MiR-146a-5p Plays an Oncogenic Role in NSCLC via Suppression of TRAF6.
ABSTRACT: Non-small cell lung cancer (NSCLC) is the most deadly cancer in the world due to its often delayed diagnosis. Identification of biomarkers with high sensitivity, specificity, and accessibility for early detection, such as circulating microRNAs, is therefore of utmost importance. In the present study, we identified a significantly higher expression of miR-146a-5p in the serum and tissue samples of NSCLC patients than that of the healthy controls. In parallel, miR-146a-5p was also highly expressed in three human NSCLC adenocarcinoma-cell lines (A549, H1299, and H1975) compared to the human bronchial epithelium cell line (HBE). By dual-luciferase reporter assay and manipulation of the expressions of miR-146a-5p and its target gene, tumor necrosis factor receptor-associated factor 6 (TRAF6), we showed that the functional effects of miR-146a-5p on NSCLC cell survival and migration were mediated by direct binding to and suppression of TRAF6. Overexpression of TRAF6 sufficiently reversed miR-146a-5p-induced cancer cell proliferation, migration, and apoptosis resistance. Our data implied that miR-146a-5p/TRAF6/NF-?B-p65 axis could be a promising diagnostic marker and a therapeutic target for NSCLC.
Project description:Background: Dysregulated microRNA (miRNA) expression in cancer can act as a key factor that modifies biological processes, including chemoresistance. Our study aimed to identify the miRNAs associated with gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC) and to explore the potential mechanisms. Methods: The miRNA microarray was used to identify miRNAs associated with GEM resistance. Quantitative real-time PCR was used to examine miR-146a-5p expression in paired PDAC and adjacent normal tissues. Bioinformatics analysis, luciferase reporter assays, and chromatin immunoprecipitation assays were used to confirm tumor necrosis factor receptor-associated factor 6 (TRAF6) as a direct target of miR-146a-5p and to explore the potential transcription factor binding and regulation by miR-146a-5p. In vitro and in vivo experiments were performed to investigate the mechanisms. Results: MiR-146a-5p expression was significantly decreased in PDAC tissues compared with adjacent normal tissues, and miR-146a-5p expression correlated with prognosis in PDAC patients. Functional studies indicated that miR-146a-5p suppressed PDAC cell proliferation and sensitized PDAC cells to GEM chemotherapy by targeting the 3'-untranslated region (3'-UTR) of TRAF6. MiR-146a-5p was also observed to downregulate the TRAF6/NF-?B p65/P-gp axis, which regulates PDAC cell growth and chemoresistance. Conclusions: Taken together, the results indicate that the miR-146a-5p/TRAF6/NF-?B p65 axis drives pancreatic chemoresistance by regulating P-gp, suggesting that miR-146a-5p may be utilized as a new therapeutic target and prognostic marker in PDAC patients.
Project description:Intervertebral disc degeneration (IDD) is associated with the deterioration of nucleus pulposus (NP) cells due to hypertrophic differentiation and calcification. Emerging studies have shown that long noncoding RNAs (lncRNAs) play critical roles in the development of IDD. Using bioinformatics prediction, we hereby sought to identify the lncRNAs that regulate the expression of microRNA-146a-5p (miR-146a-5p), an IDD-related inflammatory factor. Our study demonstrated that lncRNA HCG18 acted as an endogenous sponge to down-regulate miR-146a-5p expression in the NP cells by directly binding to miR-146a-5p. In addition, HCG18 expression was up-regulated in the patients with IDD, bulging or herniated discs, and its level was positively correlated with the disc degeneration grade. In vitro, miR-146a-5p up-regulation HCG18 retarded the growth of NP cells by decreasing S phase of cell cycle, inducing cell apoptosis, recruitment of macrophages and hypercalcification. Conversely, down-regulation of miR-146a-5p exerted opposite effects. Furthermore, we elucidated that TRAF6, a target gene by miR-146a-5p, was modulated by HCG18 expression. Restore of TRAF6 expression by virus infection reserved the effect of HCG18 on the NP cells. Altogether, our data indicated that HCG18 suppressed the growth of NP cells and promoted the IDD development via the miR-146a-5p/TRAF6/NF?B axis.
Project description:Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.
Project description:BACKGROUND Lupus nephritis (LN) is a major complication of systemic lupus erythematosus (SLE). This study tested miR-146a and its target gene TRAF6 expression in LN patients and discussed their relationship with LN. MATERIAL AND METHODS One hundred twenty-eight LN patients and 30 healthy controls were enrolled in this study. MiR-146a and TRAF6 expression in peripheral blood mononuclear cells (PBMCs) were detected. Serum cytokines content was determined by ELISA. The diagnostic role of miR-146a and TRAF6 in LN activity was evaluated by ROC curve. The impact of miR-146a and TRAF6 on end-stage renal disease (ESRD) was compared by survival curve. The effect of miR-146a and TRAF6 on LN recurrence was analyzed. RESULTS Compared with healthy controls, miR-146a expression was significantly reduced and TRAF6 was upregulated in LN patients. The expression was related to LN activity. MiR-146a expression was negatively correlated, whereas TRAF6 was positively correlated with serum IL-1?, IL-6, IL-8, and TNF-? activity. The area under the ROC curve (AUC) of miR-146a and TRAF6 on the diagnosis of LN was 0.821 and 0.897, respectively. The AUC of miR-146a and TRAF6 on LN activity differentiation was 0.921 and 0.872, respectively. Downregulation of miR-146a and upregulation of TRAF6 increased the incidence of ESRD progression. Downregulation of miR-146a and upregulation of TRAF6 elevated the possibility of recurrence within one year. CONCLUSIONS MiR-146a declined, while TRAF6 increased in LN patients compared with healthy controls. Their expression can be used to effectively differentiate LN and evaluate activity. MiR-146a reduction and TRAF6 upregulation increased the possibility of ESRD progress and recurrence within one year.
Project description:Lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway is demonstrated to be involved in the hepatic fibrosis. MicroRNA (miR)-146a-5p is a key regulator of the innate immune response. The functional significance of miR-146a-5p during the LPS/TLR4 mediated hepatic fibrosis process remains unclear. In this study, we found that TLR4 and ?-smooth muscle actin (?-SMA) were up-regulated and miR-146a-5p was down-regulated in human hepatic stellate cell (HSC) line LX2 after LPS stimulation. Overexpression of miR-146a-5p inhibited LPS induced pro-inflammatory cytokines secretion through down-regulating the expression levels of TLR-4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor associated factor-6 (TRAF6) and phosphorylation of nuclear factor-kappa B (NF-?B). Knockdown of IRAK1 and TRAF6 also suppressed pro-inflammatory cytokine production by inhibiting NF-?B phosphorylation. In addition, miR-146a-5p mimic blocked LPS induced TRAF6 dependent c-Jun N-terminal kinase (JNK) and Smad2 activation as well as ?-SMA production. Taken together, these results suggest that miR-146a-5p suppresses pro-inflammatory cytokine secretion and cell activation of HSC through inhibition of TLR4/NF-?B and TLR4/TRAF6/JNK pathway.
Project description:Elevated toll-like receptor 4 (TLR4) expression is associated with a high risk of radiation-induced liver disease (RILD). MicroRNA (miR)-146a-5p is a key regulator of lipopolysaccharide (LPS)/TLR4 signaling, but its role in modulation of RILD remains unclear. Here, we found that irradiation and LPS stimulation induced TLR4 and miR-146a-5p expression in the human hepatic stellate cell (HSC) line LX2. Ectopic expression of miR-146a-5p in LX2 inhibited irradiation-induced and LPS-induced pro-inflammatory cytokine secretion and cell proliferation, and promoted cell apoptosis by down-regulating the expression levels of TLR4, interleukin-1 receptor associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6) and phosphorylation of nuclear factor-kappa B. In addition, the culture medium from the irradiated and LPS-stimulated HSCs transfected with miR-146a-5p significantly attenuated apoptosis in irradiated hepatocytes. Overexpression of miR-146a-5p reduced ?-smooth muscle actin production in irradiated and LPS-stimulated LX2 cells, which was associated with inhibition of TRAF6-mediated JNK and Smad2 phosphorylation. Knockdown of TRAF6 or IRAK1 mimicked the effects of miR-146a-5p on HSC function. Furthermore, miR-146a-5p treatment alleviated irradiation-induced and endotoxin-induced hepatic inflammatory response and fibrogenesis in mice through inhibition of the TLR4 signaling pathway. Collectively, this study reveals the anti-pro-inflammatory and anti-fibrotic effects of miR-146a-5p on liver injury, and suggests a potential application of miR-146a-5p in the therapeutic prevention of RILD.
Project description:Non-small cell lung cancer (NSCLC) is a complex disease in need of new methods of therapeutic intervention. Recent interest has focused on using microRNAs (miRNAs) as a novel treatment method for various cancers. miRNAs negatively regulate gene expression post-transcriptionally, and have become attractive candidates for cancer treatment because they often simultaneously target multiple genes of similar biological function. One such miRNA is miR-146a-5p, which has been described as a tumor suppressive miRNA in NSCLC cell lines and tissues. In this study, we performed RNA-Sequencing (RNA-Seq) analysis following transfection of synthetic miR-146a-5p in an NSCLC cell line, A549, and validated our data with Gene Ontology and qRT-PCR analysis of known miR-146a-5p target genes. Our transcriptomic data revealed that miR-146a-5p exerts its tumor suppressive function beyond previously reported targeting of EGFR and NF-?B signaling. miR-146a-5p mimic transfection downregulated arachidonic acid metabolism genes, the RNA-binding protein HuR, and many HuR-stabilized pro-cancer mRNAs, including TGF-?, HIF-1?, and various cyclins. miR-146a-5p transfection also reduced expression and cellular release of the chemokine CCL2, and this effect was mediated through the 3' untranslated region of its mRNA. Taken together, our work reveals that miR-146a-5p functions as a tumor suppressor in NSCLC by controlling various metabolic and signaling pathways through direct and indirect mechanisms.
Project description:BACKGROUND:microRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. METHODS:The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. RESULTS:We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. CONCLUSIONS:miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.
Project description:A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-?B P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.
Project description:Epidermal growth factor receptor (EGFR), a cancer-driven gene, plays an important role in tumorigenesis of lung cancer. Cryptotanshinone (CT) is the main constituent of salia miltiorrhiza and has been found to affect tumor progression. However, the mechanism of CT on lung cancer is still not clear. Here we found that CT could suppress the proliferation of non-small cell lung cancer (NSCLC) by inhibiting EGFR. We further confirmed that knockdown of EGFR also suppressed cell proliferation and arrested cell cycle progression. Furthermore, we evaluated EGFR was a direct target gene of miR-146a-5p which was upregulated by CT. In general, our results proved that CT could restrain NSCLC via miR-146a-5p/EGFR axis. CT and miR-146a-5p have the potential to be positive candidates in drug development of NSCLC.