miR-363-3p inhibits migration, invasion, and epithelial-mesenchymal transition by targeting NEDD9 and SOX4 in non-small-cell lung cancer.

miR-363-3p is downregulated in lung adenocarcinoma and can inhibit tumor growth. Here, we aimed to investigate the effect of miR-363-3p on non-small-cell lung cancer (NSCLC) metastasis. In our study, miR-363-3p overexpression inhibited cell migration and invasion via epithelial-mesenchymal transition inhibition, while miR-363-3p knockdown exhibited the opposite effects. Further studies demonstrated that miR-363-3p bound to 3'-untranslated regions of NEDD9 and SOX4, and negatively regulated their levels. Interestingly, NEDD9 or SOX4 knockdown rescued the metastasis-promoting effects of antagomiR-363-3p. The inhibitory effects of agomiR-363-3p were also blocked by NEDD9 or SOX4 overexpression. Moreover, lentivirus particles carrying pre-miR-363 (LV-pre-miR-363) significantly decreased, while LV-miR-363-3p inhibitor increased metastatic nodule numbers and the levels of NEDD9 and SOX4 in lungs. In conclusion, tumor suppressor miR-363-3p may be a potential target in NSCLC therapy.

MiR-495 regulates proliferation and migration in NSCLC by targeting MTA3.

Our previous studies have showed that metastasis-associated protein 3 (MTA 3) is overexpressed in non-small cell lung cancer (NSCLC) tissue, and increased MTA3 mRNA levels is a risk factor of lymph node metastasis. Using bioinformatics analyses, we found that MTA3 was a potential target of miR-495. However, the pathophysiological role of miR-495 and its relevance to the growth and development of NSCLC have yet to be investigated. The purpose of this study was to elucidate the molecular mechanisms by which miR-495 acts as a tumor suppressor in NSCLC. qRT-PCR data showed significant downregulation of miR-495 in 56 NSCLC tissue samples and 5 lung cancer cell lines, compared with their adjacent normal tissue; furthermore, western blotting analysis revealed MTA3 protein was overexpressed in the tumor samples compared with the matched adjacent normal tissue. MiR-495 was shown to not only inhibit the proliferation of lung cancer cells (A549 and Calu-3) but also to inhibit cell migration in vitro. Using western blotting and luciferase assays, MTA3 was identified as a target of miR-495. These findings suggest the importance of miR-495 targeting of MTA3 in the regulation of lung cancer growth and migration.

Serum miR-21 and miR-155 expression in idiopathic pulmonary fibrosis.

OBJECTIVE: To investigate the expression of serum miRNA-21(miR-21) and miRNA-155 (miR-155) in idiopathic pulmonary fibrosis (IPF). METHODS: A study including 65 patients with IPF and 65 similar age and gender healthy controls was performed. Serum specimens were collected from all subjects. Total RNA was extracted and the quantitative reverse transcription-polymerase chain reaction was used to measure serum miR-21 and miR-155 in both groups. Clinicopathologic features were assessed to determine associations with serum miR-21 and miR-155 concentrations. RESULTS: Serum miR-21 expression was significantly higher in IPF samples than in healthy controls (p < 0.01), while serum miR-155 expression did not show a statistically significant difference (p > 0.05). Forced vital capacity (FVC) and radiologic features were associated with miR-21 and miR-155 expression in serum (p < 0.05). Neither miR-21 nor miR-155 expression was statistically significantly associated with clinicopathologic parameters, such as gender (p > 0.05) and age (p > 0.05). CONCLUSION: These findings suggest that serum miR-21 is associated with IPF and the degree of damage indicated by FVC and radiologic examinations could correlate with miR-21 and miR-155 expression in serum. From another perspective, our study confirmed serum miRNA can be stable and detectable in serum of patients with IPF, which could prove useful as it could be considered as a new biomarker in serum for diagnosis and assessment of prognosis of IPF in the future.

Neutrophil gelatinase-associated lipocalin in gastric carcinoma cells and its induction by TPA are controlled by C/EBPß.

Neutrophil gelatinase-associated lipocalin (NGAL) expression has been found to be upregulated in a variety of tumors, but the mechanism of NGAL elevation in gastric carcinoma remains unknown. Here, immunohistochemistry was applied to analyze NGAL expression in gastric carcinoma patients. Reverse transcription PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate NGAL mRNA and protein levels before and after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction. Luciferase reporter assay was carried out to identify the core cis element in NGAL promoter. The binding ability and specificity of transcription factors were analyzed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP), respectively. Results showed that NGAL was overexpressed in gastric tumor tissues. Gastric cancer cells treated with TPA resulted in the transactivation of NGAL promoter and the upregulation of its mRNA and protein levels. We identified the -110 to -79 sequence segment upstream from the transcription initiation site of NGAL as a TPA responsive element (TRE) and confirmed that C/EBPß was able to bind to the -87 to -79 segment. Forced expression of C/EBPß significantly increased the promoter activity of NGAL as well as its mRNA level. These results suggest that NGAL is overexpressed in gastric cancer, the binding of C/EBPß to the TRE of its gene promoter mediates its TPA-induced overexpression in gastric carcinoma cells.

The Orphan Nuclear Receptor Gene NR0B2 Is a Favorite Prognosis Factor Modulated by Multiple Cellular Signal Pathways in Human Liver Cancers.

BACKGROUND: Liver cancer is a leading cause of cancer death worldwide, and novel prognostic factor is needed for early detection and therapeutic responsiveness monitoring. The orphan nuclear receptor NR0B2 was reported to suppress liver cancer development in a mouse model, and its expression levels were reduced in liver cancer tissues and cell lines due to hypermethylation within its promoter region. However, it is not clear if NR0B2 expression is associated with cancer survival or disease progression and how NR0B2 gene expression is regulated at the molecular level. METHODS: Multiple cancer databases were utilized to explore NR0B2 gene expression profiles crossing a variety of human cancers, including liver cancers, on several publicly assessable bioinformatics platforms. NR0B2 gene expression with or without kinase inhibitor treatment was analyzed using the qPCR technique, and NR0B2 protein expression was assessed in western blot assays. Two human hepatocellular carcinoma cell lines HepG2 and Huh7, were used in these experiments. NR0B2 gene activation was evaluated using NR0B2 promoter-driven luciferase reporter assays. RESULTS: NR0B2 gene is predominantly expressed in liver tissue crossing human major organs or tissues, but it is significantly downregulated in liver cancers. NR0B2 expression is mostly downregulated in most common cancers but also upregulated in a few intestinal cancers. NR0B2 gene expression significantly correlated with patient overall survival status in multiple human malignancies, including lung, kidney, breast, urinary bladder, thyroid, colon, and head-neck cancers, as well as liposarcoma and B-cell lymphoma. In liver cancer patients, higher NR0B2 expression is associated with favorite relapse-free and progression-free survival, especially in Asian male patients with viral infection history. In addition, NR0B2 expression negatively correlated with immune infiltration and PIK3CA and PIK3CG gene expression in liver cancer tissues. In HepG2 and Huh7 cells, NR0B2 expression at the transcription level was drastically reduced after MAPK inhibition but was significantly enhanced after PI3K inhibition. CONCLUSION: NR0B2 gene expression is altered mainly in most human malignancies and significantly reduced in liver cancers. NR0B2 is a prognosis factor for patient survival in liver cancers. MAPK and PI3K oppositely modulate NR0B2 expression, and NR0B2 gene upregulation might serve as a therapeutic responsiveness factor in anti-PI3K therapy for liver cancer.

MiRNA-192-5p attenuates airway remodeling and autophagy in asthma by targeting MMP-16 and ATG7.

Asthma is a chronic lung inflammatory disease with high incidence. MicroRNA-192-5p (miR-192-5p) was down-regulated in asthmatics. However, the role of miR-192-5p in asthma is still unclear. In current study, in vitro, the overexpression of miR-192-5p, matrix metalloproteinase (MMP)-16 and autophagy related 7 (ATG7) was conducted in airway smooth muscle cells (ASMCs). We found that miR-192-5p suppressed cell proliferation, and decreased MMP-16 and ATG7 expression. MMP-16 and ATG7 promoted cell proliferation, and further alleviated the down-regulation of miR-192-5p on proliferation of ASMCs. in vivo, miR-192-5p was down-regulated in asthma mice, and involved in improvement of asthma mice. MiR-192-5p was demonstrated to alleviate inflammation in asthma mice, including decreasing the level of ovalbumin (OVA)-specific IgE, interleukin (IL)-4, IL-5, IL-13, iNOS and COX-2. Moreover, the attenuation of airway remodeling induced by miR-192-5p in asthma mice were expressed by the reduction of fibroblast growth factor-23 (FGF-23) level, decrease in concentrations of MMP-2 and MMP-9 as well as down-regulation of collagen I deposition. Further, miR-192-5p also caused the suppression of autophagy in asthma mice, exhibiting a decrease in LC3II/I, beclin-1 and ATG7, and an increase in p62. Importantly, MMP-16 and ATG7 were confirmed to be targets of miR-192-5p. Therefore, our results indicate that miRNA-192-5p may attenuate airway remodeling and autophagy in asthma via targeting MMP-16 and ATG7.

Xanthatin alleviates airway inflammation in asthmatic mice by regulating the STAT3/NF-κB signaling pathway.

Here, we aimed to investigate the role of Xanthatin in asthma and its underlying mechanism. BALB/c mice were treated with ovalbumin (OVA) to establis a mouse model of asthma. Our results showed that OVA injection significantly increased inflammatory cell infiltration and goblet cell hyperplasia in lung issues, while Xanthatin treatment and STAT3 inhibitor C188-9 administration relieved these symptoms. Moreover, OVA-induced OVA-specific immunoglobulin E level in serum and the number of total cell, macrophages, lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid (BALF) were markedly reduced by Xanthatin treatment and signal transducer and activator of transcription 3 (STAT3) inhibition. Additionally, Xanthatin treatment and STAT3 inhibition was also significantly decreased the levels of inflammatory cytokines in BALF in asthmatic mice. We further demonstrated that the STAT3/nuclear factor-kappaB (NF-κB) pathway was blocked by Xanthatin in asthmatic mice. Overall, we conclude that Xanthatin attenuates airway inflammation in asthmatic mice through blocking the STAT3/NFκB signaling pathway, indicating the potential of Xanthatin as a useful therapeutic agent for asthma.

MicroRNA-206 promotes lipopolysaccharide-induced inflammation injury via regulation of IRAK1 in MRC-5 cells.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play crucial role in the airway inflammatory diseases. However, the involvement of miR-206 in airway inflammatory diseases is still uninvestigated. The study aimed to explore the effect of miR-206 on lipopolysaccharide (LPS)-induced inflammation injury in MRC-5 cells, and point out a potential relevance for chronic obstructive pulmonary disease (COPD). METHODS: LPS was utilized to expose MRC-5 cells, then cell viability, cell migration, apoptosis, apoptosis-associated factors, as well as the concentrations and protein levels of IL-6 and IL-8 were explored. After transfected with miR-206 mimic and inhibitor, above parameters were reassessed in LPS-injured cells. Expression level of IRAK1 was examined in miR-206 mimic/inhibitor transfected cells by using RT-qPCR. The effect of IRAK1 on LPS-induced inflammation injury was investigated in MRC-5 cells after transfection with pc-IRAK1 and sh-IRAK1. The effects of miR-206 and IRAK1 on MEK/ERK and JNK pathways were determined by western blot assay. RESULTS: LPS significantly triggered inflammation injury in MRC-5 cells by inhibiting cell viability, suppressing the healing of scratches, inducing cell apoptosis, down-regulating Bcl-2 expression and up-regulating Bax, cleaved-Caspase-3 and cleaved-Caspase-9 expression, and concurrently increasing the concentrations and the protein levels of IL-6 and IL-8. MiR-206 overexpression aggravated LPS-induced inflammation injury in MRC-5 cells. Up-regulation of IRAK1 was observed in miR-206 mimic-transfected cells. Moreover, IRAK1 overexpression promoted LPS-induced inflammation injury in MRC-5 cells. MiR-206 activated MEK/ERK and JNK pathways by regulating IRAK1. CONCLUSIONS: MiR-206 promotes LPS-induced inflammation injury through regulation of IRAK1 in MRC-5 cells.

Potential diagnostic value of miR-155 in serum from lung adenocarcinoma patients.

Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. The expression profile of miR-21 and miR-155 was evaluated in the present study, since miR-21 is frequently reported as highly expressed in several types of cancers, while miR-155 was also found to be significantly expressed in lung cancer cell lines. Using in vitro studies, we found that miR-155 could be a candidate plasmatic biomarker for diagnosing lung cancer. We assessed the differences in levels of miR-21, miR-155, carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA-125) expression in serum samples between lung cancer patients and healthy controls. We estimating the clinical diagnostic value of miR-155 independently and combined with CA-125 and/or CEA levels. The present study consisted of three parts: i) confirmation of the stable expression of miR-155 in the patient serum samples using quantitative PCR; ii) confirmation of higher miR-155, CEA and CA-125 levels in the patient serum samples when compared with levels in the normal controls by quantitative PCR; iii) evaluation of miR-155, CEA and CA-125 concentrations in serum sampes for tumor diagnosis of lung adenocarcinoma via ROC (receiver-operating characteristic) analysis. The results showed that i) expression of miR-155 was significantly higher in the serum of lung adenocarcinoma patients than that in normal controls (P<0.05); ii) testing results of serum miR-155 levels showed a much higher sensitivity (0.722) than that for CA-125 or CEA; iii) CEA associated with CA-125 had the highest Youden's index (0.639) in all terms of combinations; and iv) combined with CA-125 testing, miR-155 received a competitive sensitivity (0.889) and specificity (0.688) for diagnosing lung adenocarcinoma (OOP=14.88). In conclusion, endogenous miR-155 stably existed in patient serum and could be sensitively and specifically measured. Overexpression of miR-155 in serum specimens could constitute a diagnostic marker for the early detection of lung adenocarcinoma.

Effects of lentivirus-mediated RNAi knockdown of NEDD9 on human lung adenocarcinoma cells in vitro and in vivo.

The aim of the present study was to investigate the biological behavior of lung adenocarcinoma A549 cells following transfection with NEDD9-specific lentiviral particles in vitro and in vivo. NEDD9-specific lentiviral particles were chemically synthesized and transfected into the human lung adenocarcinoma A549 cell line. NEDD9 mRNA and protein levels were determined by fluorescence quantitative RT-PCR and western blotting. Cell proliferation was evaluated using soft agar colony formation assays and flow cytometric analysis. Migration and invasion were evaluated by wound-healing and transwell assays and xenograft animal models. Transfection was successful, and expression levels of NEDD9 mRNA and protein in the lentivirus-NEDD9-siRNA group were downregulated. As indicated by soft agar colony formation assays, the number of clones in the siRNA group were significantly lower than the number of colonies in the blank and negative control groups (P<0.01). In addition, the percentage of cells in the S phase in the siRNA group was significantly lower than the percentages in the blank and negative control groups (P<0.05). Furthermore, as detected by cell migration and invasion assays, values of wound healing were increased and the number of invading cells were decreased in the siRNA group (both P<0.05). We also showed that lentivirus-mediated NEDD9-siRNA decreased the growth potential of subcutaneous A549 xenografts in vivo. These data imply that knockdown of the NEDD9 gene results in suppression of tumor cell proliferation, migration, invasion and cell growth in vitro and in vivo. Lentivirus-mediated NEDD9-siRNA may have potential therapeutic utility for human lung adenocarcinoma.

Computed tomography image analysis before and after treatment of anti-neutrophil cytoplasmic antibody-associated pulmonary interstitial fibrosis in 8 patients.

PURPOSE: The purpose of this study was to observe the treatment response of anti-neutrophil cytoplasmic antibody (ANCA)-associated pulmonary interstitial fibrosis in 8 patients before and after glucocorticoid or immunosuppressive therapy. METHODS: The clinical features and computed tomography imaging findings of the 8 patients in our hospital from October 2011 to October 2013, were retrospectively analyzed. FINDINGS: Mean age of the 8 patients was 72.6 (range 60-80) years. Five patients exhibited cough, sputum, and chest tightness, including 2 patients with fever. One patient developed hemoptysis, 1 patient exhibited abnormal urinalysis and developed renal insufficiency, and 1 patient developed limb pain. Two patients exhibited high urine erythrocytes and 2 patients had renal dysfunction and urinary abnormalities. One of the latter patients, upon renal biopsy, had focal proliferative necrotizing glomerulonephritis (consistent with vasculitis damage) with stage II to III mild nephropathy. Seven cases were anti-myeloperoxidase-ANCA, and 1 case was anti-proteinase 3-ANCA. All 8 cases exhibited streaks and grid shadows in chest imaging; 2 cases exhibited limited ground-glass patches; 1 case displayed multiple large patches of exudative shadows, indicating diffuse alveolar hemorrhage; 2 cases exhibited obvious honeycomb manifestations; and 1 case exhibited significant traction bronchiectasis. The ground-glass opacities disappeared after corticosteroid or immunosuppressive therapy; however, for streaks and grid shadows, no significant changes in the images were observed after treatment from 2 weeks to 10 months. IMPLICATIONS: ANCA-associated pulmonary interstitial fibrosis most often in elderly patients with many complications. In these patients ground-glass opacities in computed tomography images, corticosteroid or immunosuppressant therapy may be effective. Clinicians should consider the poor efficacy and side effects of these therapies in the fibrosis stage of the disease.

Expression analysis of serum microRNAs in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology with considerable morbidity and mortality. Seeking informative diagnostic markers with greater clinical significance is essential for the early diagnosis of IPF. microRNAs (miRNAs or miRs) have emerged as novel serum diagnostic biomarkers for various diseases. In this study, we performed microarray analysis of the miRNA expression profile in the serum of patients with IPF compared to that of control subjects. We then performed a preliminary analysis of biological functions for the most differentially expressed miRNAs. Some of the microarray results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results from this study provide evidence to link the biological role of miRNAs to IPF, and suggest that miRNAs may undertake a variety of functions. Additionally, we found that the altered expression levels of miR-21, miR-155 and miR-101-3p were associated with forced vital capacity (FVC) and radiological features in IPF. Our data may serve as a basis for further investigation, preferably in large prospective studies, before miRNA can be used as a non-invasive screening tool for IPF in routine clinical practice.

Expression and clinical significance of NEDD9 in lung tissues.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) acts as a scaffold protein and belongs to a family of CAS (Crk-associated substrate) that regulates protein complexes controlling invasion and differentiation. Preclinical research for this gene was predominantly reported in melanomas, glioblastoma, and lymphoma. So we investigated the expression and significance of NEDD9 mRNA and protein in lung tissues. Specifically, we immunohistochemically compared NEDD9 expression and localization in 24 formalin-fixed and paraffin-embedded lung adenocarcinoma tissues with that of surrounding nonneoplastic tissue and five microscopically normal lungs. NEDD9 mRNA levels were quantitatively analyzed by fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR) in frozen tissue specimens of all tumors and 24 matched nonneoplastic lung parenchymas, and protein expression in 16 homogenates of matched neoplastic/nonneoplastic specimens was evaluated by Western blotting. The three techniques showed that NEDD9 is weakly expressed in nonneoplastic lung parenchyma and upregulated in lung adenocarcinoma. Moreover, FQ-PCR indicated a statistically significant correlation between NEDD9 upregulation and higher disease stages (I+II versus III+IV, p=0.001; high and middle versus low differentiation, p<0.001). Our results provide evidence that NEDD9 is upregulated in lung adenocarcinoma, and overexpression of NEDD9 protein has been strongly correlated with staging and differentiation grade and tumor size in lung adenocarcinoma, which demonstrated a poor prognosis.

Role of NEDD9 in invasion and metastasis of lung adenocarcinoma.

Treatment failure for lung adenocarcinoma is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate the invasion- and metastasis-related gene, neural precursor cell expressed, developmentally downregulated 9 (NEDD9), in lung adenocarcinoma tissues and cell lines. The expression of NEDD9 was analyzed by the SP method of immunohistochemistry for 60 formalin-fixed and paraffin-embedded (FFPE) lung adenocarcinoma tissues in which 32 cases were metastastic and 28 were without metastases. NEDD9 mRNA expression and protein levels were quantified by fluorescence quantitative reverse transcription-polymerase chain reaction (FQ-PCR) and western blotting in the highly invasive lung adenocarcinoma cell lines A549 and 95D as well as in SPC-A-1 cells with low invasive potential. The immunostaining scores revealed a statistically significant difference between metastatic and non-metastatic lung adenocarcinomas (p<0.001). FQ-PCR and western blotting demonstrated that NEDD9 expression was higher in A549 and 95D compared to SPC-A-1 cells (P=0.003). Our results provide evidence that NEDD9 is upregulated in metastatic lung adenocarcinoma and in highly invasive lung adenocarcinoma cell lines, suggesting its potential involvement in regulating cell migration and invasion.

Construction and characterization of a eukaryotic expression vector for small interfering RNA targeting the NEDD9 gene.

The aim of this study was to construct a eukaryotic expression vector for a small interfering RNA (siRNA) targeting the neural precursor cell expressed, developmentally downregulated 9 (NEDD9) gene, and to investigate the effects of RNA interference (RNAi) on NEDD9 expression in human lung adenocarcinoma A549 cells. We used the siRNA design and analysis software to determine the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Four siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into the pRNAT-CMV3.2 plasmid to construct the recombinant plasmids. These were transformed into the E. coli strain DH5α. The plasmids, after identification by PCR and DNA sequencing, were transfected into the A549 cell line via the liposome method. NEDD9 mRNA and protein in the cells were determined by fluorescence quantitative RT-PCR (FQ-PCR) and western blotting, respectively. The pRNAT-CMV3.2-transfected plasmid was used as a control. Four recombinant plasmids were identified by PCR and sequence analysis, which contained the correct insertion of the designed sequences in the plasmids. FQ-PCR and western blotting showed substantially decreased mRNA and protein expression of the NEDD9 gene in the transfected cells, compared with the control group. In conclusion, the recombinant plasmids expressing the siRNA targeting the NEDD9 gene were successfully constructed, and the siRNA expression vectors inhibited the expression of NEDD9 in A549 cells.

Regulation of neutrophil gelatinase-associated lipocalin expression by C/EBPß in lung carcinoma cells.

Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, has been found to be overexpressed in a variety of tumors, including lung adenocarcinomas. However, the mechanism by which NGAL expression is regulated in lung carcinoma needs further evaluation. In this study, immunohistochemistry was employed to analyze the expression of NGAL in lung carcinoma tissue samples, including lung squamous carcinomas, adenocarcinomas, adenosquamous carcinomas and bronchial alveolar cell carcinomas. The results showed that NGAL was expressed in 82.61% (19/23) of the samples. RT-PCR and immunofluorescent staining showed that NGAL was localized to the cytoplasm in lung carcinoma cell lines. To explore the transcriptional regulation mechanism of NGAL basal expression in lung carcinoma, a 1515-bp fragment (-1431 to +84) of the NGAL promoter region was cloned and a series of deletion and mutation constructs were generated. These constructs were analyzed using the luciferase reporter assay. The results indicated that the cis-acting elements important for the basal activity of NGAL transcription were likely located between -152 and -141. Further analysis using site-directed mutagenesis and the luciferase reporter assay suggested that the C/EBP binding sites were responsible for the activity of the NGAL promoter. Finally, the binding ability and specificity of the transcription factors were determined by electrophoretic mobility-shift assay (EMSA). The results showed that C/EBPß was able to bind to the -152 and -141 segments. Taken together, these findings suggest that NGAL is expressed in lung carcinomas and that NGAL expression is mediated by the binding of C/EBPß to the -152 and -141 segment of the NGAL promoter.