Brain-derived neurotrophic factor promotes airway smooth muscle cell proliferation in asthma through regulation of transient receptor potential channel-mediated autophagy.

OBJECTIVE: Increased proliferation of airway smooth muscle cells (ASMCs) is a key feature of airway remodeling in asthma. This study aims to determine whether brain-derived neurotrophic factor (BDNF) regulates ASMC proliferation and airway remodeling via the transient receptor potential channels (TRPCs)/autophagy axis. METHODS: Human ASMCs were isolated and passively sensitized with human asthmatic serum. Protein levels of BDNF and its receptor TrkB, TRPC1/3/6, autophagy markers, intracellular Ca2+ concentration ([Ca2+]i), LC3 immunofluorescence, cell proliferation, cell cycle population were examined. Wistar rats were sensitized with OVA to establish asthma models. RESULTS: In asthmatic serum-sensitized human ASMCs, BDNF overexpression or recombinant BDNF (rhBDNF) increased TrkB/TRPC1/3/6 axis, [Ca2+]i, autophagy level, cell proliferation, cell number in the S+G2/M phase and decreased cell number in the G0/G1 phase, whereas BDNF knockdown exerted the opposite effects. Furthermore, TRPC channel blocker SKF96365 and TRPC1/3/6 knockdown reversed the effects of the rhBDNF-mediated induction of [Ca2+]i, autophagy level, cell proliferation and cell number in the S+G2/M phase. Moreover, the autophagy inhibitor (3-MA) rescued the rhBDNF-mediated induction of cell proliferation and cell number in the S+G2/M phase. Further in vivo assays revealed that BDNF altered the pathology of airway remodeling, promoted the infiltration of inflammatory cells, promoted the proliferation of ASMCs, and upregulated the protein levels of TrkB, TRPC1/3/6, and autophagy markers in asthma model rats. CONCLUSION: We conclude that BDNF promotes ASMCs proliferation in asthma through TRPC-mediated autophagy induction.

Amygdalin alleviated TGF-ß-induced epithelial-mesenchymal transition in bronchial epithelial cells.

OBJECTIVE: Transforming growth factor-beta TGF-ß-induced epithelial-mesenchymal transition (EMT) in bronchial epithelial cells contributes to airway wall remodeling in asthma. This study aims to explore the role of amygdalin, an active ingredient in bitter almonds, in TGF-ß-induced EMT in bronchial epithelial cells and to elucidate the possible mechanisms underlying its biological effects. METHODS: An asthmatic mouse model was established through ovalbumin induction. Primary mouse bronchial epithelial cells and a human bronchial epithelial cell line were incubated with transforming growth factor-beta (TGF-ß) to induce EMT, whose phenotype of cells was evaluated by the expressions of EMT markers [alpha-smooth muscle actin (α-SMA), vimentin, and fibronectin] and cell migration capacity. A co-immunoprecipitation assay was performed to assess the ubiquitination of heparanase (HPSE). RESULTS: In asthmatic model mice, amygdalin treatment relieved airway wall remodeling and decreased expressions of EMT markers (α-SMA and vimentin). In TGF-ß-treated bronchial epithelial cells, amygdalin treatment decreased the mRNA and protein levels of EMT markers (α-SMA, vimentin, and fibronectin) without impairing cell viability. Through the Swiss Target Prediction database, HPSE was screened as a candidate downstream target for amygdalin. HPSE overexpression further promoted TGF-ß-induced EMT while the HPSE inhibitor suppressed TGF-ß-induced EMT in bronchial epithelial cells. In addition, HPSE overexpression reversed the inhibitory effect of amygdalin on TGF-ß-induced EMT in bronchial epithelial cells. The following mechanism exploration revealed that amygdalin downregulated HPSE expression by enhancing ubiquitination. CONCLUSION: Our study showed that amygdalin inhibited TGF-ß-induced EMT in bronchial epithelial cells and found that the anti-EMT activity of amygdalin might be related to its regulatory effect on HPSE expression.

SRSF1 promotes ASMC proliferation in asthma by competitively binding CCND2 with miRNA-135a.

BACKGROUND: Asthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. Abnormal proliferation of airway smooth muscle cells (ASMCs) is the main pathological feature of asthma. This study investigated the function and mechanism of serine arginine-rich splicing factor 1 (SRSF1) in ASMC proliferation in asthma. METHODS: SRSF1 expressions in the bronchi of ovalbumin-induced asthmatic mice and IgE-treated mouse ASMCs (mASMCs) were evaluated using quantitative real-time PCR and Western blot. The localization and expression of SRSF1 in the bronchi of asthmatic mice were assessed by immunohistochemistry. Functionally, gain- and loss-of-function assays, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted. Mechanistically, RNA degradation assay, RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays were carried out. RESULTS: SRSF1 was highly expressed in the bronchi of ovalbumin-induced asthma mice and IgE-treated mASMCs and was mainly located in the nucleus. Experiments on the function of SRSF1 showed that the silencing of SRSF1 induced the cell cycle of mASMC arrest and restrained mASMC proliferation. Investigations into the mechanism of SRSF1 revealed that SRSF1 and miR-135a are competitively bound to the 3'UTR region of Cyclin D2 (CCND2). SRSF1 overexpression repressed the degradation of CCND2 mRNA, and miR-135a negatively regulated CCND2 expression. Furthermore, SRSF1 knockdown inhibited ASMC proliferation in asthma mouse models by regulating the levels of miR-135a and CCND2. CONCLUSION: SRSF1 knockdown repressed ASMC proliferation in asthma by regulating miR-135a/CCND2 levels.

Exosomal transfer of activated neutrophil-derived lncRNA CRNDE promotes proliferation and migration of airway smooth muscle cells in asthma.

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs), thereby aggravating the airway wall remodeling during asthma; however, the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS + si-CRNDE (a siRNA targets long non-coding RNA CRNDE), respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO, and cell viability, proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE), inhibitor of nuclear factor kappa B kinase subunit beta (IKKß) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically, CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKKß phosphorylation, thereby activating the nuclear factor kappa B (NF-κB) pathway. Functionally, silencing CRNDE in LPS-EXO, an IKKß inhibitor, and an NF-κB inhibitor all removed the upregulation of cell viability, proliferation and migration induced by LPS-EXO in ASMCs. In the end, the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-κB pathway by enhancing IKKß phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma.

LincRNA-p21 promotes classical macrophage activation in acute respiratory distress syndrome by activating NF-κB.

Background: Previous studies have revealed the important role of alveolar macrophages (AMs) in the pathogenesis of acute respiratory distress syndrome (ARDS) and potential anti-inflammatory properties of lincRNA-p21. This study aims to study the association between lincRNA-p21 and active AMs to understand the molecular mechanisms of AMs-mediated inflammatory responses in ARDS.Methods: This study was mainly investigated in mice with the intratracheal instillation of lipopolysaccharide (LPS) or LPS-treated AMs. The expression of lincRNA-p21 and classical macrophage markers, IL-12ß and iNOS, was detected by quantitative RT-PCR, while NF-κB p65 translocation was measured by western blotting analysis. And, NF-κB activity was analyzed through luciferase report assays. Gain- and loss-of-function studies were also performed for further investigations.Results: Elevated lincRNA-p21 levels were observed in both LPS-induced ARDS mice and LPS-treated AMs, with upregulated expression of IL-12ß and iNOS, namely M1 activation, and p65 nuclear translocation. Further in vitro studies showed that LPS-induced M1 activation could be counteracted by both lincRNA-p21 inhibition and inhibited NF-κB activation. Moreover, both p65 nuclear translocation and NF-κB activity were promoted by lincRNA-p21 overexpression, while lincRNA-p21 inhibition showed a negative effect on LPS-induced p65 nuclear translocation and increase of NF-κB activity. Additionally, LPS-induced lung injuries could be attenuated by lincRNA-p21 inhibition in vivo.Conclusion: This study revealed elevated lincRNA-p21 levels in LPS-induced ARDS and investigated the potential role of lincRNA-p21 in LPS-induced pro-inflammatory response via NF-κB/p65 mediated pathways, suggesting the potential application of lincRNA-p21 for ADRS therapy.

Involvement of E-cadherin/AMPK/mTOR axis in LKB1-induced sensitivity of non-small cell lung cancer to gambogic acid.

Liver kinase B1 (LKB1) is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival, and polarity. Patients with NSCLC possessing mutated LKB1 respond to chemotherapy differently from those with wild-type LKB1. Gambogic acid (GA), a small molecule from natural product, has been established as an anti-tumor agent due to its potent activity and low toxicity. Here, we find out that NSCLC cells with wild-type LKB1 are more sensitive to GA in vitro and in vivo. Mechanistic studies pinpoint that the selective inhibition of mTOR signaling confers the stronger suppression of NSCLC in presence of wild-type LKB1, which is involved in the enhancement of p-AMPK. Further studies reveal that GA increases p-AMPK levels through up-regulation of E-cadherin associated with LKB1. In addition, induction of E-cadherin by GA may be through down-regulation of ZEB1, which is independent with LKB1 status. Hence, our findings support that enhanced E-cadherin by GA cooperates LKB1, leading to up-regulation of p-AMPK, and thus blocking of mTOR signaling pathway, which provide theoretical foundation for utilization of GA as a potential targeted drug against NSCLC harboring wild-type LKB1.

Diagnostic accuracy of serum dickkopf-1 protein in diagnosis hepatocellular carcinoma: An updated meta-analysis.

BACKGROUND: To verify the accuracy of serum dickkopf-1 protein (DKK-1) in the diagnosis of hepatocellular carcinoma (HCC) by an updated meta-analysis. METHODS: We searched potential eligible studies in PubMed and Embase before July 8, 2018. Sensitivity (SN), specificity (SP), positive likelihood ratio (PLR), negative likelihood ratio (NLR), summary receiver operating characteristics curve (sROC), and diagnostic odds ratio (DOR) were pooled with their 95% confidence intervals CIs) using a bivariate random-effects model. RESULTS: A total of 8 articles contained 10 studies on diagnosis of HCC with DKK-1 alone,7 articles contained 9 studies on diagnosis of HCC with a-fetoprotein (AFP) alone and 5 articles contained 7 studies on diagnosis of HCC with DKK-1 + AFP were identified. The pooled SN, SP, PLR, NLR, and DOR of DKK-1 alone, AFP alone and DKK-1 + AFP were 0.72 (95% CI: 0.70-0.75), 0.62 (95% CI:0.59-0.64) and 0.80 (95% CI:0.78-0.83), 0.86 (95% CI: 0.84-0.87), 0.82 (95% CI:0.80-0.84) and 0.87 (95% CI: 0.85-0.88), 4.91 (95% CI: 2.73-8.83), 3.60 (95% CI:2.01-6.44) and 6.18 (95% CI: 4.68-8.16), 0.32 (95% CI: 0.22-0.47), 0.49 (95% CI:0.40-0.60) and 0.20 (95% CI: 0.15-0.26), and 17.21 (95% CI: 9.10-32.57), 7.45 (95% CI:3.69-15.01) and 31.39 (95% CI: 23.59-43.20), respectively. The area under the sROC was 0.88, 0.70, and 0.92 for the 3 diagnostic methods. CONCLUSIONS: Serum DKK-1 + AFP showed a high accuracy for diagnosis of HCC, and serum DKK-1 alone had moderate accuracy as compared to a previous meta-analysis, while AFP alone owned an unsatisfied diagnostic behavior for HCC. Due to the limitations of the current analysis, further well-designed studies are needed to confirm the diagnostic value of DKK-1 and DKK-1 + AFP in HCC diagnosis.

Upregulated microRNA miR-21 promotes the progression of lung adenocarcinoma through inhibition of KIBRA and the Hippo signaling pathway.

PURPOSE: In this study, we aimed to identify the key pathways and hub genes in lung adenocarcinoma (LAD)1 through bioinformatics analysis and to identify the miRNAs that targeted the selected hub gene. The present study was conducted to explore the effect of the hub gene KIBRA, the Hippo signaling pathway and miR-21 on LAD progression. METHODS: Through gene set enrichment analysis (GSEA), the enriched KEGG pathways involved in LAD were identified. Weighted correlation network analysis (WGCNA) was employed to screen out hub genes. The differentially expressed miRNAs related to the hub gene were then screened by the network analysis. The mRNA expression levels of miR-21 and KIBRA were detected by qRT-PCR. The protein expression levels of KIBRA and the pathway related proteins LATS2 and YAP were determined by Western blot assay. The target relationship between miR-21 and KIBRA was confirmed by the dual luciferase reporter assay. Through colony formation assay, the viability of the LAD cells was determined. In addition, the mobility of LAD cells was detected by wound healing assays, and flow cytometry was employed to detect apoptotic cancer cells. RESULTS: The hub gene identified in the black module was KIBRA, and suppression of the Hippo signaling pathway was detected in LAD. KIBRA was downregulated and miR-21 was upregulated in LAD tissues and cells; moreover, miR-21 was found to target KIBRA. KIBRA reduced the proliferative and invasive ability of LAD cells and induced apoptosis. KIBRA also activated the Hippo signaling pathway in LAD. The role of MiR-21 was opposite that of KIBRA in LAD. CONCLUSION: MiR-21 suppressed the Hippo signaling pathway and promoted the progression of LAD through targeting KIBRA.

GAS5 promotes airway smooth muscle cell proliferation in asthma via controlling miR-10a/BDNF signaling pathway.

AIMS: To explore the role of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in the cell proliferation of airway smooth muscle cells (ASMCs) in asthma. MATERIALS AND METHODS: An asthma rat model was established by ovalbumin sensitization and challenge. The expression of GAS5, miR-10a and BDNF mRNA and protein was determined with qRT-PCR and western blot, separately. The targeting relationship between GAS5 and miR-10a was examined with RNA immunoprecipitation and RNA pull-down assay; the interaction between miR-10a and BDNF was evaluated by luciferase reporter assay. Cell Proliferation Assay (MTS) was used for ASMC proliferation detection. Knock-down of GAS5 was performed in asthmatic rats to determine the effects of GAS5 in vivo. KEY FINDINGS: Compared with control group, the inspiratory resistance and expiratory resistance were increased in asthma group; and the expression of GAS5, miR-10a and BDNF was higher, lower and higher, respectively. The expression of GAS5 and miR-10a was elevated and repressed, respectively, by platelet-derived growth factor-BB (PDGF-BB). GAS5 functioned as a bait of miR-10a. GAS5 regulates BDNF expression through miR-10a. PDGF-BB promotes the cell proliferation of ASMCs through miR-10a/BDNF. Knock-down of GAS5 significantly decreased airway hyperresponsiveness in asthmatic rats. SIGNIFICANCE: The lncRNA GAS5/miR-10a/BDNF regulatory axis played an important role in promoting ASMCs proliferation, thus contributing to asthma.

The effects of transient receptor potential channel (TRPC) on airway smooth muscle cell isolated from asthma model mice.

This study aimed to validate whether transient receptor potential channel1 (TRPC1) and TRPC3 participate in the regulation the proliferation of airway smooth muscle cells (ASMCs) through modulating calcium ion (Ca2+ ) influx in vitro. Chronic model of murine asthma was induced and ASMCs isolated from asthmatic mice were used in this whole study. TRPC1 and TRPC3 were upregulated in asthmatic mouse ASMCs and selected for further investigation. Ca2+ concentration and the cell viability of asthmatic mouse ASMCs were significantly higher than that from non- asthma mice, however, TRPC channels blocker SKF96365 alleviated these effects. Furthermore, TRPC1 or TRPC3 overexpression markedly increased Ca2+ concentration and significantly induced the viability of ASMCs; whereas TRPC1 or TRPC3 knockdown exerted the completely conversed effects. Moreover, knockdown of TRPC1 and TRPC3 also exerted different effects on the protein expression of growth-related proteins p-p38, p-JNK, cleaved caspase-3 and Bcl-2, as well as on cell cycle. Finally, we found Ca2+ chelator EGTA or BAPTA-AM significantly diminished the effects of si-TRPC1 and si-TRPC3 on the cell viability, cell cycle, and the protein expression of p-p38, p-JNK, cleaved caspase-3, and Bcl-2 in asthmatic mouse ASMCs. Our findings demonstrated that the effects of TRPC1 and TRPC3 on the cell viability and cell cycle of ASMCs were, at least partially, through regulating Ca2+ influx.

Schisandrin B down-regulated lncRNA BCYRN1 expression of airway smooth muscle cells by improving miR-150 expression to inhibit the proliferation and migration of ASMC in asthmatic rats.

OBJECTIVE: The mechanism of Schisandrin B on the proliferation and migration of airway smooth muscle cells (ASMCs) in asthmatic rats was explored. METHODS: SD rats were divided into three groups: control (group 1), model (group 2) and model + Schisandrin B (group 3). miR-150 and lncRNA BCYRN1 levels were measured by qRT-PCR. The combination of BCYRN1 and miR-150 was detected by RNA pull down. ASMCs' viability/proliferation/migration were examined by WST-1 assay and 24-well Transwell system. RESULTS: Schisandrin B up-regulated miR-150 expression and down-regulated BCYRN1 expression in sensitized rats. Schisandrin B reversed the expression of miR-150 and BCYRN1 in MV-treated ASMCs. In addition, Schisandrin B inhibited the viability, proliferation and migration of MV-induced ASMCs. We also found miR-150 inhibited BCYRN1 expression which was proved by experiments using ASMCs transfected with miR-150 inhibitor. CONCLUSION: Schisandrin B increased miR-150 expression and decreased BCYRN1, and BCYRN1 expression was inhibited by miR-150, which indicated that Schisandrin B could regulate BCYRN1 through miR-150.

LncRNAs BCYRN1 promoted the proliferation and migration of rat airway smooth muscle cells in asthma via upregulating the expression of transient receptor potential 1.

BACKGROUND: Long noncoding RNAs (lncRNAs) played important roles in several biological processes through regulating the expression of protein. However, the function of lncRNA BCYRN1 in airway smooth muscle cells (ASMCs) has not been reported. METHODS: Male Sprague-Dawley (SD) rats were divided into control and asthma groups and the ovalbumin (OVA) model was constructed. The expression of BCYRN1 and transient receptor potential 1 (TRPC1) were detected in the ASMCs separated from these rats. Then 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay, Roche real-time cell analyzer (RTCA) DP assay and Transwell cell migration assay were performed to detect the effect of BCYRN1 on the viability/proliferation and migration of ASMCs. RNA pull-down assays and RNA immunoprecipitation assay were used to identify and verify the binding between BCYRN1 and TRPC1. Inspiratory resistance and expiratory resistance were measured in OVA challenged rats with BCYRN1 knockdown. RESULTS: We foundthe high expression of BCYRN1 and TRPC1 in asthma groups and ASMCs treated with PDGF-BB. Overexpression of BCYRN1 greatly promoted the proliferation and migration of ASMCs. In addition,TRPC1 overexpression reversed the function of si-BCYRN1 indecreasing the viability/proliferation and migration of ASMCs treated with PDGF-BB. BCYRN1 could up-regulate the protein level of TRPC1 through increasing the stability of TRPC1. Finally, we found that BCYRN1 knockdown reduced the inspiratory resistance and expiratory resistance in OVA challenged rats. CONCLUSION: Our study indicated that BCYRN1 promotedthe proliferation and migration of rat ASMCs in asthma via upregulating the expression of TRPC1.

Schisandrin B inhibits the proliferation of airway smooth muscle cells via microRNA-135a suppressing the expression of transient receptor potential channel 1.

Airway smooth muscle cell (ASMC) was known to involve in the pathophysiology of asthma. Schisandrin B was reported to have anti-asthmatic effects in a murine asthma model. However, the molecular mechanism involving in the effect of Schisandrin B on ASMCs remains poorly understood. Sprague-Dawley rats were divided into three groups: rats as the control (Group 1), sensitized rats (Group 2), sensitized rats and intragastric-administrated Schisandrin B (Group 3). The expression of miR-135a and TRPC1 was detected in the rats from three groups. Platelet-derived growth factor (PDGF)-BB was used to induce the proliferation of isolated ASMCs, and the expression of miR-135a and TRPC1 was detected in PDGF-BB-treated ASMCs. Cell viability was examined in ASMCs transfected with miR-135a inhibitor or si-TRPC1. The expression of TRPC1 was examined in A10 cells pretreated with miR-135a inhibitor or miR-135a mimic. In this study, we found that Schisandrin B attenuated the inspiratory and expiratory resistances in sensitized rats. Schisandrin B upregulated the mRNA level of miR-135a and decreased the expression of TRPC1 in sensitized rats. In addition, Schisandrin B reversed the expression of miR-135a and TRPC1 in PDGF-BB-induced ASMCs. Si-TRPC1 abrogated the increasing proliferation of ASMCs induced by miR-135a inhibitor. We also found that miR-135a regulated the expression of TRPC1 in the A10 cells. These results demonstrate that Schisandrin B inhibits the proliferation of ASMCs via miR-135a suppressing the expression of TRPC1.