Pathogenesis Of Non-small Cell Lung Cancer Research Articles

Elevated Circulating CD4+CD25-Foxp3+ Regulatory T Cells in Patients with Nonsmall Cell Lung Cancer.

Purpose: CD4+CD25+Foxp3+ regulatory T (Treg) cell-mediated immunosuppression has been implicated as a crucial mechanism of tumor immune cell escape in nonsmall cell lung cancer (NSCLC). However, little is known concerning the specific role of CD4+CD25-Foxp3+ Treg cells in NSCLC. The aim of this study was to investigate the frequency of circulating CD4+CD25-Foxp3+ Treg cells and their role in NSCLC. Methods: The frequencies of Treg, T helper (Th)1, Th2, and Th17 cells in peripheral blood were separately measured in 36 NSCLC patients and 20 healthy controls (HCs) using flow cytometry. Serum cytokine concentrations were determined using cytometric bead arrays. Results: The frequencies of circulating CD4+CD25+ T cells and CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg cells were significantly higher in advanced-stage NSCLC patients compared with patients with limited-stage NSCLC. The frequencies of circulating CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg cells were negatively correlated with interleukin (IL)-17, but positively correlated with serum IL-10 levels. In addition, the Th17/CD4+CD25-Foxp3+ Treg cell ratios were negatively correlated with serum cytokeratin 19 fragment (CYFRA 21-1) concentrations in patients with NSCLC. Moreover, coculturing CD4+CD25-Foxp3+ Treg cells and CD14+ monocytes in vitro resulted in a higher frequency of CD206+CD14+ M2-like monocytes compared with CD14+ monocytes. Conclusions: Elevated circulating CD4+CD25-Foxp3+ Treg cells may be involved in the pathogenesis of NSCLC.

Investigation into the underlying molecular mechanisms of non-small cell lung cancer using bioinformatics analysis

Development of new therapeutic strategies has proven to be of little impact on non-small cell lung cancer (NSCLC), and unfortunately, its molecular mechanisms still remain poorly understood. Therefore, the current study aimed to diagnose the genes associated in the pathogenesis of NSCLC. The differentially expressed genes (DEGs) were diagnosed using the limma software package. The WebGestalt was used to perform pathway and Gene Ontology (GO) enrichment analysis of the DEGs. Protein-protein interaction (PPI) networks, extracted modules, miRNA-target genes regulatory network and miRNA-target genes regulatory network were used to obtain insight into the actions of DEGs. Survival analysis for DEGs carried out. A total of 118 DEGs, including 74 up-regulated and 44 down-regulated genes were diagnosed between serum starved Calu-6 lung cancer cells transfected with control siRNA and serum starved Calu-6 lung cancer cells transfected with siRNA against the LAPTM4B gene. The up-regulated genes were enriched in PPAR signalling pathway, angiogenesis, positive regulation of neuron projection development and extracellular space. The down-regulated genes were enriched in FoxO signalling pathway, plasminogen activating cascade, cardiac chamber morphogenesis and cell surface. The current study screened the genes in PPI network, extracted modules, miRNA-target genes regulatory network and TF-target genes regulatory network with higher degrees as hub genes, which included FGFR1, HEY1, SPP1, FOS, NDRG1, TFF3, CDK15, PDGFRB, IL7R, LYPD6B, IQGAP2, CEACAM1, VEGFA, SGK1, GPRC5B and WNT5A. Survival and expression analysis suggested that low expressed CACNA1C and GATA2, and high expressed MANSC1 and MSX2 in patients with NCSLC was linked with a positive prognosis for overall survival and all individual stages of NSCLC. In conclusion, the current study diagnosed DEGs between serum starved Calu-6 lung cancer cells transfected with control siRNA and serum starved Calu-6 lung cancer cells transfected with siRNA against the LAPTM4B gene, which could improve our understanding of the molecular mechanisms of small interference RNA (siRNA) against LAPTM4B in NSCLC, and up- and down-regulated genes, over and under expressed genes in NSCLC patients indicated a positive prognosis for overall survival and may be used as a prognostic biomarker for patients with NSCLC. Thus, we developed a signature based on seven DEGs with prognostic value for NSCLC patients using multi-steps bioinformatics methods, which may provide new insights and potential biomarkers for prognosis, as well as possibly serving as new therapeutic targets in clinical applications.

Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer.

(1) The TBX2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) are markedly down-regulated in human non-small cell lung cancer (NSCLC) and exert tumor suppressor effects in lung malignancy. Yet, mechanisms underlying suppressed expression of the TBX2 subfamily in NSCLC are elusive. Here, we interrogated probable epigenetic mechanisms in suppressed expression of the TBX2 subfamily in human NSCLC. (2) TBX2 subfamily gene expression and methylation levels in NSCLC and normal lung tissues were surveyed using publicly available RNA-sequence and genome-wide methylation datasets. Methylation β-values of the four genes were statistically compared between NSCLCs and normal lung tissues, correlated with gene expression levels, and interrogated with clinicopathological variables. Expression and methylation levels of TBXs were quantified in NSCLC cells using real-time PCR and methylation-specific PCR assays, respectively. Effects of the DNA methyltransferase inhibitor 5-azacytidine (Aza) on TBX2 subfamily expression were assessed in NSCLC cells. Impact of TBX2 subfamily expression on Aza-treated cells was evaluated by RNA interference. (3) All four TBXs were significantly hypermethylated in NSCLCs relative to normal lung tissues (p < 0.05). Methylation β-values of the genes, with exception of TBX2, were significantly inversely correlated with corresponding mRNA expression levels (p < 0.05). We found no statistically significant differences in hypermethylation levels of the TBX2 subfamily by clinicopathological features including stage and tobacco history. Expression levels of the TBX genes were overall suppressed in NSCLC cells relative to normal alveolar cells. Members of the subfamily were significantly hypermethylated in all tested NSCLC cell lines relative to normal alveolar cells. Treatment with Aza induced the expression of the TBX2 subfamily concomitant with NSCLC cell growth inhibition. Further, simultaneous knockdown of the four TBX genes markedly reduced anti-growth effects of Aza in NSCLC cells. (4) Our study sheds light on new epigenetic profiles in the molecular pathogenesis of human NSCLC.

Down-regulation of microRNA-144-3p and its clinical value in non-small cell lung cancer: a comprehensive analysis based on microarray, miRNA-sequencing, and quantitative real-time PCR data

BackgroundPrevious studies have shown that miR-144-3p might be a potential biomarker in non-small cell lung cancer (NSCLC). Nevertheless, the comprehensive mechanism behind the effects of miR-144-3p on the origin, differentiation, and apoptosis of NSCLC, as well as the relationship between miR-144-3p and clinical parameters, has been rarely reported.MethodsWe investigated the correlations between miR-144-3p expression and clinical characteristics through data collected from Gene Expression Omnibus (GEO) microarrays, the relevant literature, The Cancer Genome Atlas (TCGA), and real-time quantitative real-time PCR (RT-qPCR) analyses to determine the clinical role of miR-144-3p in NSCLC. Furthermore, we investigated the biological function of miR-144-3p by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Protein-protein interaction (PPI) network was created to identify the hub genes.ResultsFrom the comprehensive meta-analysis, the combined SMD of miR-144-3p was − 0.95 with 95% CI of (− 1.37, − 0.52), indicating that less miR-144-3p was expressed in the NSCLC tissue than in the normal tissue. MiR-144-3p expression was significantly correlated with stage, lymph node metastasis and vascular invasion (all P < 0.05). As for the bioinformatics analyses, a total of 37 genes were chosen as the potential targets of miR-144-3p in NSCLC. These promising target genes were highly enriched in various key pathways such as the protein digestion and absorption and the thyroid hormone signaling pathways. Additionally, PPI revealed five genes—C12orf5, CEP55, E2F8, STIL, and TOP2A—as hub genes with the threshold value of 6.ConclusionsThe current study validated that miR-144-3p was lowly expressed in NSCLC. More importantly, miR-144-3p might function as a latent tumor biomarker in the prognosis prediction for NSCLC. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of NSCLC.

MRTF-A Regulates the Proliferation and Migration of Non-small Cell Lung Cancer Cells of A549 through HOTAIR

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）作为肺癌的一种，由于其高发病率一直备受关注。准确揭示其发病机制对于NSCLC的诊断以及治疗具有重要的指导意义。MATF-A作为转录调控因子在多种肿瘤的发生发展过程中发挥着重要作用，可以调控多种肿瘤细胞的迁移过程。HOTAIR是近年研究发现的一个长链非编码RNA，在多种肿瘤中异常表达并参与多种肿瘤的增殖及迁移的进程。本研究旨在探究MRTF-A通过HOTAIR调控NSCLC的增殖及迁移进程。方法构建MRTF-A的过表达质粒及干扰质粒，检测对A549细胞增殖及迁移的影响。设计合成HOTAIR的siRNA检测对A549细胞增殖及迁移的作用。qRT-PCR检测MRTF-A对HOTAIR表达的调控作用。构建HOTAIR的启动子检测MRTF-A对HOTAIR启动子活性的影响。结果过表达MRTF-A促进A549细胞的增殖，沉默MRTF-A的表达抑制A549细胞的增殖及迁移。干扰HOTAIR的表达抑制A549细胞的增殖及迁移。MRTF-A能够影响HOTAIR的表达，同时能够调控HOTAIR启动子的活性。结论MRTF-A通过HOTAIR调控NSCLCA549细胞的增殖及迁移进程。

Correlations of the ZEB1 expression with the incidence and prognosis of non-small cell lung cancer.

In order to investigate the role of the zinc finger E-box-binding homeobox 1 (ZEB1) expression in the incidence of non-small cell lung cancer (NSCLC), the effects of the ZEB1 expression on the pathogenesis and prognosis of NSCLC were investigated. Correlations of the expression of ZEB1 in 88 clinical patients with NSCLC with clinicopathological features were determined. The patients were followed up for 60 months to study the correlation between the ZEB1 expression and the prognosis of NSCLC patients. To further explore the role of the ZEB1 expression in the incidence of NSCLC, the expressions of ZEB1 and E-cadherin in three NSCLC cell lines (A549, NCI-H1299 and NCI-H1975) and human normal lung epithelial BESA-2B cell line were measured, and the cell invasion ability was detected. The role of ZEB1 in NSCLC cell invasion was verified through the knockdown of ZEB1 by the short hairpin ribonucleic acids (shRNAs). In addition, its effects on the proliferation and apoptosis of NSCLC cells were confirmed by the overexpression of ZEB1. The expression of ZEB1 in NSCLC tissues was remarkably higher than that in normal tissues, and the expression level of ZEB1 was significantly related to the cancer stage and tumor size. The lower the expression of ZEB1 was, the higher the overall survival rate and the longer the survival time would be. The expression of ZEB1 was negatively correlated with that of E-cadherin in cell lines, and ZEB1-shRNAs markedly reduced the invasion ability of NSCLC cells. The overexpression of ZEB1 resulted in an increase in the proliferation activity and a significant decrease in the apoptosis of A549 cells. The expression of ZEB1 is closely related to the incidence and prognosis of NSCLC. Increased expression of ZEB1 is helpful for promoting the invasion of NSCLC and enhancing the proliferation activity of NSCLC.

Significance of miR-15a-5p and CNKSR3 as Novel Prognostic Biomarkers in Non-Small Cell Lung Cancer

In recent years, targeted cancer treatment methods at various molecular levels have been developed for Non-Small Cell Lung Cancer (NSCLC), one of two major subtypes of lung cancer. miRNAbased clinical trials are currently the preferred targeted therapeutic strategy. Also, ceRNAs (competing endogenous RNA) would be the newest and the most effective approach to uncover novel interactions between mRNAs and miRNAs in NSCLC carcinogenesis. There are many factors influencing the efficiency of a miRNA to suppress or silence translation of the target mRNA. The most effective event is the presence of other RNAs showing ceRNA activity. These RNAs contain binding sites for specific miRNAs and enable miRNAs to bind these pseudo targets, instead of the original binding sites on the target mRNA. Therefore, the mRNA of the target gene is less affected by this miRNA, while the amount of miRNA remains the same in the media. For this project, we determined that five clinically important different oncogenes (PDL1, FGFR1, DDX3X, SLC1A5, FXR1 ) are involved in the pathogenesis of NSCLC. For this purpose, we transfected model NSCLC cell line, A549, with miRNAs (miR-150-5p, miR-15a-5p, miR-503-5p) targeting these oncogenes to investigate whether these oncogenes will be suppressed at the mRNA level and also how the suppression efficiency of these miRNA on the oncogenes will be affected by possible ceRNA (CNKSR3, POU2F1, HIPK2) activities. miR-15a-5p was determined to have the most suppressive effect on the five genes and three potential ceRNAs (p<0.05). Furthermore, CNKSR3 was the ceRNA most affected by all three miRNAs (p<0.05). CNKSR3 was affected more than the oncogenes known to act on NSCLC and this might make it a stronger and novel marker for use in possible treatment regimens designed using miR-15a-5p silencing effect on oncogenes in NSCLC pathogenesis. According to the literature, this is the first study associating NSCLC with miR-15a-5p and CNKSR3.

MicroRNA-497-5p inhibits proliferation and invasion of non-small cell lung cancer by regulating FGF2.

Increasing number of microRNAs (miRNAs) have been reported to play an important role in the development and progression of non-small cell lung cancer (NSCLC). In particular, microRNA-497-5p (miR-497-5p) has been proposed as a tumor suppressor miRNA in human cancers. However, the role of miR-497-5p and its potential molecular mechanism associated with NSCLC are less studied. Therefore, the role of miR-497-5p in the pathogenesis of NSCLC was investigated. In the present study, the expression of miR-497-5p was significantly downregulated in NSCLC. Moreover, overexpression of miR-497-5p inhibited the proliferation and invasion of NSCLC cells by suppressing FGF2. In addition, FGF2 was a downstream target of miR-497-5p in NSCLC. FGF2 was upregulated in NSCLC promoting cell proliferation and invasion. Overexpression of FGF2 impaired the inhibitory effect of miR-497-5p in NSCLC. Taken together, these results demonstrate that miR-497-5p is a tumor suppressor miRNA and demonstrate its potential for future use in the treatment of human NSCLC.

High expression of lncRNA MEG3 participates in non-small cell lung cancer by regulating microRNA-7-5p.

This study was made to investigate whether long noncoding RNA (lncRNA) MEG3 could participate in the occurrence and development of non-small cell lung cancer (NSCLC) by regulating the expression of BRCA1 through competitive binding to microRNA-7-5p. We used quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) to explore the expression of lncRNA MEG3 and BRCA1 in NSCLC tissues and adjacent normal tissues, as well as NSCLC cell lines. The dual luciferase reporter gene assay was used to detect the binding of microRNA-7-5p to lncRNA MEG3 and BRCA1. Meanwhile, the expression of BRCA1, B-cell lymphoma-2 (Bcl-2) and BCL2-associated X (Bax) was detected by Western blot after the cells were overexpressed or knocked down of lncRNA MEG3. All these experiments were designed to investigate whether lncRNA MEG3 participated in the pathogenesis of NSCLC through inhibiting the expression of BRCA1 and Bcl-2 and promoting Bax expression. The expressions of lncRNA MEG3 and BRCA1 in NSCLC tissues and A549 and HCC823 cell lines were significantly lower than those in the normal group. Overexpression of lncRNA MEG3 and BRCA1 in A549 and HCC823 cell lines resulted in increased apoptosis of lung cancer cells. Dual luciferase reporter assay demonstrated that lncRNA MEG3 can regulate the expression of BRCA1 through competitively binding to microRNA-7-5p to form the lncRNA MEG3/microRNA-7-5p/BRCA1 regulatory network. Besides, lncRNA MEG3 could inhibit the apoptosis inhibitory protein Bcl-2 and promote the expression of apoptosis-promoting factor Bax. LncRNA MEG3 was significantly downregulated in NSCLC, and it could regulate the BRCA1 expression by competitive binding to microRNA-7-5p.

Regulation by Pink1 on the mitochondrial dysfunction in endothelial cells post the hypoxia mimetic agent CoCl2 treatment.

To explore the role of miR-451a in the migration and invasion of non-small cell lung cancer (NSCLC) cells. Quantitative Real time-polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the levels of miR-451a and activating transcription factor 2 (ATF2) in NSCLC. Transwell assay was employed to analyze the migratory and invasive abilities in NSCLC cells. Dual-luciferase reporter assay was applied to confirm the binding condition of miR-451 and its target gene in NSCLC cells. MiR-451a was downregulated in NSCLC tissues and lung cancer cell lines A549 and NCI-H460, while ATF2 was upregulated. The mRNA level of miR-451a was negatively correlated to ATF2. Additionally, miR-451a regulated cell migration and invasion through targeting ATF2. Furthermore, ATF2 could reverse the inhibitory migration and invasion of A549 cells induced by miR-451a. MiR-451a inhibits the migratory and invasive abilities of NSCLC cells through ATF2 regulation. The newly identified miR-451a/ATF2 axis provides a novel insight into the pathogenesis of NSCLC.

MiR-409 Inhibits Human Non-Small-Cell Lung Cancer Progression by Directly Targeting SPIN1

Lung cancers, the leading cause of cancer mortality worldwide, are characterized by a high metastatic potential. Growing evidence reveals that Spindlin 1 (SPIN1) is involved in tumor progression and carcinogenesis. However, the role of SPIN1 in non-small-cell lung cancer (NSCLC) and the molecular mechanisms underlying SPIN1 in human NSCLC remain undetermined. Here we examined the function of SPIN1 in human NSCLC and found that the expression of SPIN1 was closely correlated with the overall survival and poor prognosis of NSCLC patients. Aberrant regulation of microRNAs (miRNAs) has an important role in cancer progression. We revealed that miR-409 inhibits the expression of SPIN1 by binding directly to the 3′ UTR of SPIN1 using dual-luciferase reporter assays. Overexpression of miR-409 significantly suppressed cell migration, growth, and proliferation by inhibiting SPIN1 in vitro and in vivo. SPIN1 overexpression in miR-409-transfected NSCLC cells effectively rescued the suppression of cell migration, growth, and proliferation regulated by miR-409. miR-409 regulates the PI3K/AKT (protein kinase B) pathway in NSCLC. Moreover, clinical data showed that NSCLC patients with high levels of miR-409 experienced significantly better survival. miR-409 expression was also negatively associated with SPIN1 expression. Taken together, these findings highlight that the miR-409/SPIN1 axis is a useful pleiotropic regulatory network and could predict the metastatic potential in NSCLC patients early, indicating the possibility that miR-409 and SPIN1 might be attractive prognostic markers for treating NSCLC patients.

Abstract 5380: Whole-exome sequencing of Brazilian non-small cell lung cancer

Abstract Background: Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer with low survival rates. NSCLC patients harboring EGFR mutations are benefited by targeted therapy with tyrosine kinase inhibitors. The mutational profiling and the knowledge about recurrent mutations in Brazilian NSCLC is limited. Therefore, the detection of the mutation landscape of Brazilian NSCLC tumors is crucial for tailored therapy. Aim: To evaluate the mutational profiling of NSCLC of a Brazilian series. Methods: We analyzed a series of 40 NSCLC patients from Barretos Cancer Hospital. Clinical staging and exposure data were collected from all patients. Exome sequencing was performed in paired tumor/blood tissue DNA using the Illumina HiSeq2500™ System. Germline variations were excluded and the Genome Analysis Toolkit and VarScan were used for variants annotation and mutation detection. Results: Overall, 29 (72.5%) of cases were adenocarcinomas and 11 (27.5%) were squamous cell carcinoma (SCC) and the series was enriched by initial stages cases (I, n=16; II, n=12; III, n=6; IV, n=6). Regarding tobacco exposure, 33 patients were current smokers or former smokers and 7 patients were never smokers. In accordance, most of the cases presented the mutation signature 4, related to tobacco exposure, which is characterized by C&amp;gt;A substitutions and signature 29, related to tobacco chewing habit, which is characterized by C&amp;gt;A and C&amp;gt;T substitutions. All SCC patients were current or former smokers. TP53 gene was the most recurrently mutated gene observed in 48% (n=14) of the adenocarcinomas and 100% (n=11) of SCC cases (p=0,003). In adenocarcinomas, besides of TP53 gene, recurrent mutations were also found in KRAS (28%; n=8), STK11(21%; n=6), USP36 and APOB (14%; n=4/each), POLQ and CHD5 (10% n=3/each), EGFR and ANO3 (7%; n=2/each) and PDGFRA, BRAF, ERBB2, ERBB4, NRAS, RB1, CD22, CTNNA2 and ATR mutations were identified in only one adenocarcinoma case (3%). In SCC, besides of TP53 gene, recurrent mutations were also found in RB1, CD22, CTNNA2 and CHD7 (27%; n=3/each), ATR and APOB (18%; n=2/each), KRAS, PDGFRA, ERBB2, ERBB4, POLQ, CHD5 and ANO3 were identified in only one SCC case (9%). EGFR, STK11, BRAF, NRAS and USP36 mutations were exclusively found in adenocarcinomas and CHD7 mutations were exclusively found in SCC. Conclusions: The mutational landscape of lung adenocarcinoma and SCC are clearly different. A noteworthy number of pathogenic mutations was identified in this initial stages-enriched series. Our results may contribute to improve the knowledge about the molecular pathogenesis of NSCLC to better guide the clinical management with targeted therapies. Citation Format: Leticia Ferro Leal, Adriane Feijo Evangelista, Pedro R. de Marchi, Ysadhora Christiane Camargo Rodrigues, Eduardo Caetano Albino da Silva, Rui Manuel Reis. Whole-exome sequencing of Brazilian non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5380.

DNA methylation of microRNA‐coding genes in non‐small‐cell lung cancer patients

Deregulated DNA methylation leading to transcriptional inactivation of certain genes occurs frequently in non‐small‐cell lung cancers (NSCLCs). As well as protein‐coding genes, microRNA (miRNA)‐coding genes may be targets for methylation in NSCLCs; however, the number of known methylated miRNA genes is still small. Thus, we investigated methylation of miRNA genes in primary tumour (TU) samples and corresponding non‐malignant lung tissue (NL) samples of 50 NSCLC patients by using methylated DNA immunoprecipitation followed by custom‐designed tiling microarray analyses (MeDIP‐chip), and 252 differentially methylated probes between TU samples and NL samples were identified. These probes were annotated, which resulted in the identification of 34 miRNA genes with increased methylation in TU samples. Some of these miRNA genes were already known to be methylated in NSCLCs (e.g. those encoding miR‐9‐3 and miR‐124), but methylation of the vast majority of them was previously unknown. We selected six miRNA genes (those encoding miR‐10b, miR‐1179, miR‐137, miR‐572, miR‐3150b, and miR‐129‐2) for gene‐specific methylation analyses in TU samples and corresponding NL samples of 104 NSCLC patients, and observed a statistically significant increase in methylation of these genes in TU samples (p < 0.0001). In silico target prediction of the six miRNAs identified several oncogenic/cell proliferation‐promoting factors (e.g. CCNE1 as an miR‐1179 target). To investigate whether miR‐1179 indeed targets CCNE1, we transfected miR‐1179 gene mimics into CCNE1‐expressing NSCLC cells, and observed downregulated CCNE1 mRNA expression in these cells as compared with control cells. Similar effects on cyclin E1 expression were seen in western blot analyses. In addition, we found a statistically significant reduction in the growth of NSCLC cells transfected with miR‐1179 mimics as compared with control cells. In conclusion, we identified many methylated miRNA genes in NSCLC patients, and found that the miR‐1179 gene is a potential tumour cell growth suppressor in NSCLCs. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Long non-coding RNA SNHG15 promotes CDK14 expression via miR-486 to accelerate non-small cell lung cancer cells progression and metastasis.

Long non‐coding RNAs (lncRNAs) have been validated to play important role in multiple cancers, including non‐small cell lung cancer (NSCLC). In present study, our team investigate the biologic role of SNHG15 in the NSCLC tumorigenesis. LncRNA SNHG15 was significantly upregulated in NSCLC tissue samples and cells, and its overexpression was associated with poor prognosis of NSCLC patients. In vitro, loss‐of‐functional cellular experiments showed that SNHG15 silencing significantly inhibited the proliferation, promoted the apoptosis, and induced the cycle arrest at G0//G1 phase. In vivo, xenograft assay showed that SNHG15 silencing suppressed tumor growth of NSCLC cells. Besides, SNHG15 silencing decreased CDK14 protein expression both in vivo and vitro. Bioinformatics tools and luciferase reporter assay confirmed that miR‐486 both targeted the 3′‐UTR of SNHG15 and CDK14 and was negatively correlated with their expression levels. In summary, our study conclude that the ectopic overexpression of SNHG15 contribute to the NSCLC tumorigenesis by regulating CDK14 protein via sponging miR‐486, providing a novel insight for NSCLC pathogenesis and potential therapeutic strategy for NSCLC patients.

Alteration of DACH1 methylation patterns in lung cancer contributes to cell proliferation and migration.

Lung cancer is the most common cause of cancer-related death. Non-small cell lung cancer (NSCLC) accounts for 80%-85% of total lung cancer cases. Dachshund homolog 1 (DACH1), is a protein encoded by the DACH1 gene in humans. DACH1 inhibits lung adenocarcinoma invasion and tumor growth but has a lower expression in NSCLC. To investigate the mechanisms of decreased DACH1 expression, its DNA methylation patterns were investigated. The results showed a higher methylation rate in NSCLC compared with the adjacent normal lung tissues. Cell transfection experiments showed that increased methylation impaired transcription factor transactivation. In vivo demethylation treatment and overexpression of DACH1 increased apoptosis and decreased migration and invasion in NSCLC A549 cells. Our research provides new insight into NSCLC pathogenesis and identifies a new therapeutic target.

IL‑17 induces NSCLC A549 cell proliferation via the upregulation of HMGA1, resulting in an increased cyclin D1 expression.

Non-small cell lung cancer (NSCLC) is considered to be an inflammation-associated carcinoma. Although interleukin‑17 (IL‑17) production contributes to the proliferation and growth of NSCLC, the mechanisms underlying IL‑17-induced NSCLC cell proliferation have not been fully elucidated. In the present study, by using ELISA and immunohistochemical analyses, we first found that the expression levels of IL‑17, IL‑17 receptor (IL‑17R), high-mobility groupA1 (HMGA1) and cyclinD1 were elevated in the samples of patients with NSCLC. Subsequently, by RT-qPCR, western blot analysis and cell proliferation assay in vitro, we revealed that stimulation with recombinant human IL‑17 (namely IL‑17A) markedly induced the expression of HMGA1 and cyclinD1 in the A549 cells (a human lung adenocarcinoma cell line) and promoted cell proliferation. Furthermore, luciferase reporter and ChIP assays confirmed that upregulated HMGA1 directly bound to the cyclinD1 gene promoter and activated its transcription. Notably, the response element of HMGA1 binding to the cyclinD1 promoter was disclosed for the first time, at least to the best of our knowledge. Taken together, our findings indicate that the IL‑17/HMGA1/cyclinD1 axis plays an important role in NSCLC cell proliferation and may provide new insight into NSCLC pathogenesis and may thus aid in the development of novel therapeutic targets for NSCLC.

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ1) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ1 can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ1 treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ1 treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ1-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ1, in the clinical progression of NSCLC.

RETRACTED ARTICLE: Dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal and PTEN-suppressing Wnt/\u03b2-catenin signaling in non-small-cell lung cancer

MiR-374a appears to play a complex role in non-small-cell lung cancer (NSCLC). Here, we demonstrate a dual role for miR-374a in NSCLC pathogenesis. The effects and modulatory mechanisms of miR-374a on cell growth, migration, invasion, and in vivo tumorigenesis and metastasis in nude mice were also analyzed. The expression of miR-374a was examined in NSCLC and non-cancerous lung tissues by quantitative real-time reverse transcription-PCR (qRT-PCR), and in situ hybridization, respectively. miR-374a directly targets CCND1 and inactivates PI3K/AKT and Ras-mediated cell cycle signalings, as well as epithelial–mesenchymal transition (EMT). This not only dramatically suppressed cell growth, migration, invasion,and metastasis, but also elevated A549 and pc-9 NSCLC cell sensitivity to cisplatin (DDP) while increasing survival time of tumor-bearing mice. Interestingly, miR-374a serves an inverse function in SPCA-1 and H1975 NSCLC cells by directly targeting PTEN to activate Wnt/β-catenin and Ras signalings and its downstream cascade signals. Surprisingly, transcription factor c-Jun bound to the promoter region of human miR-374a and suppressed miR-374a in A549 and pc-9 cells while inducing it in SPCA-1 and H1975 cells. Increased levels of miR-374a appeared to serve a protective role by targeting CCND1 in early-stage NSCLC (Stages I and II). Inversely, increased miR-374a was an unfavorable factor when targeting PTEN in more advanced staged NSCLC patients. Our studies are the first to demonstrate that miR-374a plays divergent roles in NSCLC pathogenesis at different stages of the disease and implicate the potential application of miR-374a targeting for cancer therapy.

Expression Signature and Role of miR-30d-5p in Non-Small Cell Lung Cancer: a Comprehensive Study Based on in Silico Analysis of Public Databases and in Vitro Experiments

Background/Aims: The purpose of this study was to probe the clinico-pathological significance and the underlying mechanism of miR-30d-5p expression in non-small cell lung cancer (NSCLC). Methods: We initially examined the level of miR-30d-5p expression in NSCLC and non-cancer tissues using RT-qPCR. Then, a series of validation analyses including a meta-analysis of data from microarray chips in Gene Expression Omnibus (GEO), data mining of the cancer genome atlas (TCGA) and an integrated meta-analysis incorporating GEO microarray chips, TCGA data, in-house RT-qPCR and literature studies were performed to examine the clinico-pathological value of miR-30d-5p expression in NSCLC. In vitro experiments were further conducted to investigate the impact of miR-30d-5p on NSCLC cell growth. The molecular mechanism by which miR-30d-5p regulates the pathogenesis of NSCLC was probed through a bioinformatics analysis of its target genes. Moreover, dual luciferase reporter assay was conducted to verify the targeting regulatory relationship between miR-30d-5p and CCNE2. Results: Based on results from RT-qPCR, GEO meta-analysis, TCGA data mining and the integrated meta-analysis incorporating GEO microarray chips, TCGA data, in-house RT-qPCR and literature studies, miR-30d-5p expression was decreased in NSCLC tissues, and patients with NSCLC who presented with lower miR-30d-5p expression tended to display an advanced clinical progression. Significant pathways including the Mucin type O-glycan biosynthesis pathway, cell cycle pathway and cysteine and methionine metabolism pathway (all P< 0.05) revealed potential roles of the target genes of miR-30d-5p in the oncogenesis of NSCLC. Results from in vitro experiments indicated that miR-30d-5p could attenuate proliferation and viability of NSCLC cells. Among the 12 identified hub genes, nine genes including E2F3, CCNE2, SKP2, CDK6, TFDP1, LDHA, GOT2, DNMT3B and ST6GALNAC1 were validated by Pearson’s correlation test and the human protein atlas (HPA) database as targets of miR-30d-5p with higher probability. Specifically, dual luciferase reporter assay confirmed that CCNE2 was directly targeted by miR-30d-5p. Conclusion: In summary, miR-30d-5p expression is decreased in NSCLC, and it might play the role as tumor suppressor in NSCLC by regulating target genes.

Non-small-cell lung cancer pathological subtype-related gene selection and bioinformatics analysis based on gene expression profiles.

Lung cancer is one of the most common malignant diseases and a major threat to public health on a global scale. Non-small-cell lung cancer (NSCLC) has a higher degree of malignancy and a lower 5-year survival rate compared with that of small-cell lung cancer. NSCLC may be mainly divided into two pathological subtypes, adenocarcinoma and squamous cell carcinoma. The aim of the present study was to identify disease genes based on the gene expression profile and the shortest path analysis of weighted functional protein association networks with the existing protein-protein interaction data from the Search Tool for the Retrieval of Interacting Genes. The gene expression profile (GSE10245) was downloaded from the National Center for Biotechnology Information Gene Expression Omnibus database, including 40 lung adenocarcinoma and 18 lung squamous cell carcinoma tissues. A total of 8 disease genes were identified using Naïve Bayesian Classifier based on the Maximum Relevance Minimum Redundancy feature selection method following preprocessing. An additional 21 candidate genes were selected using the shortest path analysis with Dijkstra's algorithm. The AURKA and SLC7A2 genes were selected three and two times in the shortest path analysis, respectively. All those genes participate in a number of important pathways, such as oocyte meiosis, cell cycle and cancer pathways with Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The present findings may provide novel insights into the pathogenesis of NSCLC and enable the development of novel therapeutic strategies. However, further investigation is required to confirm these findings.