Target Gene Of miR-146a Research Articles

MiR-34a suppression targets Nampt to ameliorate bone marrow mesenchymal stem cell senescence by regulating NAD+-Sirt1 pathway

BackgroundExpansion-mediated replicative senescence and age-related natural senescence have adverse effects on mesenchymal stem cell (MSC) regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that microRNA-34a (miR-34a) has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined.MethodsMiR-34a expression in MSCs was evaluated utilizing RT-qPCR. The functional effects of miR-34a exerting on MSC senescence were investigated via gene manipulation. Relevant gene and protein expression levels were analyzed by RT-qPCR and western blot. Luciferase reporter assays were applied to confirm that Nampt is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting Nampt in MSC senescence was further explored by measuring intracellular NAD+ content, NAD+/NADH ratio and Sirt1 activity.ResultsIn contrast to Nampt expression, miR-34a expression incremented in senescent MSCs. MiR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-β-galactosidase activity, and upregulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. In addition, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression.ConclusionThis study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, carrying great implications for optimal strategies for MSC therapeutic applications.

MiR-20a/TCF4 axis-mediated inhibition of hepatocytes proliferation impairs liver regeneration in mice PHx model by regulating CDC2 and CDC6.

MicroRNAs have emerged as essential regulators in the biological process of liver regeneration by modulating the post‐transcriptional expression of the target genes. In the present study, we found miR‐20a expression is decreased remarkably in three rodent liver regeneration models using miRNA PCR array and Venn diagram analysis. Inhibition of miR‐20a expression enhanced hepatocytes proliferation in vivo and in vitro. In contrast, overexpression of miR‐20a reduces hepatocytes proliferation and subsequently impaired liver regeneration in the mouse PHx model. Moreover, we have identified TCF4 as a target gene of miR‐20a using the PCR Array and luciferase assay. Next, mice with TCF4 deficiency were used to establish the PHx model and subjected to the examination of liver regeneration capacity. We found TCF4‐deficient mice exhibited impaired liver regeneration compared with control. Given that TCF4 acts as a transcription factor, we sort to elucidate the downstream genes involved in liver regeneration. Promoter analysis and Chip assay confirmed that TCF4 enhances CDC2 and CDC6 expression through binding to the promoter region and leads to the proliferation and cell cycle progression in hepatocytes. In conclusion, this study provides evidence that the miR20a‐TCF4‐CDC2/6 axis plays an essential role during liver regeneration.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

BACH1 is transcriptionally inhibited by TET1 in hepatocellular carcinoma in a microRNA-34a-dependent manner to regulate autophagy and inflammation

Hepatocellular carcinoma (HCC), one of the main contributors to cancer-associated deaths globally, is characterized by high invasiveness. Herein, we studied the molecular mechanisms underlying ten-eleven translocation 1 (TET1)-mediated autophagy in HCC. Following data mining using GSE101728, GSE14520 and GSE138178, TET1 was screened out, and the differential expression of TET1 was verified by bioinformatics analysis. TET1, one of the prognostic markers in HCC, was poorly expressed in HCC. Through functional experiments, we determined that upregulation of TET1 inhibited the proliferation, migration, invasion, tumorigenesis, metastasis and inflammatory factors of HCC cells, and promoted cell autophagy and apoptosis. Mechanistically, TET1 activated miR-34a by demethylating miR-34a. BTB domain and CNC homology 1 (BACH1) was identified as the target gene of miR-34a. Notably, Downregulation of miR-34a increased cellular inflammatory factors and decreased autophagy in the presence of TET1, while declines in BACH1 suppressed cellular inflammatory factors and enhanced autophagy in the presence of miR-34a inhibitor. BACH1 negatively regulated the p53 pathway. In conclusion, TET1 is a tumor suppressor in the progression of HCC by regulating the miR-34a/BACH1/p53 axis, and may contribute to the improvement of HCC prognosis and therapy.

Administration of simvastatin halts progression of cirrhosis via up-regulating expression of miR-34a and interleukin-10 in rats

IntroductionSimvastatin (SIM) treatment has been found to be able to reduce the expression of miR-34a, and we found that interleukin-10 (IL-10) is a potential target gene of miR-34a by searching the online microRNA (miRNA) database. Furthermore, it has been shown that IL10 up-regulation could halt the progression of cirrhosis. The objective of this study was to explore the underlying mechanism of Simvastatin/miR-34a/IL-10 involved in HBV associated cirrhosis.Material and methodsReal-time PCR, western-blot analysis, immunohistochemistry, computational analysis, luciferase assay was carried out to explore the underlying mechanism of miR-34a involved in HBV associated cirrhosis.ResultsSIM treatment dose-dependently decreased the levels of miR-34a while increasing the levels of IL-10 mRNA and protein. Levels of IL-10 mRNA and protein were remarkably decreased, while miR-34a mRNA level and active caspase-3 protein level was apparently increased in Cirrhosis group compared with sham group. Accordingly, SIM treatment obstructed the dysregulated miR-34a expression and IL-10 expression in cirrhosis animals. By performing computational analysis, we identified that a complementary binding site of miR-34a was located in IL-10 3’ untranslated region (3’UTR), and miR-34a reduced luciferase activity of wild-type IL-10 3’UTR.ConclusionsOur data also suggested that SIM may become a new therapeutic strategy for HBV-associated cirrhosis via targeting the miR-34a/IL-10 axis.

Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.

The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPβ) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression.

Extracellular vesicle-encapsulated microRNA-23a from dorsal root ganglia neurons binds to A20 and promotes inflammatory macrophage polarization following peripheral nerve injury.

Extracellular vesicles (EVs) are capable of transferring microRNAs (miRNAs or miRs) between two different types of cells and also serve as vehicles for delivery of therapeutic molecules. After peripheral nerve injury, abnormal expression patterns of miRNAs have been observed in dorsal root ganglia (DRG) sensory neurons. We hypothesized that sensory neurons secrete miRs-containing EVs to communicate with macrophages. We demonstrated that miR-23a was upregulated in DRG neurons in spared nerve injury (SNI) mouse models. We also found that miR-23a was enriched in EVs released by cultured DRG neurons following capsaicin treatment. miR-23a-containing EVs were taken up into macrophages in which increased intracellular miR-23a promoted pro-inflammatory phenotype. A20 was verified as a target gene of miR-23a. Moreover, intrathecal delivery of EVs-miR-23a antagomir attenuated neuropathic hypersensitivity and reduced the number of M1 macrophages in injured DRGs by targeting A20. In conclusion, these results demonstrate that sensory neurons transfer EVs-encapsulated miR-23a to activate M1 macrophages and enhance neuropathic pain following the peripheral nerve injury. The study highlighted a new therapeutic approach to alleviate chronic neuropathic pain after nerve trauma by targeting detrimental miRNA in sensory neurons.

MiR‐34a in extracellular vesicles from bone marrow mesenchymal stem cells reduces rheumatoid arthritis inflammation via the cyclin I/ATM/ATR/p53 axis

Extracellular vesicles (Evs) participate in the development of rheumatoid arthritis (RA), but the mechanisms remain unclear. This study aimed to determine the mechanism by which microRNA‐34a (miR‐34a) contained in bone marrow mesenchymal stem cell (BM‐MSC)‐derived Evs functions in RA fibroblast‐like synoviocytes (RA‐FLSs). BM‐MSC‐derived Evs and an Evs inhibitor were extracted. A rat model of RA was established. miR‐34a gain‐ and loss‐of‐function experiments were performed, and the inflammation in rat synovial fluid and tissues was detected. The role of miR‐34a in RA‐FLSs was also measured in vitro. The target gene of miR‐34a was predicted using the online software TargetScan and identified using a dual‐luciferase reporter gene assay, and the activation of the ATM/ATR/p53 signalling pathway was assessed. BM‐MSC‐derived Evs mainly elevated miR‐34a expression, which reduced RA inflammation in vivo and inhibited RA‐FLS proliferation and resistance to apoptosis in vitro, while inhibited miR‐34a expression enhanced RA development. In addition, miR‐34a could target cyclin I to activate the ATM/ATR/p53 signalling pathway, thus inhibiting abnormal RA‐FLS growth and RA inflammation. Our study showed that miR‐34a contained in BM‐MSC‐derived Evs could reduce RA inflammation by inhibiting the cyclin I/ATM/ATR/p53 signalling pathway.

Apoptotic effects of valproic acid on miR-34a, miR-520h and HDAC1 gene in breast cancer

Identifying miRNAs involved in cancer and devising strategies to control their expression is a new therapeutic approach. Valproic acid (VPA) has attracted a lot of interest in cancer research. We evaluated the impact of VPA on the expression of miR-34a, miR-520h, and their target gene histone deacetylase 1 (HDAC1), as well as their relationship with apoptosis in breast cancer. First, through bioinformatics analyses, the possible target genes of miR-34a and miR-520h and their roles in apoptosis regulation were investigated. Then, miR-34a, miR-520h, and HDAC1 gene expression in tissues of breast cancer patients were determined using the qRT-PCR method. The anticancer impact of VPA on apoptosis and the expression levels of miR-34a, miR-520h, and HDAC1 gene were measured in MCF-7 and MDA-MB-231 cell lines. The bioinformatics analyses indicated that miR-34a and miR-520h might make a unique contribution in regulating the apoptosis pathway. The relative expression of miR-34a and miR-520h significantly decreased in cancer tissues, while the relative expression of HDAC1 increased. In the in vitro study, VPA led to apoptosis induction and increased lipid peroxidation products in breast cancer cells. Moreover, VPA increased the expression of miR- 34a and miR-520h and decreased HDAC1 expression in MCF-7 cells. In MDA-MB-231 cells, VPA decreased the expression of these miRNAs and increased the expression of HDAC1. It can be concluded that miR-34a and miR-520h are implicated in the apoptosis pathways, and thus, VPA can recruit as a possible option in breast cancer research due to its interference with epigenetic processes.

TUG1 promotes the expression of IFITM3 in hepatocellular carcinoma by competitively binding to miR-29a.

Purpose: Numerous studies have demonstrated the important relationship of TUG1 with tumorigenesis. The present study investigated the role of TUG1 and its downstream genes miR-29a and IFITM3 in the occurrence and development of hepatocellular carcinoma (HCC). We found that both TUG1 and IFITM3 genes are highly expressed in HCC, whereas the expression of miR-29a is low in HCC. Downregulation of TUG1 reduces cell invasion, metastasis, and cell proliferation ability and promotes cell apoptosis. Simultaneous downregulation of miR-29a reverses this effect. Moreover, IFITM3, as the target gene of miR-29a, is positively regulated by TUG1. However, the adjustment relationship between these three components is still unknown and thus warrants further investigation. The objective of this study was to investigate the regulatory relationship between TUG1, miR-29a, and IFITM3 in human liver cancer.Patients and methods: The expression of TUG1 and miR-29a in tumor tissues and adjacent non-tumor tissues of 65 patients with HCC was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The migration and invasion of liver cancer cells were studied by the wound healing assay and the Transwell method, respectively. The apoptosis rate of HCC cells was detected by flow cytometry, and the proliferation rate of hepatoma cells was detected by the 5-ethynyl-2'-deoxyuridine (EdU) method. Immunofluorescence was used to detect the expression of TUG1 and IFITM3 in HCC-LM3 and HL-7702 cell lines. The relationship between TUG1 and miR-29a was detected using a double luciferase reporter assay and fluorescence in situ hybridization (FISH). Tumors were established in vivo by subcutaneous injection of HCC cells into nude mice and injection of these cells into the tail vein. Western blotting was used to quantify the biomarkers.Results: The expression of TUG1 increased significantly in tumor tissues and HCC cells. Moreover, the expression of miR-29a in liver cancer tissues was significantly lower than that in normal human liver tissues. The expression of TUG1 in liver cancer tissue was negatively correlated with miR-29a. Knockdown of TUG1 weakened the invasion, migration, and proliferation of HCC cells, and enhanced their apoptosis. A simultaneous knockdown of miR-29a enhanced cell invasion, metastasis, and cell proliferation, whereas the apoptosis ability decreased. As a target gene of miR-29a, IFITM3 is not only negatively regulated by miR-29a, but also positively regulated by TUG1. Therefore, TUG1 regulates IFITM3 in HCC cells by competitively binding to miR-29a, thus affecting cell invasion, migration, proliferation, and apoptosis.Conclusion: As a CeRNA, TUG1 competitively binds to miR-29a to regulate IFITM3 and promote the development of liver cancer. Downregulation of TUG1 can significantly inhibit the migration, invasion, and proliferation of liver cancer cells. Based on these results, we conclude that TUG1 could serve as a key gene to improve the prognosis of patients with HCC.

MicroRNA-20a Targeting LASS2 Promotes the Proliferation, Invasiveness and Migration of Bladder Cancer.

Abnormal expression of miR 20a is reported in various types of malignancy neoplasms. However, its function is not consistent in different tumors. This study aims to explore the potential functions of miR 20a and its underlying mechanisms in bladder cancer. Ninety-six patients diagnosed with bladder cancer were recruited into the study. The expression levels of miR-20a in bladder cancer samples and adjacent non-tumor samples were investigated by qRT-PCR. Wound healing, CCK8, and transwell migration assays were carried out for determining the functions of miR20a. Bioinformatics analysis was used for predicting the downstream gene of miR-20a. Western blot, qRT-PCR, and fluorescent reporter assays were used to verify the target gene. MiR-20a was significantly increased in bladder cancer tissues, and its rising level was closely correlated with histological grade, clinical stage, recurrence and metastasis in bladder cancer. Exogenous upregulation of miR-20a expression obviously enhanced the aggressive biological functions of bladder cancer in vitro. LASS2 was verified to be a target gene of miR-20a. Moreover, miR-20a can negatively regulate LASS2 at protein and mRNA levels. Increasing miR-20a is closely related to aggressive clinicopathological features. MiR 20a plays an oncogenic role in bladder cancer, which contributes to target LASS2 directly.

MiR-20a attenuates acute lung injury in septic rats via targeting TLR4

Background: Sepsis is most likely to cause lung damage in patients, and the detection rate and mortality rate are high. Here, we investigated the expression of miR-20a in sepsis-induced acute lung injury (ALI) rats and its effect on inflammatory response, and reveal its possible molecular mechanism. Method: The model of acute lung injury caused by sepsis in rats was established by cecal ligation and puncture. The expression of miR-20a in lung tissue was determined by RT-qPCR. Acute lung injury rats were injected with 5 nmol miR-20a agomir or agomir NC every day for 3 days. Rats were sacrificed by arterial bleeding and lung tissues were removed. Serum interleukin (IL) -1β, IL-6, and tumor necrosis factor alpha (TNF-α) were detected by ELISA. HE staining was used to observe the pathology of lung tissue and calculate the pathological score of lung injury. Western blot to determine the level of TLR4 and nuclear transcription factor κB p65 (NF-κB p65) protein in lung tissue. The luciferase reporter assay was used to verify the binding effect of miR-20a on the 3 non-coding TLR4. Results: We found that compared with that in Normal group, the expression of miR-20a in lung tissues of rats with ALI was decreased (p < 0.05). In miR-20a agomir group, the plasma level of IL-1β, IL-6, and TNF-α was significantly lower than that in agomir NC group and ALI group (p < 0.05), while higher than those in Normal group (p < 0.05). The HE staining results showed that the pathological score of lung injury in rats in miR-20a agomir group was lower than that of agomir NC group and ALI group (p < 0.05). Compared with agomir NC group and ALI group, the expression of TLR4 and NF-κB p65 in miR-20a agomir group was decreased (p < 0.01). The luciferase reporting experiment confirmed that TLR4 was a target gene of miR-20a. Conclusion: To sum up, miR-20a exerts a protective effect on sepsis-induced ALI rats through its anti-inflammatory effect. The targeting of TLR4 by miR-20a may be an effective method to reduce the inflammatory response in sepsis-induced ALI.

Long non-coding RNA small nucleolar RNA host gene 1 alleviates the progression of epilepsy by regulating the miR-181a/BCL-2 axis in vitro

PurposeLong non-coding RNAs (lncRNAs) have been reported to be involved in regulating epilepsy. The purpose of this study is to investigate the possibly regulatory mechanism of small nucleolar RNA host gene 1 (SNHG1) on epilepsy. MethodsQuantitative real-time PCR was utilized to detect the expression of SNHG1, microRNA (miR)-181a, and B-cell lymphoma-2 (BCL-2). Through an enzyme-linked immunosorbent assay, the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cyclooxygenase-2 (COX-2) were determined. The viability and apoptosis of CTX-TNA2 cells were measured using MTT assay and flow cytometry analysis, respectively. Western blot assay was performed to analyze the protein levels of Bcl-2, BCL2-associated X, and Caspase-3. The relationships between miR-181a and SNHG1/BCL-2 were confirmed by the dual-luciferase reporter assay. ResultsSNHG1 expression was down-regulated in EP tissues and kainic acid (KA)-induced CTX-TNA2 cells. The apoptosis and release of inflammatory factors (TNF-α, IL-1β, IL-6, and COX-2) in KA-induced CTX-TNA2 cells were suppressed by SNHG1 overexpression and promoted by miR-181a up-regulation. In addition, we confirmed that SNHG1 targeted miR-181a, whereas BCL-2 was a target gene of miR-181a. Negative correlations between SNHG1 and miR-181a, as well as miR-181a and BCL-2 were exhibited. Both the up-regulation of miR-181a and down-regulation of BCL-2 reversed the inhibiting effects of SNHG1 on apoptosis and inflammatory response of KA-induced CTX-TNA2 cells, and the promoting effect upon cell viability. ConclusionsSNHG1 alleviated the progression of EP by modulating the miR-181a/BCL-2 axis in vitro, thus SNHG1 could act as a possible therapeutic target for treating EP.

MiR-146a functions as a potential tumor suppressor in retinoblastoma by negatively regulate neuro-oncological ventral antigen-1.

MicroRNAs (miRNAs) are dysregulated in many tumors and have been found to play crucial roles in cancer biology. Retinoblastoma is a rare tumor that develops rapidly from a malignant tumor of immature cells in the retina known as photoreceptor progenitors. Our study aimed to explore the role of miR-146a in the pathology of retinoblastoma. Potential target gene of miR-146a was predicted by Targetscan. Reverse transcription quantitative polymerase chain reaction (RT-PCR) showed that miR-146a was downregulated and ventral nerve tumor antigen 1 (Neuro - oncological ventral antigen 1, NOVA1) was upregulated in retinoblastoma. Luciferase assay confirmed that miR-146a directly target NOVA1. MiR-146a knockdown and overexpression experiments were performed and found that miR-146a could regulate the expression of NOVA1. The miR-146a knockdown and overexpression experiments were conducted to investigate the biological function of miR-146a. MiR-146a was found inhibited the viability, proliferation and invasion of retinoblastoma cell by MTT, EdU, and transwell assays. Flow cytometry was performed for the apoptosis analysis and miR-146a increased the apoptosis of retinoblastoma cell was found. Above phenomenon can be rescued by overexpression of NOVA1. In conclusion, these results suggest that miR-146a acts as a tumor suppressor and can act as a potential therapeutic target for retinoblastoma in the future.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

BackgroundEsophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored.MethodsWe divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN−) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN− cells.ResultsCell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients.ConclusionsThis is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells. Methods: The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3' terminal untranslated region (3' UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively. Results: The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) (P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group (P<0.01) and (1.70±0.20)% of blank control group (P<0.01). The percentage of cells in G(1) phase [(44.74±6.76)%] was decreased while the G(2)/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group (P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups (P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group (P<0.01). Conclusions: The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G(2)/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.

The Mediation of miR-34a/miR-449c for Immune Cytokines in Acute Cold/Heat-Stressed Broiler Chicken.

Simple SummaryIn the intensive and scale poultry industry, the level of heat stress (HS) directly affects the growth, development, and production performance of poultry. To alleviate the adverse effects of stress in broilers, microRNA (miRNA) was regarded as a potential regulator of immune cytokines. In this study, through the sequencing analysis of spleens after cold/heat stress, we found that 33 and 37 miRNA were differentially expressed in the heat stress group compared with the normal (NS) group and cold stress (CS) group, respectively. The differential miRNA were mainly involved in biological processes such as the cytokine–cytokine receptor interaction. To further understand the miRNA-mediated effect of heat stress on the immune level of chickens, we selected miR-34a and miR-449c as the research objects, predicted and verified that interleukin 2 (IL-2) and interleukin 12α (IL-12α) were the target genes of miR-34a and miR-449c. Coupled with the analysis of the expression of other cytokines, we found that miRNA could change the expression of immune cytokines directly or indirectly. This discovery provides a new insight into the mediation of miRNA for immune cytokines in acute cold/heat stressed broiler chicken.An increasing amount of evidence has revealed that microRNAs (miRNAs) participated in immune regulation and reaction to acute cold and heat stresses. As a new type of post-transcriptional regulatory factor, miRNA has received widespread attention; However, the specific mechanism used for this regulation still needs to be determined. In this study, thirty broilers at the same growth period were divided into three groups and treated with different temperature and humidity of CS (10–15 °C and 90% Relative Humidity (RH)), HS (39 °C and 90% RH), and NS (26 °C and 50–60% RH) respectively. After 6 h, splenic tissues were collected from all study groups. miRNA sequencing was performed to identify the differentially expressed miRNAs (DEMs) between HS, CS, and NS. We found 33, 37, and 7 DEMs in the HS-NS, HS-CS, CS-NS group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEMs were significantly enriched in cytokine–cytokine receptor interaction and functioned as the cellular responders to stress. We chose two miRNA, miR-34a and miR-449c, from the same family and differential expressed in HS-CS and HS-NS group, as the research objects to predict and verify the target genes. The dual-luciferase reporter assay and quantitative real-time PCR (qRT-PCR) confirmed that two cytokines, IL-2 and IL-12α, were the direct target genes of miR-34a and miR-449c. To further understand the mediation mechanism of miRNAs in acute cold/heat-stressed broiler chicken, a splenic cytokines profile was constructed. The results showed that IL-1β was strongly related to acute heat stress in broiler chicken, and from this we predicted that the increased expression of IL-1β might promote the expression of miR-34a, inducing the upregulation of interferon-γ (INF-γ) and IL-17. Our finds have laid a theoretical foundation for the breeding of poultry resistance and alleviation of the adverse effects of stress.

MiR-146a regulates insulin sensitivity via NPR3

The pathogenesis of obesity-related metabolic diseases has been linked to the inflammation of white adipose tissue (WAT), but the molecular interconnections are still not fully understood. MiR-146a controls inflammatory processes by suppressing pro-inflammatory signaling pathways. The aim of this study was to characterize the role of miR-146a in obesity and insulin resistance. MiR-146a−/− mice were subjected to a high-fat diet followed by metabolic tests and WAT transcriptomics. Gain- and loss-of-function studies were performed using human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes. Compared to controls, miR-146a−/− mice gained significantly more body weight on a high-fat diet with increased fat mass and adipocyte hypertrophy. This was accompanied by exacerbated liver steatosis, insulin resistance, and glucose intolerance. Likewise, adipocytes transfected with an inhibitor of miR-146a displayed a decrease in insulin-stimulated glucose uptake, while transfecting miR-146a mimics caused the opposite effect. Natriuretic peptide receptor 3 (NPR3) was identified as a direct target gene of miR-146a in adipocytes and CRISPR/Cas9-mediated knockout of NPR3 increased insulin-stimulated glucose uptake and enhanced de novo lipogenesis. In summary, miR-146a regulates systemic and adipocyte insulin sensitivity via downregulation of NPR3.

Hypoxia-induced miR-92a regulates p53 signaling pathway and apoptosis by targeting calcium-sensing receptor in genetically improved farmed tilapia (Oreochromis niloticus).

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed that CaSR (encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated and CaSR was down-regulated in a time-dependent manner. Knocked-down CaSR expression led to inhibited expression of p53, TP53INP1, and caspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation of CaSR, p53, TP53INP1, and caspase-3/8 genes in the liver, resulting in lower serum aspartate aminotransferase and alanine aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targeting CaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.

MicroRNA-146a controls age-related bone loss.

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA‐146a (miR‐146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR‐146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR‐146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR‐146a‐deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR‐146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR‐146a‐deficient mice are protected from ovariectomy‐induced bone loss. In humans, the levels of miR‐146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR‐146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR‐146a might be a powerful therapeutic target to prevent age‐related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.