Normal Bronchial Epithelial Cell Line Research Articles

MiR-216a-5p inhibits malignant progression in small cell lung cancer: involvement of the Bcl-2 family proteins.

ObjectivemicroRNAs are regulatory molecules regarded as important in the pathogenesis of different types of tumors. microRNA-216a (miR-216a-5p) has been identified as a tumor suppressor in multiple malignancies. However, the role of miR-216a-5p in the pathogenesis of small cell lung cancer (SCLC) remains obscure. The objective of this study was to investigate the role of the miR-216a-5p/Bcl-2 axis in SCLC pathogenesis.Materials and methodsAll the experimental methods used were as follows: microarray analysis, cell culture, transient, and stable gene transfection; real-time fluorescence PCR; Western blot; flow cytometry for cell cycle analysis; in vitro proliferation assay; in vitro wound healing experiment; in vivo xenograft model in nude mice; and dual luciferase reporter assay. All statistical analyses were carried out using GraphPad Prism 7 software. Statistical significance was analyzed by Student’s t-test or one-way ANOVA. P <0.05 (typically compared with the negative control group) was considered as significant and is marked with an asterisk in the figures.ResultsIn this study, we observed that miR-216a-5p is downregulated in SCLC cell lines compared to that in the normal human bronchial epithelial cell line 16-HBE. In vitro and in vivo experiments demonstrate that upregulation of miR-216a-5p significantly decreased cell growth and migration and its downregulation increased SCLC cell proliferation and migration and influenced the cell cycle. Using bioinformatics analyses, we predicted that the important antiapoptotic gene Bcl-2 is targeted by miR-216a-5p, and we identified a functional miR-216a-5p binding site in the 3′-UTR of Bcl-2 using luciferase reporter assay. Furthermore, we determined that suppression of miR-216a-5p modulated the expression of Bcl-2, Bax, and Bad proteins (Bcl-2 family proteins), while Bcl-2 knockdown abrogated the effect of miR-216a-5p downregulation on cell proliferation, cell migration, and the cell cycle.ConclusionTaken together, these findings suggest that miR-216a-5p regulates SCLC biology via Bcl-2 family proteins. Therefore, our study highlights the role of the miR-216a-5p/Bcl-2 axis in SCLC pathogenesis.

BLACAT1 predicts poor prognosis and serves as oncogenic lncRNA in small-cell lung cancer.

Bladder cancer-associated transcript 1 (BLACAT1) is a novel identified long noncoding RNA (lncRNA) in bladder cancer, and has been suggested to function as an oncogenic lncRNA in several types of human cancer. However, its involvement in the progression of small-cell lung cancer (SCLC) remained unknown. The aim of our study was to investigate the clinical value and biological function in SCLC. In our results, BLACAT1 expression was increased in SCLC tissues and cell lines compared with paired adjacent normal tissues and bronchial epithelial cell lines, respectively. In addition, BLACAT1 high-expression was obviously associated with advanced clinical stage, large tumor size, more lymph node metastasis, present distant metastasis, and poor prognosis. Furthermore, multivariate analysis indicated that high-expression of BLACAT1 acted as an independent poor prognostic factor for overall survival in SCLC cases. The loss-of-function studies suggested that of BLACAT1 suppressed SCLC cell proliferation, migration, and invasion, and induced G0/G1 phase arrest. In conclusion, BLACAT1 is associated with the malignant status and prognosis in patients with SCLC, and functions as an oncogenic lncRNA in regulating cell proliferation and motility, suggesting BLACAT1 may act as a potential target for SCLC prevention and treatment.

Triptolide exhibits antitumor effects by reversing hypermethylation of WIF‑1 in lung cancer cells.

Triptolide (TP) exhibits numerous biological activities, including immunosuppressive, anti‑inflammatory and antitumor effects. The aim of the present study was to investigate the role of TP as a potent therapeutic drug for the treatment of lung cancer and to investigate the underlying therapeutic mechanisms. Western blot analyses and reverse transcription‑quantitative polymerase chain reaction (PCR) were performed to investigate the expression of genes at transcriptional and translational levels, respectively. Methylation‑specific PCR assays were conducted to investigate whether TP affects the Wnt inhibitory factor‑1 (WIF‑1) methylation status and subsequently affects apoptosis, migration or the invasion of lung cancer cells. The results of the present study revealed that the methylation status of WIF‑1 in lung cancer cell lines A549 and H460 was significantly enhanced compared with the human normal bronchial epithelial cell line HBE, whereas treatment with TP was revealed to induce the demethylation of WIF‑1. The present study aimed to investigate whether the biological activities of TP are regulated by inhibiting the Wnt signaling pathway via an increase in WIF‑1 expression levels. The results of the present study revealed that Wnt signaling was suppressed in cells following treatment with TP, which was concluded by the downregulation of Axin 2 and β‑catenin expression. Further investigation demonstrated that the silencing of WIF‑1 expression with small interfering RNA reversed the TP‑induced upregulation of WIF‑1 expression, upregulated Axin 2 and β‑catenin expression and enhanced the activation of Wnt signaling. Notably, an upregulation of cellular tumor antigen p53 expression, and downregulation of matrix metalloproteinase‑9 (MMP‑9) and phosphorylated‑nuclear factor‑κB (NF‑κB) P65 (p‑P65) levels was observed following TP treatment. These results suggest that the Wnt, p53 and NF‑κB signaling pathways mediate the potent antitumor effects of TP. Notably, the silencing of WIF‑1 did not completely recover the levels of p53, MMP‑9 and p‑P65 in cells treated with TP compared with the control cells, thus suggesting that TP exhibits further functions in addition to the targeting of WIF‑1.

Estrogen-related receptor alpha triggers the proliferation and migration of human non-small cell lung cancer via interleukin-6.

Human non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen-related receptor alpha (ERRα), while not ERRβ or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS-2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si-ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL-6, while not IL-8, IL-10, IL-22, VEGF, TGF-β, or TNF-α, in NSCLC cells. Silence of IL-6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF-κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL-6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF-κB/IL-6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.

MicroRNA-216b regulated proliferation and invasion of non-small cell lung cancer by targeting SOX9.

Micro (mi)RNAs are small, evolutionarily conserved and endogenous noncoding RNA molecules between 19 and 24 nucleotides in length. The potential roles of miRNAs in the carcinogenesis and progression of non-small cell lung cancer (NSCLC) have been studied previously. In the present study, it was revealed that miRNA-216b (miR-216b) expression was lower in NSCLC tissue and cell lines compared with that in adjacent healthy lung tissue samples and the normal bronchial epithelial 16HBE cell line, respectively. The ectopic expression of miR-216b inhibited the proliferation and invasion of NSCLC cells in vitro. SRY-Box 9 (SOX9) was identified as a direct target of miR-216b in NSCLC. In addition, SOX9 small interfering RNA was able to mimic the effects of miR-216b overexpression on cell proliferation and invasion in NSCLC. Therefore, the data reported in the present study demonstrate that miR-216b is an important tumor suppressor in NSCLC. These data may contribute to the understanding of the molecular mechanism underlying the carcinogenesis and progression of NSCLC, and provide novel therapies for patients with NSCLC.

The lncRNA MALAT1 contributes to non-small cell lung cancer development via modulating miR-124/STAT3 axis.

lncRNAs can exert many biological effects in several cancer types. MALAT1 is a kind of lncRNA which is greatly overexpressed in several tumors including non-small cell lung cancer (NSCLC). However, the mechanism of MALAT1 in NSCLC still remains unclear. In our current study, we concentrated on the biological mechanism of MALAT1 in NSCLC. It was observed that MALAT1 was significantly upregulated in five human NSCLC cells including A549, H23, H522, H1299, and H460 cells compared to normal bronchial epithelial cell line 16HBE cells. On the contrary, miR-124 was remarkably downregulated, which indicated a potential negative correlation between miR-124 and MALAT1. MALAT1 inhibition can increase miR-124 expression in A549 and H460 cells. In addition, miR-124 mimics were able to repress MALAT1 expression and miR124 inhibitors can promote MALAT1 levels. Then it was found that shMALAT1 can inhibit NSCLC cell proliferation, colony formation and apoptosis, which can be reversed by miR-124 inhibitors. Bioinformatic analysis predicted the correlation between miR-124 and MALAT1. In addition, STAT3 was found to be a novel mRNA target of miR-124. Downregulation of MALAT1 can inhibit NSCLC development by enhancing miR-124 and decreasing STAT3 expression. We speculated that MALAT1can act as a competing endogenous lncRNA (ceRNA) to modulate miR-124/STAT3 in NSCLC. Taken these together, we revealed that MALAT1/miR-124/STAT3 was involved in NSCLC development.

Epithelial cell-basophil interactions stimulate the production of IL-5 in innate allergic inflammation

Epithelial cell-derived interleukin (IL)-33 plays an important role in the initiation and activation of innate allergic inflammation by stimulating the production of Th2 cytokines. It act as a cytokine through the transmembrane receptor ST2L, which is expressed in most of the inflammatory cells, and its activity is neutralized by the soluble spliced variant of ST2 (sST2). Basophil expresses ST2L and produces IL-5 in response to IL-33, however, the role of epithelial cell-basophil interactions in allergic inflammation is still unclear. Cultured Normal Human bronchial epithelial (NHBE) cells and human basophil cell line KU812 cells were used to study the epithelial cell-basophil interactions in the house dust mite (HDM)-induced production of IL-5. Production of IL-5, IL-33 and sST2 was evaluated by ELISA. HDM stimulated the rapid release of IL-33 from cultured NHBE cells at 1 hour after the incubation. Basophil cell line KU812 cells produced IL-5 in response to IL-33, and the conditioned medium of HDM-stimulated NHBE cells stimulated IL-5 production from KU812 cells. Co-culture of NHBE cells with KU812 cells stimulated the production of sST2 time-dependently in response to HDM, and HDM did not stimulate the production of IL-5 in co-cultured cells. These results indicate that epithelial cell-derived IL-33 stimulates the production of IL-5 from basophils, and sST2 is an important regulator of IL-33 in innate allergic inflammation.

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

TIFA Promotes Cell Survival and Migration in Lung Adenocarcinoma

Background/Aims: TNF-α receptor-associated factor (TRAF)-interacting protein with a forkhead-associated (FHA) domain (TIFA) may mediate the impact of TRAF on the development of lung cancer. The current study was conducted to investigate the expression of TIFA in lung adenocarcinoma and its potential role in the regulation of cancer cell proliferation and migration, and its influence on patient survival. Methods: Specimens of lung adenocarcinoma tissues and their adjacent normal lung tissues were obtained from 116 patients who underwent surgical resection of lung cancer. The expression of TIFA in the lung tissues was examined by immunohistochemistry, immunoblotting, and real-time RT-PCR in four different lung cancer cell lines and one normal bronchial epithelial cell line (BEAS-2B). TIFA was silenced by RNAi technique, and cell proliferation was then assessed by the CCK8 method. Furthermore, cell migration was determined by wound-healing trans-well and wound-healing migration assays. Additionally, cell-cycle arrest and apoptosis were assessed by flow cytometry analysis. Results: TIFA was positively detected in 63 (54.3%) out of 116 lung adenocarcinoma specimens, which was significantly higher than the respective rate established in normal tissues adjacent to the tumor (30.1%, p < 0.05). The overall survival rate was significantly lower in the patients with positive TIFA expression than that in the patients with negative TIFA expression (p < 0.05). TIFA was also highly expressed in the lung cancer cell lines (H1299, H1975, and HCC827) tested. It is noteworthy that siRNA suppressed the expression of TIFA, which contributed to the attenuation of cell proliferation and migration, but promoted cell-cycle arrest and apoptosis. Furthermore, the silencing of TIFA caused upregulation of p53, p21, and cleaved-caspase-3, but downregulation of Bcl-2, cyclin D1, and CDK4, as well as phosphorylation of IKKß, IκB, and p65. Conclusions: TIFA may serve as a biomarker in the prediction of lung adenocarcinoma. Furthermore, TIFA may modulate lung cancer cell survival and proliferation through regulating the synthesis of apoptosis-associated proteins.

MicroRNA-4465 suppresses tumor proliferation and metastasis in non-small cell lung cancer by directly targeting the oncogene EZH2

MicroRNA-26 (miR-26) has been reported to be connected with tumor progression. MicroRNA-4465 (miR-4465) was one member of miR-26 family, however, the role of miR-4465 in non-small cell lung cancer (NSCLC) was unknown. This study was aimed to explore the function of miR-4465 and investigate whether miR-4465 can be a potential target for treating human NSCLC. QRT-PCR was applied to evaluate the miR-4465 expression levels in NSCLC cells. Then, we demonstrated the role of miR-4465 in NSCLC cells biological characteristics through detecting proliferation, migration and invasion. Luciferase reporter assay and TargetScan were applied to explore the potential target gene of miR-4465. In this study, we found that the miR-4465 expression levels in NSCLC cell lines were significantly reduced when compared to the normal human bronchial epithelial cell lines. And, over expression of miR-4465 could restrain the proliferation, migration and invasion of NSCLC. Moreover, MiR-4465 reduced EZH2 protein expression through the binding sites in 3′ -UTR of the EZH2 mRNA, indicating EZH2 may be a direct target gene of miR-4465. Conclusively, miR-4465 suppressed cancer cells proliferation and metastasis by directly targeting the oncogene EZH2 and it may serve as a new potential therapeutic target in NSCLC.

Downregulation of microRNA-301a inhibited proliferation, migration and invasion of non-small cell lung cancer by directly targeting DLC1.

Increasing evidence has indicated that the abnormal expression of microRNAs contributes to tumorigenesis and tumor development. Understanding the roles of microRNAs in non-small cell lung cancer (NSCLC) might provide valuable information for therapeutic strategies in the therapy for patients with NSCLC. In the present study, significant upregulation of microRNA (miR)-301a was observed in NSCLC tissues and cell lines compared with normal adjacent tissues and a normal human bronchial epithelial cell line. The inhibition of miR-301a suppressed proliferation, migration and invasion of NSCLC cells. Functional analyses indicated that DLC1 was a direct target of miR-301a in NSCLC. Inhibiting miR-301a expression decreased DLC1 expression at mRNA and protein levels. Moreover, DLC1 knockdown partially reversed the inhibition of proliferation, migration and invasion induced by miR-301a knockdown in NSCLC cells. Therefore, these findings may provide novel insights into the molecular mechanisms of miR-301a in proliferation, migration and invasion of NSCLC cells. The findings also indicated that miR-301a may act as a novel potential therapeutic target for patients with NSCLC.

AEG-1 mRNA expression in non-small cell lung cancer is associated with increased tumor angiogenesis

Astrocyte-elevated gene-1 (AEG-1) is implicated in the oncogenesis and angiogenesis of various types of human malignant disease. However, the angiogenesis roles of AEG-1 in non-small cell lung cancer (NSCLC) remain to be further elucidated. In the present study, the expression level of AEG-1 mRNA in seven human lung cell lines and 89 paired tissue samples (tumor tissues (TTs) and pair-matched normal adjacent tissues (PMNATs)) from NSCLC patients was detected by real-time PCR. Staining of vascular endothelial growth factor (VEGF) and intratumoral microvessel density (iMVD, labeled by CD105) were assessed by immunohistochemistry. Furthermore, cell migration and invasion were evaluated by wound healing assay and transwell assays. AEG-1 mRNA level was significantly higher in human lung cancer cells and TTs than that in human normal bronchial epithelial cell line 16HBE and PMNATs, respectively (P<0.001). Higher AEG-1 mRNA level in patients with NSCLC was correlated with clinical stages (P=0.028), differentiation (P=0.042), and lymph node metastasis (P=0.004). Moreover, Upregulated AEG-1 mRNA expression level was associated with higher tumor angiogenesis, reflected by the increase of VEGF expression and iMVD counting (P=0.021, P<0.001). However, 95D cell line transfected with AEG-1 siRNA oligos (siAEG-1) exhibited no significant decrease of cell invasion or migration capacities when compared with the control cells (P>0.05).These results suggested that AEG-1 may play important roles at the transcription level in malignant transformation and tumor angiogenesis in NSCLC, and anti-AEG-1 mRNA expression may be a novel potential strategy for anti-angiogenic therapy of NSCLC.

MiR-155 inhibits proliferation and invasion by directly targeting PDCD4 in non-small cell lung cancer.

BackgroundMicroRNAs are often abnormally expressed in human non‐small cell lung cancer (NSCLC) and are thought to play a critical role in the emergence or maintenance of NSCLC by binding to its target messenger RNA. We assessed the effects of miR‐155 on cell proliferation and invasion to elucidate the role played by miR‐155/PDCD 4 in NSCLC.MethodsQuantitative reverse transcription‐PCR, Western blotting, and cell counting kit‐8, luciferase, and transwell invasion assays were conducted on a normal human bronchial epithelial cell line (BEAS‐2B) and three NSCLC cell lines (SPC‐A‐1, A549, and H2170).ResultsWe confirmed that miR‐155 was upregulated, while PDCD 4 messenger RNA and protein levels were downregulated in NSCLC cell lines. miR‐155 negatively regulated PDCD 4 at both transcriptional and post‐transcriptional levels. Moreover, PDCD 4 was forecast as an assumed target of miR‐155 using bioinformatic methods and we demonstrated that PDCD 4 was a direct target of miR‐155 using luciferase reporter assays. Furthermore, PDCD 4 overexpression could restrain NSCLC proliferation and invasion induced by miR‐155.ConclusionOur results collectively demonstrate that miR‐155 exerts an oncogenic role in NSCLC by directly targeting PDCD 4.

Extracellular heat shock protein 90\u03b1 mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling

BackgroundThe disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported.MethodsTo investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90β respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and β-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting.ResultsThe epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and β-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90β that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells.ConclusionsThe eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

The lncRNA XIST exhibits oncogenic properties via regulation of miR-449a and Bcl-2 in human non-small cell lung cancer.

Long non-coding RNAs (lncRNAs) are associated with the occurrence, development and prognoses of non-small cell lung cancer (NSCLC). In the present study, we investigated the functional mechanisms of the lncRNA XIST in two human NSCLC cell lines, A549 and NCI-H1299. In all the 5 NSCLC cell lines (NL9980, NCI-H1299, NCI-H460, SPC-A-1 and A549) tested, the expression levels of XIST were significantly elevated, as compared with those in normal human bronchial epithelial cell line BEAS-2B. In A549 and NCI-H1299 cells, knockdown of XIST by siRNA significantly inhibited the cell proliferation, migration and invasion, and promoted cell apoptosis. Furthermore, XIST knockdown elevated the expression of E-cadherin, and suppressed the expression of Bcl-2. Moreover, knockdown of XIST significantly suppressed the tumor growth in NSCLC A549 xenograft mouse model. Bioinformatic analysis and luciferase reporter assays revealed that XIST was negatively regulated by miR-449a. We further identified reciprocal repression between XIST and miR-449a, which eventually influenced the expression of Bcl-2: XIST functioned as a miRNA sponge of miR-449a, which was a negative regulator of Bcl-2. These data show that expression of the lncRNA XIST is associated with an increased growth rate and metastatic potential in NSCLC A549 and NCI-H1299 cells partially through miR-449a, and suggest that XIST may be a potential prognostic factor and therapeutic target for patients with NSCLC.

SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth

SIRT2 is a NAD-dependent deacetylase and inhibition of SIRT2 has a broad anticancer activity. Here we report that SPOP binds to SIRT2 and mediates its degradation by the 26S proteasome, which can be blocked by MG132 treatment. We also found that the levels of SPOP significantly decreased, while the levels of SIRT2 significantly increased in non-small cell lung cancer (NSCLC) cell lines, compared to normal bronchial epithelial cell line and NSCLC specimens, compared to the paired non-tumor lung tissue. Furthermore, SPOP can suppress NSCLC cell growth. Notably, mutations in NSCLC inhibit the abilities of SPOP to degrade SIRT2 and suppress NSCLC cell growth. These results reveal a novel regulation of SIRT2 by SPOP mediated degradation, which is important for the growth of lung tumor cells.

Increased Expression of Long Non-Coding RNA BCAR4 Is Predictive of Poor Prognosis in Patients with Non-Small Cell Lung Cancer.

Lung cancer is the most common human cancer, and the majority of lung cancer cases are categorized as non-small cell lung cancer (NSCLC). Long non-coding RNAs (lncRNAs) play key roles in the development and progression of human cancers. LncRNA breast cancer anti-estrogen resistance 4 (BCAR4) has been identified as an oncogenic lncRNA involved in the progression of breast cancer and osteosarcoma. However, the clinical significance of the lncRNA BCAR4 in NSCLC remains largely unclear. In the present study, real-time quantitative reverse transcriptase-polymerase chain reaction was used to examine the relative level of lncRNA BCAR4 in 68 cases of NSCLC tissues and their adjacent non-tumor tissues. Our data showed that the expression level of lncRNA BCAR4 was significantly higher in NSCLC tissues compared to their matched non-tumor tissues. Moreover, BCAR4 expression was significantly upregulated in NSCLC cell lines, when compared to the normal human bronchial epithelial cell line BEAS-2B. In addition, the BCAR4 expression was associated with the lymph node metastasis, distant metastasis and clinical stage, but not with the age, sex, tumor size, histological grade, and histological type. The increased expression of BCAR4 was significantly associated with poorer 5-year overall survival rate of NSCLC patients. Multivariate survival analysis indicated that BCAR4 was an independent prognostic factor for NSCLC patients. Taken together, our study suggests that the upregulation of lncRNA BCAR4 expression plays a promoting role in the malignant progression of NSCLC. Thus, BCAR4 is a potential biomarker for NSCLC progress and a therapeutic target for NSCLC.

P3.01-042 Lung Cancer Cells Can Stimulate Functional and Genotypic Modifications in Normal Bronchial Epithelial Cells: Topic: Functional Biology in Lung Cancer

Normal lung epithelium cells may act in concert with tumor cells, given that bystander effects may exist between the two. This interaction may lead to inappropriate activation of pro-oncogenic signaling pathways, which may result in high mutational load and tumor heterogeneity. The aim of this project is to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalized normal bronchial epithelial cell line. A normal bronchial epithelial cell line (HBEC4) was exposed to A549 (adenocarcinoma), H460 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) NSCLC cell lines in a trans-well co-culture system. Cellular characteristics were examined using a Cytell Cell Imaging System (cell number, viability, apoptosis, cell cycle). The gene expression profile was also determined in terms of inflammatory mediators, stem cell markers (RT-PCR) and miRNA profiling (Nanostring). The proliferative effect of NSCLC cancer exosomes was also examined (BrdU ELISA) on the HBEC4 cell line. A number of functional and gene modifications were observed in the HBEC4 cell line after seven days of co-culture. While patterns were similar amongst all NSCLC subtypes, SK-MES-1 elicited the most significant effects in terms of cell number, viability, cell cycle progression and proliferative potential of isolated cancer exosome fraction. Promotion of both inflammatory mediators and stem cell marker expression was evident at the mRNA level. There was no apparent consensus between NSCLC subtypes and miRNA expression, as exposure to each cell line resulted in distinct profiles of miRNAs in HBEC4 cells. Bioinformatic analysis of miRNA target genes, demonstrated that pathways such as p53, MAPK, VEGF, TLR and Wnt were amongst those altered. Cancer cells may promote significant genotypic and phenotypic alterations within the normal lung epithelium though multiple mechanisms. These modifications may, in part, contribute to the heterogeneity of lung cancer tumors and influence response to both chemotherapeutics and targeted agents.

P2.03b-024 microRNA-330-3p Promotes Brain Metastasis of Non-Small Cell Lung Cancer (NSCLC) by Activating MAPK/MEK/ERK Signaling Pathway: Topic: Biomarkers

Brain metastasis (BM) is associated with poor prognosis, recurrence, and death in patients with non-small cell lung cancer (NSCLC). MicroRNAs (miRNAs) play an essential role in the development of NSCLC. We investigated miRNAs that serve as biomarkers to differentiate NSCLC patients with and without BM, and explored the underlying mechanism. The significant association for BM of NSCLC was analyzed by univariate and multivariable logistic regression analysis of the clinical features of NSCLC patients with BM and without BM. The effects of miR-330-3p on NSCLC cells (A549 and HCC827) were investigated using assays of cell viability, migration, invasion, cell cycle, Western blot, and a tumor xenograft and brain metastatic xenografts models. The miR-330-3p targets were identified using microarray analysis and verified by luciferase reporter assay. Female gender (OR = 3.15，P = .003), age under 60 (OR = 4.54, P < .001), adenocarcinoma (OR = 3.57, P = .01), EGFR19 exon mutation (OR = 3.76, P = .05), N2 (OR = 8.23, P = .01) or N3 (OR = 29.38, P < .001) were highly associated with BM in NSCLC patients. The miR-330-3p was expressed at a higher level in BM+ than in BM- NSCLC serum samples, and also higher in NSCLC cell lines than the normal human bronchial epithelial cell line. Cell proliferation, migration and invasiveness of 2 representative NSCLC cell lines (A549 and HCC827) were increased by over-expressing miR-330-3p using a lentivirus carrying a sequence of miR-330-3p, and decreased by knockdown of miR-330-3p. In nude mice receiving subcutaneous A549 and HCC827 cell inoculation, tumor growth were significantly faster in mice receiving A549 and HCC827 cell permanently expressing exogenous miR-330-3p, and slower in cells permanently expressing an anti- miR-330-3p sequence. The mice receiving cancer cells stably expressing exogenous miR-330-3p injection directly into the brain almost developed multiple metastatic foci, while developed a smaller orthotopic tumor in mice receiving injection of cells expressing an anti- miR-330-3p sequence. GRIA3 was identified as a direct target of miR-330-3p using luciferase reporter assays. Real-time PCR and Western blot confirmed that miR-330-3p downregulated GRIA3 expression. MEK inhibition suggested that GRIA3 was regulated by miR-330-3p via MAPK/MEK/ERK signaling pathway. These results support the oncogenic role of miR-330-3p in NSCLC brain metastasis, providing a rationale for miRNA-targeted therapeutic strategies.

MiR-382 inhibits tumor progression by targeting SETD8 in non-small cell lung cancer.

Previous studies showed that miR-382 plays important roles in several types of cancers. Nevertheless, its expression and function in non-small cell lung cancer (NSCLC) remains largely unknown. In this study, we found that miR-382 expression was evidently downregulated in NSCLC tissue and cell lines in comparison with the adjacent normal tissues and human bronchial epithelial cell line (16HBE). Moreover, the expression levels of miR-382 were significantly associated with last-stage and tumor metastasis in NSCLC patients. In addition, exogenous miR-382 evidently inhibited NSCLC cell proliferation, migration and invasion in vitro. We also revealed SETD8 as a direct target of miR-382 in NSCLC, and restored SETD8 partially reversed the negative effects miR-382 on NSCLC cells. In total, our study demonstrated that miR-382 dysregulated in NSCLC and involved in NSCLC tumorigenesis and metastasis by suppressing SETD8 expression, which may help to identify effective therapies for NSCLC treatment.