR-1299/NOTCH3/TUG1 feedback loop in the development of OC. Collectively, our findings improve the understanding of NOTCH3-mediated regulation in OC pathogenesis and facilitate the development of miRNA- and lncRNA-directed diagnostics and therapeutics against this disease.

MicroRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation, migration, and invasion, while promoting apoptosis of colorectal cancer cells by downregulating ITGA2.

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2.

Molecular mechanism of estrogen-mediated neuroprotection in the relief of brain ischemic injury

BackgroundThis study aimed to explore the molecular mechanism of estrogen-mediated neuroprotection in the relief of cerebral ischemic injury. The gene expression profiles were downloaded from Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using limma package in R software. Further, DEGs were subjected to Gene Ontology (GO) cluster analysis using online Gene Ontology Enrichment Analysis Software Toolkit and to GO functional enrichment analysis using DAVID software. Using the Gene Set Analysis Toolkit V2, enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways was performed. In addition, protein-protein interaction (PPI) network was constructed using STRING database, and submodule analysis of PPI network. Lastly, the significant potential target sites of microRNAs (miRNAs) were predicted using Molecular Signatures Database, and the function analysis of targets of predicted miRNA was also performed using DAVID software.ResultsIn total, 321 DEGs were screened in the estrogen-treated sample. The DEGs were mainly associated with intracellular signaling and metabolic pathways, such as calcium channel, calcineurin complex, insulin secretion, low-density lipoprotein reconstruction, and starch or sugar metabolism. In addition, GO enrichment analysis indicated an altered expression of the genes related to starch and sucrose metabolism, retinol metabolism, anti-apoptosis (eg., BDNF and ADAM17) and response to endogenous stimulus. The constructed PPI network comprised of 243 nodes and 590 interaction pairs, and four submodules were obtained from PPI network. Among the module d, four glutamate receptors as Gria4, Gria3, Grin3a and Grik4 were highlighted. Further, 5 novel potential regulatory miRNAs were also predicted. MIR-338 and MIR520D were closely associated with cell cycle, while the targets of MIR-376A and MIR-376B were only involved in cell soma.ConclusionsThe DEGs in estrogen-treated samples are closely associated with calcium channel, glutamate induced excitotoxicity and anti-apoptotic activity. In addition, some functionally significant DEGs such as BDNF, ADAM17, Gria4, Gria3, Grin3a, Grik4, Gys2 and Ugtla2, and new miRNAs like MIR-338 and MIR-376A were identified, which may serve as potential therapeutic targets for the effective treatment of cerebral ischemic injury.

The mRNA and miRNA transcriptomic landscape of Panax ginseng under the high ambient temperature

BackgroundGinseng is a popular traditional herbal medicine in north-eastern Asia. It has been used for human health for over thousands of years. With the rise in global temperature, the production of Korean ginseng (Panax ginseng C.A.Meyer) in Korea have migrated from mid to northern parts of the Korean peninsula to escape from the various higher temperature related stresses. Under the high ambient temperature, vegetative growth was accelerated, which resulted in early flowering. This precocious phase change led to yield loss. Despite of its importance as a traditional medicine, biological mechanisms of ginseng has not been well studied and even the genome sequence of ginseng is yet to be determined due to its complex genome structure. Thus, it is challenging to investigate the molecular biology mechanisms at the transcript level.ResultsTo investigate how ginseng responds to the high ambient temperature environment, we performed high throughput RNA sequencing and implemented a bioinformatics pipeline for the integrated analysis of small-RNA and mRNA-seq data without a reference genome. By performing reverse transcriptase (RT) PCR and sanger sequencing of transcripts that were assembled using our pipeline, we validated that their sequences were expressed in our samples. Furthermore, to investigate the interaction between genes and non-coding small RNAs and their regulation status under the high ambient temperature, we identified potential gene regulatory miRNAs. As a result, 100,672 contigs with significant expression level were identified and 6 known, 214 conserved and 60 potential novel miRNAs were predicted to be expressed under the high ambient temperature.ConclusionCollectively, we have found that development, flowering and temperature responsive genes were induced under high ambient temperature, whereas photosynthesis related genes were repressed. Functional miRNAs were down-regulated under the high ambient temperature. Among them are miR156 and miR396 that target flowering (SPL6/9) and growth regulating genes (GRF) respectively.

Integrative analysis of mRNA and miRNA expression profiles in oral lichen planus: preliminary results

Oral lichen planus (OLP), a chronic inflammatory disease of unknown etiology, is considered a potentially malignant oral disorder. The aim of the present study was to analyze candidate microRNAs (miRNAs) and genes from patients with OLP and healthy controls (HCs). Biopsy specimens of the oral mucosa were collected from patients with OLP (n=9) and from HCs (n=4). Differentially expressed miRNAs and differentially expressed genes were screened by using next-generation sequencing with DESeq and edgeR software algorithms. A total of 94 differentially expressed miRNAs and 599 differentially expressed genes were detected in OLP. Potential regulatory miRNAs and genes were obtained by analyzing miRNA-messenger RNA networks. Of these, 5 downregulated miRNAs-Hsa-miR-135 a-5 p (P=.33), hsa-miR-128-3 p (P=.03), hsa-miR-218-5 p (P=.01), hsa-miR-125 a-5 p (P=.01), and hsa-let-7 e-5 p (P=.04)-were the most promising biomarkers in patients with OLP compared with HCs. The identified differentially expressed genes were significantly enriched in "inflammatory" events and immune-related terms through Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis. The integrative analysis of messenger RNA and miRNA profiles provides important information to elucidate gene expression mechanisms and a comprehensive perspective to study the etiology and pathogenesis of OLP.

MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae.

BackgroundMicroRNAs (miRNAs) are critical regulators responding to acute environmental stresses in both plants and animals. By modulating gene expression, miRNAs either restore or reconstitute a new expression program to enhance cell tolerance to stresses. Cold shock is one of the stresses that can induce acute physiological responses and transcriptional changes in aquatic creatures. Previous genomic studies have revealed many cold-affected genes in fish larvae and adults, however, the role of miRNAs in acute cold response is still ambiguous. To elucidate the regulatory roles of miRNAs in the cold-inducible responses, we performed small RNA-seq and RNA-seq analyses and found potential cold regulatory miRNAs and genes. We further investigated their interactions and involvements in cold tolerance.ResultsSmall RNA-seq and RNA-seq identified 29 up-/26 down-regulated miRNAs and 908 up-/468 down-regulated genes, respectively, in responding to cold shock for 4 h at 18 °C. miRNA and transcriptomic analyses showed these miRNAs and mRNAs are involved in similar biological processes and pathways. Gene ontology enrichment analyses revealed the cold-induced genes were enriched in pathways, including melanogenesis, GnRH pathway, circadian rhythm, etc. We were particularly interested in the changes in circadian clock genes that affect daily metabolism. The enrichment of circadian clock genes was also observed in previous fish cold acclimation studies, but have not been characterized. To characterize the functional roles of circadian clock genes in cold tolerance, we individually overexpressed selected clock genes in zebrafish larvae and found one of the core clock genes per2 resulted in better recovery from cold shock. In addition, we validated the interaction of per2 with its associate miRNA, dre-mir-29b, which is also cold-inducible. It suggests the transcription of per2 can be modulated by miRNA upon cold shock.ConclusionsCollectively, our observations suggest that miRNAs are fine turners for regulating genomic plasticity against cold shock. We further showed that the fine tuning of core clock gene per2 via its associated miRNA, dre-mir-29b, can enhance the cold tolerance of zebrafish larvae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3239-4) contains supplementary material, which is available to authorized users.