Relationship of TLR2, TLR4 and tissue remodeling in chronic rhinosinusitis

In order to explore the role of innate immunity in the remodeling of CRS (chronic rhinosinusitis), we investigated the correlation between TLR2, TLR4 and remodeling involved cytokines and histopathological features. Immunohistochemical staining was applied to detect the expression of TLR2, TLR4 and TGF-β1. Masson staining was used for observing the collagen deposition. The other histopathologic features of remodeling were observed by hemotoxylin and eosin (HE) staining. Nasal epithelial cell culture was used to elucidate the effect of TLR2, TLR4 agonists and inhibitors on the expression of TGF-β1 and MMP-9. The association study showed that the significantly higher expression of TLR2, TLR4, TGF-β1 and collagen appeared in CRSsNP (chronic rhinosinusitis without nasal polyps) patients compared with CRSwNP (chronic rhinosinusitis with nasal polyps) patients. In CRSsNP, patients with a severe epithelial damage (score 3) had a significantly higher expression of TLR2 than patients with mild epithelial damage (score ≤ 2) (P < 0.05). Moreover the expression of TLR2 correlated negatively with squamous hyperplasia in CRSsNP, and positively with gland hyperplasia in CRSwNP. The expression of TLR2 and TLR4 was closely related to neutrophil infiltration in CRSsNP (P < 0.01). TGF-β1 was downregulated by TLR2 agonist in CRSwNP and upregulated by TLR4 agonist in CRSsNP (P < 0.05). MMP-9 was upregulated by TLR4 agonist in CRSwNP (P < 0.05). TLR2 and TLR4 had close relationship with TGF-β1 and the histologic features of remodeling, especially collagen deposition and neutrophil infiltration in CRSsNP. The innate immunity could influence the histologic characteristics and involved cytokines through TLR2 and TLR4 in the remodeling of CRS.     

p21活化激酶6对人非小细胞肺癌A549细胞侵袭及迁移能力的影响

 目的：探讨p21活化激酶6（PAK6）对非小细胞肺癌侵袭及迁移能力的影响及机制研究。方法：实时荧光定量PCR检测人非小细胞肺癌A548细胞、人支气管上皮细胞、非小细胞肺癌组织及癌旁组织中PAK6 mRNA的表达。A549细胞分别转染siRNA-PAK6（siPAK6组）和阴性对照（对照组）,实时荧光定量PCR技术检测PAK6 mRNA,Western blotting检测PAK6表达量;并行体外迁移及侵袭实验,检测转染siRNA-PAK6对A549细胞侵袭及迁移能力的影响;共聚焦显微镜观察细胞骨架的改变。结果：A549细胞中PAK6 mRNA的表达量明显高于人支气管上皮细胞中PAK6 mRNA的表达量（3.50±1.16 vs 1.12±0.42,P<0.05）,非小细胞肺癌组织中PAK6 mRNA的表达量明显高于癌旁组织中PAK6 mRNA的表达量（5.13±1.33  vs 1.08±0.37,P<0.05）。A549细胞转染siPAK6后,PAK6蛋白表达量下降72%（P<0.05）,PAK6 mRNA水平明显下降（3.72±0.75 vs  0.69±0.21,P<0.05）。A549细胞转染siPAK6组侵袭及迁移出的细胞数量明显少于对照组(P<0.05)。siPAK6组A549细胞骨架应力纤维减少明显,肌动蛋白皱缩。结论：PAK6通过细胞骨架的重构影响非小细胞肺癌的侵袭及迁移能力。     

MicroRNA 144 inhibits cell migration and invasion and regulates inflammatory cytokine secretion through targeting toll like receptor 2 in non-small cell lung cancer

IntroductionMicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cancer progression. Here, we investigated the possible role of miR-144 in non-small cell lung cancer (NSCLC) development.Material and methodsThe expression of miR-144 and TLR2 in NSCLC tissue and cell lines was determined by quantitative real-time PCR (qPCR). The TargetScan database was used to predict potential target genes of miR-144. Luciferase assay was used to verify the interaction between TLR2 and miR-144. TLR2 protein expression was measured by western blot. The secretion of interleukin (IL)-1β, IL-6 and IL-8 in A549 cells was detected by an ELISA kit. Cell migration and invasion were evaluated by wound healing assay and transwell assay, respectively.ResultsOur results showed that miR-144 was downregulated in NSCLC tissue and cell lines when compared with the normal tissues and cell line (p < 0.05). The protein level of TLR2 in NSCLC tissue and cell lines was significantly higher than that in normal lung tissues. Dual luciferase reporter gene assay showed that miR-144 could bind to the 3ʹUTR of TLR2 specifically. Up-regulation of miR-144 significantly decreased the expression of TLR2. Up-regulation of miR-144 or down-regulation of TLR2 could decrease cell migration, invasion and secretion of IL-1β, IL-6 and IL-8 in A549 cells. Moreover, overexpression of TLR2 rescued the inhibitory effects of miR-144 on migration, invasion and inflammatory factor secretion of A549 cells.ConclusionsmiR-144 could inhibit the migration, invasion and secretion of IL-1β, IL-6 and IL-8 through downregulation of TLR2 expression in A549 cells.     

IL-19及其受体与慢性鼻-鼻窦炎组织重塑的相关性研究

目的:探讨白细胞介素19(interleukin-19,IL-19)及其受体(IL-20R1/IL-20R2)在不同类型慢性鼻-鼻窦炎[慢性鼻-鼻窦炎伴鼻息肉(chronic rhinosinusitis with nasal polyps,CRSw NP)和慢性鼻-鼻窦炎不伴鼻息肉(chronic rhinosinusitis without nasal polyps,CRSs NP)]患者中表达的差异,并分析其与慢性鼻-鼻窦炎组织重塑的相关性。方法:研究共分3组:CRSw NP组(30例)、CRSs NP组(15例)和对照组(鼻中隔偏曲患者15例)。实时荧光定量PCR检测IL-19及其受体(IL-20R1/IL-20R2)和组织重塑因子基质金属蛋白酶(MMP)-2、MMP-9、金属蛋白酶组织抑制物(TIMP)-1的mRNA相对表达量;免疫组化检测IL-19及其受体的蛋白表达量;ELISA检测MMP-9、TIMP-1的蛋白表达量。结果:CRSw NP组IL-19、IL-20R1、IL-20R2和MMP-9的mRNA和蛋白表达量高于CRSs NP组和对照组(P0.05),CRSs NP组TIMP-1的mRNA和蛋白的表达量均高于CRSw NP组和对照组(P0.05);MMP-2的mRNA和蛋白的表达量在各组无显著差异。CRSw NP组IL-19、IL-20R1和IL-20R2均与MMP-9的mRNA相对表达量呈正相关(P0.05);IL-19及其受体与TIMP-1的mRNA相对表达量无明显相关。结论:IL-19及其受体的mRNA在CRSw NP中高表达,且与MMP-9 mRNA表达呈正相关,提示二者可能存在交互作用,可能参与慢性鼻-鼻窦炎的组织重塑。     

Chronic rhinosinusitis with and without nasal polyps is associated with decreased expression of glucocorticoid-induced leucine zipper

Background Chronic rhinosinusitis without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP) is characterized by persistent inflammation of sinonasal mucosa. Glucocorticoid-induced leucine zipper (GILZ) is a recently described anti-inflammatory mediator.
Objective Here we analysed the expression of GILZ in CRSsNP and CRSwNP, its association with response to surgery, and its cytokine-driven expression regulation in the upper airways.
Methods The messenger RNA (mRNA) and protein expression of GILZ in 33 CRSsNP, 32 CRSwNP, and 11 control samples was assessed by means of a quantitative RT-PCR and immunohistochemistry, respectively. Nasal explant culture was used to investigate the effect of IFN-γ, IL-4, IL-13, IL-1β, and TNF-α on GILZ mRNA expression in normal sinonasal mucosa.
Results The GILZ mRNA and protein expression was significantly suppressed in both CRSsNP and CRSwNP patients compared with controls. No significant difference in GILZ expression was found between CRSsNP and CRSwNP patients. Comparing patients responsive and patients recalcitrant to surgery, a significant further decrease of GILZ expression was found in recalcitrant patients both in the CRSsNP and in the CRSwNP group. IL-1β, TNF-α, IL-4, and IL-13 reduced, whereas IFN-γ enhanced GILZ mRNA levels in the sinonasal mucosa.
Conclusion Down-regulated expression of GILZ may contribute to the pathogenesis of CRSsNP and CRSwNP and associate with response to surgery. GILZ expression in the upper airways can be regulated differentially by different cytokines.     

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues

Objective: Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues.

Methods: Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10?ng/ml) treatment for 4?h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay.

Results: Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB.

Conclusions: The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.     

Effect of rapamycin (RAPA) on the growth of lung cancer and its mechanism in mice with A549

Objective: To investigate the effects of rapamycin (RAPA) on the tumor growth of lung cancer in the mice bearing A549 and the mechanisms. Methods: 60 mice with A549 lung cancer models established were randomly divided into model group, low RAPA dose group and high RAPA dose group. The low dose group underwent intraperitoneal injection of 1.5 mg/kg RAPA, while the high dose group underwent intraperitoneal injection of 4.5 mg/kg RAPA, and the control group was given the same volume of PBS. 21 d after the administration, the changes of the tumor growth and survival rates of three groups were observed. RT-PCR and Western blot were utilized to analyze Caspase-3 mRNA and protein levels in the tumor tissues of the mice, and TUNEL staining method was used to analyze the cellular apoptosis of tumor tissues. Results: Compared with the model group, the low and high dose groups significantly inhibit tumor growth and have remarkably higher survival rates (P<0.05). The high dose group has obviously better effects on inhibiting tumors and a higher survival rate than low dose group (P<0.05). Compared with the model group, the low and high dose groups have significantly increased Caspase-3 mRNA and protein levels in tumor tissues (P<0.05), and higher cellular apoptosis rates in tumor tissues (P<0.05); Caspase-3 mRNA and protein levels and apoptosis rates of the mice’s tumor tissues of high dose group are markedly higher than those of low dose group (P<0.05). Conclusions: RAPA can significantly increase the expression of Caspase-3 in tumor tissues and promote the apoptosis of tumor tissue cells, and thus achieve good anti-tumor effects.     

Regulated expression of the IL-31 receptor in bronchial and alveolar epithelial cells, pulmonary fibroblasts, and pulmonary macrophages.

Interleukin-31 (IL-31), an IL-6 cytokine family member, is proposed to play a role in animal models of airway hyperreactivity. It is produced by activated T cells and signals via a heterodimeric receptor complex composed of IL-31Ralpha and OSMRbeta. Only low levels of IL-31Ralpha expression have been demonstrated in pulmonary epithelial cell lines, however, and little is known about the ability to regulate its expression and signaling. Therefore, primary cultures of human bronchial and alveolar epithelial cells, pulmonary fibroblasts, pulmonary macrophages, and established lines of immortalized bronchial epithelial cells (HBE) and alveolar carcinoma cells (A549) were analyzed by RT-PCR, immunoblotting, and thymidine incorporation. Distinct, cell type-specific regulation of IL-31Ralpha expression was detected. Transforming growth factor-beta (TGF-beta) enhanced IL-31Ralpha mRNA expression in primary cultures and established lines of epithelial cells, but not in macrophages. In contrast, interferon-gamma (IFN-gamma) induced IL-31Ralpha mRNA expression in macrophages. IL-31Ralpha protein expression was below detection threshold in primary epithelial cell cultures but was detectable in A549 cells and increased with TGF-beta treatment. In HBE and A549 cells, TGF-beta pretreatment increased IL-31-mediated Stat3 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. In A549 cells, TGF-beta magnified IL-31-dependent suppression of proliferation. The data suggest that increased IL-31Ralpha expression correlates with an enhanced response to IL-31.     

miR-205 regulates A549 cells proliferation by targeting PTEN

miR-205 is an epithelial-specific miRNA and has been shown to orchestrate some cellular processes such as epithelial mesenchymal transition (EMT) and differentiation fate of stem cells in mammary gland. miR-205 play a part of a tumor suppressor in human cancers. However, the role of miR-205 in lung cancer is unclear. In this study, we detected the expression level of miR-205 in 46 cases clinical lung cancer specimens and adjacent normal tissues by stem-loop RT-PCR. We found that the expression of miR-205 was significantly increased in lung cancer specimens compared to adjacent normal tissues (P < 0.01). Furthermore, we observed the expressions of PTEN protein and mRNA in lung cancer tissues and adjacent normal tissues by methods of western blot and Real time PCR respectively. We found that the expressions of PTEN protein and mRNA was significantly decreased in lung cancer specimens compared to adjacent normal tissues. And then, we found there is a negative relationship between the expression of miR-205 and PTEN mRNA in lung cancer by analyzed. To validate whether PTEN was direct targets of miR-205, a dual-luciferase reporter assay was employed, the result showed that PTEN is a target gene of MiR-205. In subsequent experiments, we examined the expressions of PTEN protein and mRNA after transfection of miR-205 mimics or inhibitor into A549 cells, and A549 cell proliferation was measured by CCK-8 tests. We found that the expression of PTEN protein and mRNA in A549 cells were significantly down-regulated or up-regulated after miR-205 mimics and miR-205 inhibitors transfected into, and miR-205 could inhibits A549 cells proliferation. These results indicate that miR-205 might inhibitor the proliferation of A549 cells by regulating the expression of PTEN.     

Aberrant SATB1 expression is associated with Epstein-Barr virus infection,metastasis and survival in human nasopharyngeal cells and endemic nasopharyngeal carcinoma

Special AT-rich sequence-binding protein 1 (SATB1) has been identified as a key factor in the progression of some cancers, functioning as a global genome organizer and chromatin regulator. We examined the levels of SATB1 mRNA expression in NPC cell lines 5-8F (high metastasis) and 6-10B (low metastasis) and immortalized human nasopharyngeal epithelial cells NP69-SV40T by quantitative real-time PCR. We also examined the protein expression levels of SATB1 in 72 cases of nasopharyngeal carcinoma (NPC) tissues and 30 cases of normal nasopharyngeal (NNP) tissues by immunohistochemistry, and then assessed the correlations between SATB1 expression and clinicopathological factors. The expression level of SATB1 mRNA in 5-8F was much higher than those in 6-10B and NP69-SV40T (P < 0.05). The expression level of SATB1 mRNA in 6-10B was higher than in NP69-SV40T, but the difference was not statistically significant (P > 0.05). The positive expression rates of SATB1 protein in NPC (38/72, 52.8%) were significantly higher than in NNP (4/30, 13.3%) (P < 0.05). SATB1 protein levels in NPC were not associated with gender, age, and T stage (P > 0.05), but positively correlated with the titers of EBVCA-IgA, metastasis (N and M stage), recurrence, and survival (P < 0.05). Multivariate analysis showed that the overexpression of SATB1 protein is an independent prognostic factor for NPC. The expression levels of SATB1 were obviously upregulated in primary NPC tissues and human NPC cell lines. Therefore, SATB1 may be a valuable predictor in assessing the metastasis, recurrence, and prognosis of NPC.     

PGRN基因沉默对人非小细胞肺癌A549细胞增殖及迁移的影响

目的:探讨小干扰RNA(small interference RNA,si RNA)介导的颗粒蛋白前体(progranulin,PGRN)基因沉默对非小细胞肺癌A549细胞增殖、迁移和凋亡的影响及其机制。方法:分别用q PCR和Western blot法检测A549细胞和正常人支气管上皮(HBE)细胞中PGRN的m RNA和蛋白表达水平。采用脂质体转染法将PGRNsi RNA转染A549细胞,采用q PCR和Western blot法验证PGRN表达的变化;应用MTT实验检测细胞活力;活细胞计数法和结晶紫染色实验检测细胞增殖能力;划痕愈合实验和Transwell实验检测细胞迁移能力;并用Western blot法检测增殖细胞核抗原(PCNA)、细胞周期蛋白D1(cyclin D1)、Bcl-2和Bax的蛋白表达水平以及PGRN下游信号通路中细胞外信号调节激酶1/2(ERK1/2)和蛋白激酶B(Akt)的磷酸化水平。结果:PGRN在A549细胞中的m RNA和蛋白水平均明显高于HBE细胞(P0.05);转染PGRN-si RNA后A549细胞中PGRN的m RNA和蛋白水平均明显下调,细胞活力、增殖能力以及迁移能力均明显降低(P0.05)。沉默PGRN基因的表达,可下调PCNA、cyclin D1和Bcl-2的蛋白表达,而上调Bax的蛋白表达,且磷酸化的ERK1/2(p-ERK1/2)和磷酸化的Akt(p-Akt)的蛋白水平明显降低(P0.05)。结论:PGRN基因沉默能明显抑制非小细胞肺癌A549细胞的增殖和迁移能力,PI3K/Akt和MAPK/ERK信号通路可能在该过程中发挥重要作用。     

miR-155 modulates the progression of neuropathic pain through targeting SGK3

This study aimed to illustrate the potential effects of miR-155 in neuropathic pain and its potential mechanism. Spragure-Dawley (SD) rats were used for neuropathic pain model of bilateral chronic constriction injury (bCCI) construction. Effects of miR-155 expression on pain threshold of mechanical stimuli (MWT), paw withdrawal threshold latency (PMTL) and cold threshold were analyzed. Target for miR-155 was analyzed using bioinformatics methods. Moreover, effects of miR-155 target gene expression on pain thresholds were also assessed. Compared with the controls and sham group, miR-155 was overexpressed in neuropathic pain rats (P<0.05), but miR-155 slicing could significantly decreased the pain thresholds (P<0.05). Serum and glucocorticoid regulated protein kinase 3 (SGK3) was predicted as the target gene for miR-155, and miR-155 expression was negatively correlated to SGK3 expression. Furthermore, SGK3 overexpression could significantly decreased the pain thresholds which was the same as miR-155 (P<0.05). Moreover, miR-155 slicing and SGK3 overexpression could significantly decrease the painthreshold. The data presented in this study suggested that miR-155 slicing could excellently alleviate neuropathic pain in rats through targeting SGK3 expression. miR-155 may be a potential therapeutic target for neuropathic pain treatment.     

Polymorphisms in thymic stromal lymphopoietin gene demonstrate a gender and nasal polyposis-dependent association with chronic rhinosinusitis

Background

Recent studies have suggested that thymic stromal lymphopoietin (TSLP), a key cytokine involved in the dendritic cell-mediated activation of Th2-mediated inflammatory responses, is significantly increased in nasal polyps from atopic individuals. Our objective was therefore to explore firstly any associations between single nucleotide polymorphisms (SNPs) in and around the TSLP gene and development of chronic rhinosinusitis (CRS; with (CRSwNP) or without (CRSsNP) nasal polyps and, and secondly the influence of nasal polyposis and gender.

Methods

A population-based case-control association analysis was performed in a Han Chinese cohort. DNA extracted from peripheral blood leukocytes from 638 subjects with CRS (306 CRSwNP and 332 CRSsNP) and 325 healthy controls was assessed for 11 SNPs in and around TSLP gene, selected from the Chinese HapMap genotyping dataset. Genetic association tests were performed using the Haploview and STATA software package.

Results

Single-locus analysis of CRS risk, showed no significant differences in allele or genotype frequencies between CRS subjects and controls. Stratified analyses of association between the selected SNPs and CRS adjusted for gender demonstrated that rs13156068 (CC genotype: P = 0.010, OR = 0.289) and rs764917 (CC genotype: P = 0.040, OR = 0.509) were significantly protective against CRS, whereas rs6886755 (GT genotype: P = 0.040, OR = 0.509) presented a risk among females. In contrast, rs764917 (CA genotype: P = 0.033, OR = 1.553) presented risk for CRS in males. Furthermore, SNPs rs252706 (AA genotype: P = 0.012, OR = 0.552) and rs764917 (CA genotype: P = 0.001, OR = 0.182) displayed protective roles among CRSwNP, but not CRSsNP, subjects.

Conclusions

This study suggests that SNPs in TSLP gene may exert a gender and/or nasal polyposis-dependent risk for development of CRS in Chinese subjects.