Paired Adjacent Normal Lung Tissues Research Articles

Analysis of miR-375-3p, miR-197-3p, and miR-15a-5p Expression and Their Clinical Relevance as Biomarkers in Lung Cancer.

Background: MicroRNAs (miRNAs) play an important regulatory role and serve as biomarkers in various human cancers. However, their role in the prognosis and predicting response to therapy in Indian lung cancer patients is not fully explored. Methods: We collected surgically resected tumors and paired adjacent normal lung tissues from 29 early-stage and tissue biopsies from 103 locally advanced and metastatic lung cancer patients in this prospective study. We quantified the expression levels of miR-375-3p, miR-197-3p, and miR-15a-5p using TaqMan Advanced miRNA Assays. We correlated miRNAs expression with response to therapy and survival outcomes. Results: The median age of lung cancer patients was 60 years. We found significant overexpression of miR-375-3p and miR-197-3p in the tumors compared to paired normal lung tissues. Higher expression of miR-375-3p was observed more frequently in responders compared to nonresponders. The expression of miR-375-3p and miR-197-3p was able to differentiate patients of lung adenocarcinoma from lung squamous cell carcinoma. We did not find any correlation between miRNAs expression and survival outcomes. Conclusion: Overexpression of miR-375-3p and miR-197-3p might contribute to lung carcinogenesis. The expression of miR-375-3p may assist in predicting therapeutic response. More prospective studies are warranted to evaluate the potential of miR-375-3p as a predictive biomarker of response to therapy.

Tumor Immune Microenvironment Characteristics and Their Prognostic Value in Non-Small-Cell Lung Cancer.

IntroductionCancer progression is determined not only by the malignant behavior of tumors but also by the immune microenvironment. The tumor immune microenvironment also plays a pivotal role in determining the clinical response of non-small-cell lung cancer (NSCLC) to immunotherapies. To understand the possible mechanisms and explore new targets in lung cancer immunotherapy, we characterized the immune profiles in NSCLC patients.MethodsSeventy-one NSCLC patients who underwent radical resection were selected. The immune cell composition in paired tumor and adjacent normal lung tissues was tested by flow cytometry. The associations of tumor immune microenvironment characteristics with clinicopathological factors and overall survival were analyzed. Kaplan–Meier curves and Cox proportional hazards models were used to determine differences in survival.ResultsCompared with adjacent normal lung tissues, an increased proportion of CD45+ hematopoietic-derived cells, CD4+ T cell subtypes, Tregs and B cells was observed in tumor samples with a reduced frequency of myeloid cell populations. There was no significant increase in total CD8+ T cells, but both PD1+ and CD38+ CD8+ T cells were significantly enriched in tumor samples and statistically significantly associated with tumor size. In addition, positive CD38 expression was highly correlated with PD1 positivity. A high proportion of CD8+ T cells and a low percentage of PD1+ CD8+ T cells were statistically significantly associated with better survival in stage II and III patients, whereas a low frequency of CD38+ CD8+ T cells was statistically significantly associated with better survival in all patients and identified as an independent prognostic factor (p=0.049).ConclusionWe profiled the immune cells in the tumor tissues of NSCLC patients using flow cytometry. The results revealed significant enrichment of infiltrating immune cells. A strong correlation was identified between CD38 and PD-1 expression on CD8+ T cells in tumors. CD8+ T cells and their subtypes play a critical role in the prediction of prognosis.

MITF functions as a tumor suppressor in non-small cell lung cancer beyond the canonically oncogenic role.

Microphthalamia-associated transcription factor (MITF) is a critical mediator in melanocyte differentiation and exerts oncogenic functions in melanoma progression. However, the role of MITF in non-small cell lung cancer (NSCLC) is still unknown. We found that MITF is dominantly expressed in the low-invasive CL1-0 lung adenocarcinoma cells and paired adjacent normal lung tissues. MITF expression is significantly associated with better overall survival and disease-free survival in NSCLC and serves as an independent prognostic marker. Silencing MITF promotes tumor cell migration, invasion and colony formation in lung adenocarcinoma cells. In xenograft mouse model, MITF knockdown enhances metastasis and tumorigenesis, but decreases angiogenesis in the Matrigel plug assay. Whole transcriptome profiling of the landscape of MITF regulation in lung adenocarcinoma indicates that MITF is involved in cell development, cell cycle, inflammation and WNT signaling pathways. Chromatin immunoprecipitation assays revealed that MITF targets the promoters of FZD7, PTGR1 and ANXA1. Moreover, silencing FZD7 reduces the invasiveness that is promoted by silencing MITF. Strikingly, MITF has significantly inverse correlations with the expression of its downstream genes in lung adenocarcinoma. In summary, we demonstrate the suppressive role of MITF in lung cancer progression, which is opposite to the canonical oncogenic function of MITF in melanoma.

Combined evaluation of the expression status of CD155 and TIGIT plays an important role in the prognosis of LUAD (lung adenocarcinoma)

The interaction between CD155 and its high-affinity ligand TIGIT is being increasingly investigated in various solid tumors. However, the prognostic significance of CD155 and TIGIT in lung adenocarcinoma (LUAD) remains unclear. In this study, immunohistochemistry was applied in 334 LUAD cases to evaluate the expression of CD155 and TIGIT. Western blotting was conducted in 5 paired primary LUAD and adjacent normal lung tissues. Our results reveal that CD155 and TIGIT are overexpressed in LUAD tissues and that aberrant overexpression is closely correlated with poor clinical outcomes (P < 0.01). The multivariate model also shows that CD155 expression is an independent risk factor for LUAD (RR, 1.34; P = 0.036). Moreover, patients expressing high CD155 and TIGIT simultaneously presented shorter overall survival (OS) (P < 0.01) and progression-free survival (PFS) (P < 0.01). These findings suggest that CD155 and TIGIT can make up a prognosticating tool to predict clinical outcomes, thereby contributing to personalized medical care in LUAD.

Genome-wide profiling reveals alternative polyadenylation of mRNA in human non-small cell lung cancer

BackgroundLung cancer is the second most common cancer with an extremely poor overall survival rate. Post-transcriptional regulation of gene expression play many important roles in human cancer, and one of the potential mechanisms underlying this is alternative mRNA maturation at its 3′ untranslated regions (3′-UTRs).MethodsCancer tissues and paired adjacent normal lung tissues from 26 patients diagnosed with non-small cell lung cancer (NSCLC) were analyzed by in vitro transcription-sequencing alternative polyadenylation sites (IVT-SAPAS). 41,773,101 reads in average were obtained from each paired sample. A potential regulation of Cleavage Stimulation Factor Subunit 2 (CSTF2) on 3′UTR length of genes was tested in H460 cells.Results1439 (10.26%) genes showed up-regulated expression and 1364 (9.72%) genes showed down-regulated expression in lung cancer tissue versus normal lung tissue, and shorten 3′UTR in cancer tissue was detected in cancer tissues collected from 96.2% (25/26) patients, indicating lung cancer tend to have shortened 3′UTRs of these identified genes. KEGG analysis showed 1855 genes with shorten 3′UTR were enriched in mTOR signaling, ubiquitin mediated proteolysis and RNA degradation. Knocking down CSTF2 expression in H460 cells results in 3′UTR elongation of genes that was identified to be with shortened length in cancer tissues.ConclusionAlternative polyadenylation (APA) site-switching of 3′UTRs is prevalent in NSCLC, and CSTF2 may serve as an oncogene regulates the 3′UTR length of cancer related genes in NSCLC.

Clinical value of microRNA-198-5p downregulation in lung adenocarcinoma and its potential pathways.

Lung adenocarcinoma (LUAD), the main subtype of non-small cell lung cancer, is known to be regulated by various microRNAs (miRs/miRNAs); however, the role of miR-198-5p in LUAD has not been clarified. In the present study, the clinical value of miR-198-5p in LUAD and its potential molecular mechanism was evaluated. miR-198-5p expression was examined by reverse transcription-quantitative PCR (RT-qPCR) in 101 paired LUAD and adjacent normal lung tissues. Subsequently, the miR-198-5p expression level was determined from microarray data from the Gene Expression Omnibus, ArrayExpress and by meta-analyses. Furthermore, the target mRNAs of miR-198-5p from 12 miRNA-mRNA predictive tools were intersected with The Cancer Genome Atlas (TCGA)-based differentially expressed genes. In addition, Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to determine the possible mechanism of miR-198-5p in LUAD. The Search Tool for the Retrieval of Interacting Genes/Proteins database was employed to construct a protein-protein interaction network among the potential target genes of miR-198-5p. The results showed that miR-198-5p expression was lower in LUAD tissues than in adjacent non-cancerous lung tissues (4.469±2.495 vs. 5.301±2.502; P=0.015). Meta-analyses, including the data from the present study and online microarray data, also verified the downregulation of miR-198-5p in 584 cases of LUAD. The expression of miR-198-5p was associated with the age, blood vessel invasion, Tumor-Node-Metastasis stage, and lymph node metastasis of patients with LUAD and served as an independent prognostic factor for survival. The hub genes of miR-198-5p were upregulated in LUAD, according to TCGA and The Human Protein Atlas. For the KEGG pathway analysis, the most enriched KEGG pathway was the p53 signaling pathway (P=1.42×10−6). These findings indicated that the downregulation of miR-198-5p may play a pivotal role in the development of LUAD by targeting various signaling pathways.

Nuclear TAZ activity distinctly associates with subtypes of non-small cell lung cancer

The transcription co-factor TAZ plays critical roles in the regulation of human carcinogenesis. However, the pathological role for TAZ in lung cancer has remained incompletely understood. TAZ expression was examined by immunohistochemistry for 163 NSCLC tissues. TAZ expression was also examined by western blotting for 20 frozen paired NSCLC and adjacent normal lung tissues. We report that TAZ is overexpressed in non-small cell lung cancer (NSCLC) tissues and correlates with shorter patient survival. Intriguingly, we find that TAZ is overexpressed primarily in lung squamous cell carcinomas (LUSC) but not lung adenocarcinomas (LUAD) compared to normal lung tissues, and that the expression levels of TAZ are significantly higher in LUSC than LUAD. The nuclear localization of TAZ correlates worse clinical outcomes in LUSC, but not LUAD, further suggesting a prognostic value for activated TAZ in LUSC. A meta-analysis of the public datasets from TCGA, Broad institute, and Oncomine shows that the TAZ gene (WWTR1) copy numbers are significantly increased in LUSC and correlate with the increase of TAZ mRNA expression, suggesting that TAZ is overexpressed in LUSC at least partly through gene amplifications. Collectively, our results suggest that TAZ expression distinctly associates with subtypes of NSCLC and may be useful for developing novel therapeutics treating LUSC.

CASTOR1 suppresses the progression of lung adenocarcinoma and predicts poor prognosis.

As a naturally occurring inhibitor of mammalian target of rapamycin (mTOR), accumulated evidence has confirmed that CASTOR1 plays a pivotal role in regulating the progression of human malignancies. However, the function of CASTOR1 in the development of lung adenocarcinoma is still unclear. Here we report that the expression of CASTOR1 is significantly reduced in lung adenocarcinoma cell lines as compared with that in normal lung epithelial cells. Cellular studies further revealed that ectopic CASTOR1 expression led to a significant suppression of the cellular proliferation, migration, and invasion of PC-9 cells. To further test the role of CASTOR1 in human patients with lung cancers, tumor tissues derived from lung cancer patients and paired adjacent normal lung tissues were collected for immunohistochemical​​​​​​, biochemical, and molecular biology analysis. Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction analysis is revealed that CASTOR1 was significantly reduced in tumor tissues as compared with that in paired normal lung tissues. With immunostaining and retrospective analysis of pathological samples from lung cancer patients further revealed that expression of CASTOR1 was negatively correlated with smoking history, TNM stage, lymphnode metastasis, and distant metastasis. Furthermore, Kaplan-Meier survival analysis indicated that patients with low CASTOR1 expression levels had a significantly worse 5-year survival rate than those with high CASTOR1 expression. Tofurther explore the molecular actions of CASTOR1 in lung cancer development, biochemical assays were performed and the results suggested that ectopic CASTOR1 expression resulted significant suppression of mTOR and downstream S6K phosphorylation, indicating that CASTOR1 might regulate the progression of lung adenocarcinoma by controlling mTOR activation. Thus, these findings demonstrated that CASTOR1 inhibits the tumorigenesis of lung adenocarcinoma cells and might serve as a potential therapeutic target or prognostic marker for human patients with lung adenocarcinoma.

Minichromosome maintenance protein 2 correlates with the malignant status and regulates proliferation and cell cycle in lung squamous cell carcinoma.

BackgroundMinichromosome maintenance protein 2 (MCM2), which is a member of MCM family, has been found to be a relevant marker for progression and prognosis in a variety of human cancers. Due to lack of effective therapeutic target in lung squamous cell carcinoma (LUSC) patients, the aim of our study was to screen and identify biomarkers which are associated to clinicopathological characteristics including prognosis in LUSC patients.MethodsThe expression status of MCM2 in lung cancer was analyzed using the publicly available Gene Expression Omnibus databases (GSE3268 and GSE10245). The mRNA and protein expression of MCM2 in lung cancer tissues and cell lines was detected by quantitative real-time PCR and Western blot, and the association between MCM2 expression and clinicopathological factors was analyzed by immunohistochemistry. The loss-of-function study of MCM2 was conducted in LUSC cell lines.ResultsIn our study, we found MCM2 expression was increased in LUSC tissues compared with paired adjacent normal lung tissues or lung adenocarcinoma tissues through analyzing microarray data sets (GSE3268 and GSE10245), which confirmed that MCM2 mRNA and protein were overexpressed in LUSC tissues and cell lines. Meanwhile, we analyzed the association between MCM2 protein expression and clinicopathological characteristics of LUSC patients, and found high expression of MCM2 protein was obviously associated with malign differentiated degree, advanced clinical stage, large tumor size, more lymph node metastasis and present distant metastasis. The survival analysis showed MCM2 overexpression was an independent unfavorable prognostic factor for LUSC patients.ConclusionMCM2 is involved in the development and progression of LUSC as an oncogene, and thereby may act as a potential therapeutic target for LUSC patients.

The clinical and prognostic value of polo-like kinase 1 in lung squamous cell carcinoma patients: immunohistochemical analysis.

Polo-like kinase 1 (PLK1) has been suggested to serve as an oncogene in most human cancers. The aim of our study is to present more evidence about the clinical and prognostic value of PLK1 in lung squamous cell carcinoma patients. The status of PLK1 was observed in lung adenocarcinoma, lung squamous cell carcinoma, and normal lung tissues through analyzing microarray dataset (GEO accession numbers: GSE1213 and GSE 3627). PLK1 mRNA and protein expressions were detected in lung squamous cell carcinoma and normal lung tissues by using quantitative real-time PCR (qRT-PCR) and immunohistochemistry. In our results, the levels of PLK1 in lung squamous cell carcinoma tissues were higher than that in lung adenocarcinoma tissues. Compared with paired adjacent normal lung tissues, the PLK1 expression was increased in lung squamous cell carcinoma tissues. Furthermore, high expression of PLK1 protein was correlated with differentiated degree, clinical stage, tumor size, lymph node metastasis, and distant metastasis. The univariate and multivariate analyses showed PLK1 protein high expression was an unfavorable prognostic biomarker for lung squamous cell carcinoma patients. In conclusion, high expression of PLK1 is associated with the aggressive progression and poor prognosis in lung squamous cell carcinoma patients.

Expression and clinicopathological significance of S100 calcium binding protein A2 in lung cancer patients of Chinese Han ethnicity

BackgroundS100 family of calcium-binding proteins plays a significant role in the process of many kinds of tumors, including lung cancer. As an important member of this family, S100 calcium binding protein A2 (S100A2) has been confirmed to be associated with many biological processes, and has an abnormal expression in non-small cell lung cancer (NSCLC). However, the S100A2 status in lung cancer is still controversial and undefined. MethodsWe evaluated the pattern and distribution of S100A2 in 109 cases of lung cancer, including five histological types (47 adenocarcinoma, 46 squamous cell carcinoma, 7 small cell carcinoma, 3 large cell carcinoma, and 6 atypical carcinoid), and 30 cases of paired adjacent normal lung tissues by means of immunohistochemistry. ResultsCompared with the normal tissues (0/30), S100A2 experienced a dramatically upward trend of positive expression in lung cancer, with a positive rate of 68/109 (P<0.001). Specifically, squamous cell carcinoma, with 34/12, had the highest expression ratio, followed by large cell carcinoma (2/1), adenocarcinoma (31/16), and atypical carcinoid (1/5) respectively, while no S100A2 protein was detected in small cell carcinoma. Meanwhile, we firstly demonstrated that the high expression of S100A2 was significantly associated with the incidence of lymph node metastasis in adenocarcinoma (P=0.013). ConclusionsThe association between high S100A2 expression and NSCLC at the level of tissue, and S100A2 may serve as an effective biomarker for the diagnosis and prognosis of NSCLC in future.

Role of AXL expression in non-small cell lung cancer.

The present study aimed to investigate the expression profile of AXL in non-small cell lung cancer (NSCLC) and its clinical significance. The current study included 257 NSCLC patients, tyrosine-protein kinase receptor UFO (AXL) expression in paired lung cancer and adjacent normal lung tissues of NSCLC patients were compared by immunohistochemistry, western blot analysis and quantitative polymerase chain reaction (qPCR). These methods were used to detect the expression of the AXL gene and protein in fresh tissues from 35 patients. Small interfering RNA (siRNA) was transfected into the H1299 lung cancer cell line to knock down AXL expression; the effects of AXL-siRNA on cell proliferation and migration were examined by MTT and Transwell migration assay, respectively. It was found that AXL staining density in lung cancer tissues was significantly increased compared with adjacent normal lung tissues (55.25 vs. 26.85%; P<0.01); and the expression level of AXL in NSCLC patients was significantly associated with the degree of tumor differentiation (P<0.01) and the clinical stage of disease (P<0.01). Western blotting and qPCR showed that AXL expression was significantly higher in cancer tissues compared with that in adjacent lung tissue (P<0.05). Additionally, the current study also showed that AXL-siRNA inhibited H1299 cell proliferation and migration in vitro. The present study demonstrates the association between increased expression of AXL in NSCLC and the low differentiation phenotype, and its effects on cell proliferation and migration, suggesting its potential clinical values for the prognosis of NSCLC.

Elevated expression of WWP2 in human lung adenocarcinoma and its effect on migration and invasion

Lung cancer has been a hot area of research because of its high incidence and mortality. In this study, WWP2, an E3 ubiquitin ligase, is proposed to be an oncoprotein contributing to lung tumorigenesis. We attempted to determine if WWP2 gene expression is correlated with the development of human lung adenocarcinoma. Real-time PCR and western blotting were used to detect the expression of WWP2 in 65 paired lung adenocarcinoma and adjacent normal lung tissues. We found that WWP2 expression was elevated in lung adenocarcinoma tissues and was correlated with the tumor differentiation stage, TNM stage and presence of lymph node metastasis. We performed CCK-8 and colony formation assays and found that down-regulation of WWP2 inhibited proliferation in A549 and SPC-A-1 cells. A wound healing assay and trans-well invasion assays showed that down-regulation of WWP2 inhibited the migration and invasion of lung adenocarcinoma cells. It could be predicted from these data that elevated expression of WWP2 may play a role in facilitating the development of lung adenocarcinoma.

MDK Protein Overexpression Correlates with the Malignant Status and Prognosis of Non-small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor and is overexpressed in various types of human cancer. However, little is known about the clinical significance of MDK in non-small cell lung cancer (NSCLC). The aim of this study was to measure MDK protein levels in patients with NSCLC and to explore its clinical significance. The expression status of MDK in NSCLC at Gene Expression Omnibus (GEO accession number: GSE19804) was observed. The expression of MDK mRNA and protein was examined in NSCLC tissues and normal lung tissues through real-time PCR and Western blot. Meanwhile, the relationship of MDK protein expression levels with clinical characteristics of 186 NSCLC patients was analyzed by immunohistochemistry. MDK expression was increased in NSCLC tissues compared with paired normal lung tissues in microarray data (GSE19804). MDK mRNA and protein expression were obviously increased in NSCLC tissues than in paired adjacent normal lung tissues. Using immunohistochemistry, MDK protein overexpression was positively correlated with status of clinical stage, T classification, N classification, and M classification in NSCLC patients. In survival analysis, patients with higher MDK protein expression had a significantly shorter overall survival time than did patients with lower MDK protein expression. Multivariate analysis indicated that the MDK protein overexpression was an independent poor prognostic indicator for patients with NSCLC. MDK plays an important role in NSCLC progression and prognosis and may act as a convincing prognostic indicator for NSCLC patients.

PHLPP2 Downregulation Contributes to Lung Carcinogenesis Following B[a]P/B[a]PDE Exposure.

The carcinogenic capacity of B[a]P/B[a]PDE is supported by epidemiologic studies. However, the molecular mechanisms responsible for B[a]P/B[a]PDE-caused lung cancer have not been well investigated. We evaluated here the role of novel target PHLPP2 in lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. We used the Western blotting, RT-PCR, [(35)S]methionine pulse and immunohistochemistry staining to determine PHLPP2 downregulation following B[a]P/B[a]PDE exposure. Both B[a]PDE-induced Beas-2B cell transformation model and B[a]P-caused mouse lung cancer model were used to elucidate the mechanisms leading to PHLPP2 downregulation and lung carcinogenesis. The important findings were also extended to in vivo human studies. We found that B[a]P/B[a]PDE exposure downregulated PHLPP2 expression in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The ectopic expression of PHLPP2 dramatically inhibited cell transformation upon B[a]PDE exposure. Mechanistic studies showed that miR-205 induction was crucial for inhibition of PHLPP2 protein translation by targeting PHLPP2-3'-UTR. Interestingly, PHLPP2 expression was inversely associated with tumor necrosis factor alpha (TNFα) expression, with low PHLPP2 and high TNFα expression in lung cancer tissues compared with the paired adjacent normal lung tissues. Additional studies revealed that PHLPP2 exhibited its antitumorigenic effect of B[a]P/B[a]PDE through the repression of inflammatory TNFα transcription. Our studies not only first time identify PHLPP2 downregulation by lung carcinogen B[a]P/B[a]PDE, but also elucidate a novel molecular mechanisms underlying lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure.

The clinical significance of FSCN1 in non-small cell lung cancer.

Fascin actin-bundling protein 1 (FSCN1) plays significant roles in biological processes such as tumor cell invasion and metastasis. However, little is known about prognostic value of FSCN1 in non-small cell lung cancer (NSCLC). FSCN1 mRNA and protein expression were detected by real-time PCR and Western blot in NSCLC tissues and paired adjacent normal lung tissues. Furthermore, the association of FSCN1 protein expression with clinicopathological characteristics including the survival was explored in 156 NSCLC patients. In our results, FSCN1 mRNA and protein expression were obviously higher in NSCLC tissues than in normal lung tissues. High levels of FSCN1 protein were positively correlated with status of differentiated degree, clinical stage, N classification, and M classification in NSCLC. Meanwhile, higher FSCN1 protein expression was significantly associated with poor overall survival in patients with NSCLC. Multivariate analyses showed that increased FSCN1 protein expression was a poor independent prognostic biomarker for NSCLC patients. In conclusions, FSCN1 plays an important role on NSCLC progression and prognosis and may serve as a convictive prognostic biomarker for NSCLC patients.

Expression of long non-coding RNA DLX6-AS1 in lung adenocarcinoma.

BackgroundLung adenocarcinoma (LAC), the primary histological type of non-small cell lung cancer (NSCLC), has displayed an increasing incidence and mortality worldwide. However, therapeutic approaches were limited. Dysregulation of some lncRNAs has been shown in various types of cancers including LAC. The aim of the present study was to vertify lncRNA DLX6-AS1 expression in LAC.MethodsMicroarray assay revealed expression profile of lncRNAs in LAC. qRT-PCR ( quantitative reverse transcription PCR) was performed to identify lncRNA DLX6-AS1 expression level in 72 paired LAC and adjacent normal lung tissues. qRT-PCR and Western blotting were used to verify that down-regulation lncRNA DLX6-AS1 decreased DLX6 (distal-less homeobox 6) mRNA and protein expression.ResultsMicroarray analysis identified up-regulation of 272 lncRNAs and down-regulation of 635 lncRNAs in LAC tissues. The expression level of lncRNA DLX6-AS1 in LAC tissues was significantly higher compared to paired adjacent normal lung tissues (P< 0.05). In addition, its expression level was closed correlated with both histological differentiation (P = 0.004) and TNM stage (P = 0.033). qRT-PCR and Western blotting analysis showed that DLX6 mRNA and protein levels were lower in si-LncRNA group than in the NC (negative control) and Blank groups.ConclusionsMicroarray analysis identified that lncRNA DLX6-AS1 was up-regulated in LAC tissues. High DLX6-AS1 expression levels were significantly associated with both histological differentiation and TNM stage. Down-regulation of lncRNA DLX6-AS1 expression decreased the DLX6 mRNA and protein levels.

A chromosomal passenger complex protein signature model predicts poor prognosis for non-small-cell lung cancer.

AimThe chromosomal passenger complex (CPC) acts as a key modulator for mitosis and cell cytokinesis. High levels of CPC proteins are frequently observed in multiple cancers and are correlated with more progressive malignant behaviors. The aim of the study was to evaluate whether CPC components or their combinations could be used to assess the clinical risk of patients with non-small-cell lung cancer (NSCLC).MethodsThe expression levels of four CPC proteins – aurora B kinase (AURKB), borealin, inner centromere protein (INCENP), and survivin – were evaluated using immunohistochemistry in an independent cohort of NSCLC specimens. A molecular predictor model was developed based on the combination of the four CPC proteins.ResultsAll the CPC components were overexpressed in NSCLC tumors compared with their paired adjacent normal lung tissues. Survivin overexpression was significantly correlated with late tumor stage (P=0.0166). High expressions of AURKB, INCENP, and survivin, but not borealin, were associated with shorter survival in patients with NSCLC. The constructed 4-CPC-gene model divided the cohort into two different subgroups with significantly different prognoses (hazard ratio, HR =2.8915 [95% confidence interval, CI: 1.5187–5.5052]; P=0.0013) and was retained as an independent prognostic factor in multivariate analysis (HR =2.4398 [95% CI: 1.2631–4.7127], P=0.0082). Moreover, the 4-CPC-gene model demonstrated a higher predictive ability for overall survival than each individual CPC biomarker.ConclusionTaken together, our study suggests that a molecular prognostic model based on simultaneous detection of CPC components could serve as a complement to current clinical risk stratification approaches for patients with NSCLC.

Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer.

MicroRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, and can function either as oncogenes or tumor suppressors in tumor progression. The effects of the expression of miR-96 in non-small cell lung cancer (NSCLC) remain unclear. In our study, qRT-PCR (quantitative reverse transcription PCR) was performed to identify the miR-96 expression level in 68 paired NSCLC and adjacent normal lung tissues. Trans-well, cell counting kit-8, and apoptosis assays were used to evaluate the effects of miR-96 expression on cell invasion, proliferation, and apoptosis. Dual-luciferase reporter assay and Western blotting were used to verify whether FOXO3 was a potential major target gene of miR-96. Finally, the effect of FOXO3 on miR-96-induced cell survival was determined by transfection of the genes expressing FOXO3 lacking 3'UTR and miR-96. The expression level of miR-96 in NSCLC tissues was higher than that in adjacent normal lung tissues, and this increased expression was significantly associated with lymph node metastasis. In contrast to the cells in the blank and negative control groups, the number of cells migrating through the matrigel was significantly lower and the incidence of apoptosis was significantly higher in cells transfected with a miR-96 inhibitor. Western blotting and dual-luciferase reporter assays demonstrated that miR-96 can bind to the putative seed region in FOXO3 mRNA 3'UTR, and can significantly lower the expression of FOXO3. The introduction of FOXO3 cDNA without 3'UTR restored miR-96 induced cell apoptosis and invasion. MiR-96 is up-regulated in NSCLC tissues. Downregulation of miR-96 inhibits invasion and promotes apoptosis in NSCLC cells A549 and SPC-A-1 by targeting FOXO3. Therefore, our study improves our understanding of the mechanisms underlying NSCLC pathogenesis and may promote the development of novel targeted therapies.

An upregulated long noncoding RNA RP3‐337D23.3 in lung adenocarcinoma in never‐smokers promotes metastasis (1049.1)

Objective: The present study was designed to screen and validate lung adenocarcinoma in never smokers (LACINS) specific long noncoding RNAs (lncRNAs).Methods: A custom‐designed human lncRNA expression microarray was utilized to determine lncRNA expression profiles of LACINS and indentify LACINS specific lncRNA. Five LACINS and paired adjacent normal lung tissues were used for microarray analysis and another 10 LACINS were used for further searching of LACINS specific lncRNAs by quantitative real‐time polymerase chain reaction. Function of RP3‐337D23.3 was investigated by cell proliferation, transwell, and wound healing assay in vitro.Results: A total of 3353 lncRNAs were differentially expressed in LACINS (fold change &gt;2), 1509 upregulated and 1849 downregulated. Eleven candidate LACINS specific lncRNAs were identified and further validated in 10 pairs of LACINS and corresponding lung tissues. We identified RP3‐337D23.3 as a LACINS specific lncRNAs which is significantly up‐regulated in tumors as compared with adjacent normal tissues. Additionally, RP3‐337D23.3 is significantly overexpressed in LACINS with metastasis compared with those without metastasis (p=0.0006). In vitro siRNA mediated silencing of RP3‐337D23.3 led to remarkable decreased invasion, migration, and proliferation abilities of H1975 cell line.Conclusion: We identified the first LACINS specific lncRNA, RP3‐337D23.3, and provided preliminary evidence that RP3‐337D23.3 could promote LACINS metastasis.Grant Funding Source: National Natural Science Foundation of China (81372321)