Lung Cancer-related Genes Research Articles

Interaction research on smoking and microRNA genes SNP related to lung cancer in Fujian Han population

To investigate the interaction on smoking and the lung cancer related genes miR-196a2 rs11614913, miR-146a rs2910164, miR-300 rs12894467, miR-26a-1 rs7372209, miR-27a rs895819 in Fujian Han population. From January 2006 to January 2012, by using a hospital-based case-control study, 1 053 cases were pathologically diagnosed as primary lung cancer from the Department of Thoracic Surgery and 1 058 controls were randomly selected from the visiting relatives of patients and visiting people of Cangxia community health service of Fuzhou city according to match with age and genders. They were recruited for questionnaires survey and genotyping detection. Research objects of genders, height, weight, cultural degree, marital status, family history of cancer, lung disease history, smoking, drinking tea, drinking, and so on. After informed consent, we collected 5 ml fasting venous blood from every object, used MALDI-TOF-MS to analysis genotyping of polymorphic loci. Logistic regression model was constructed by using SNP as independent variable, and the multiple factors were constructed with different loci. The possible association between SNP and cigarette smoking was analyzed by using the crossover analysis. The relative excess risk of interaction (RERI) were used to analyze on smoking and SNP loci additive interaction of dominant and recessive genetic models. Smokers in case group who smoked P50(P25-P75)30.00 (0.00-56.00) packages in a year were higher than control group (0.00(0.00 - 20.48) pack years) (Z=14.57,P<0.001). Passive smoking index for non-smokers was 11.40(0.00-25.00), higher than the controls (0.00(0.00-13.11)) (Z=10.71,P<0.001). Site detection rate of rs11614913, rs2910164, rs12894467, rs7372209 and rs895819 in cases was 95.82%(1 009/1 053), 97.72%(1 029/1 053), 97.82% (1 030/1 053), 97.15% (1 023/1 053) and 96.01% (1 011/1 053) respectively. The controls were 98.11% (1 038/1 058), 98.96% (1 047/1 058), 98.30% (1 040/1 058), 98.68% (1 044/1 058) and 98.02% (1 037/1 058) respectively. rs11614913 dominant genetic model, TT genotype and smoking could increase the risk of primary lung cancer (OR=4.04, 95%CI: 2.67 -6.12). Recessive genetic model, CC genotype and smoking increased the incidence of primary lung cancer risk (OR=4.76, 95%CI: 3.16 -7.17). rs12894467 dominant genetic model, TT genotype and smoking could increase the risk (OR=2.98, 95%CI: 2.28 -3.90) in primary lung cancer. In recessive genetic model, CC genotype and smoking increased the incidence of primary lung cancer risk (OR=1.94, 95% CI: 1.10-3.43). Dominant genetic model of rs2910164, CC genotype and smoking could increase the risk (OR=2.18, 95% CI: 1.60 -2.98) in primary lung cancer. Recessive genetic model, GG genotype and smoking increased the incidence of primary lung cancer risk (OR=3.29, 95% CI: 2.16 -5.03). Especially rs12894467 dominant and recessive gene model and genders, smoking and there were combined effects(χ(2)=8.58, P=0.003; χ(2)=4.76, P=0.040). Rs11614913, rs12894467 and rs2910164 polymorphism were potentially associated with primary lung cancer in Fujian Han population.

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform.

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

Retraction Note to: Gene expression profiles on predicting protein interaction network and exploring of new treatments for lung cancer.

In the present study, we aimed to explore disease-associated genes and their functions in lung cancer. We downloaded the gene expression profile GSE4115 from Gene Expression Omnibus (GEO) database. Total 97 lung cancer and 90 adjacent non-tumor lung tissue (normal) samples were applied to identify the differentially expressed genes (DEGs) by paired t test and variance analysis in spectral angle mapper (SAM) package in R. Gene Ontology (GO) functional enrichment analysis of DEGs were performed with Database for Annotation Visualization and Integrated Discovery, followed by construction of protein–protein interaction (PPI) network from Human Protein Reference Database (HPRD). Finally, network modules were analyzed by the MCODE algorithm to detect protein complexes in the PPI network. Total 3,102 genes were identified as DEGs at FDR < 0.05, including 1,146 down-regulated and 1,956 up-regulated DEGs. GO functional enrichment analysis revealed that up-regulated DEGs mainly participated in cell cycle and intracellular related functions, and down-regulated DEGs might influence cell functions. There were 39,240 pairs of PPIs in human obtained from HPRD databases, 3,102 DEGs were mapped to this PPI network, in which 2,429 pairs of PPIs and 1,342 genes were identified. With MCODE algorithm, 48 modules were selected, including five corresponding modules and three modules with differences in gene expressing profiles. In addition, three DGEs, FXR2, ARFGAP1 and ELAVL1 were discovered as potential lung cancer related genes. The discovery of featured genes which were probably related to lung cancer, has a great significance on studying mechanism, distinguishing normal and cancer tissues, and exploring new treatments for lung cancer.

ERGIC3, which is regulated by miR-203a, is a potential biomarker for non-small cell lung cancer.

In a previous study, we found that ERGIC3 was a novel lung cancer-related gene by screening libraries of differentially expressed genes. In this study, we developed a new murine monoclonal antibody (mAb) against ERGIC3. This avid antibody (6-C4) is well suited for immunohistochemistry, immunoblotting and solid-phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 in a broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6-C4 reacted only in some epithelial cells, such as hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells; however, most normal human tissues were not stained. Moreover, almost all carcinomas that originated from the epithelial cells were positive for 6-C4, whereas all sarcomas were negative. Notably, 6-C4 strongly stained non-small cell lung cancer (NSCLC) cells but did not react with normal lung tissues. Hence, ERGIC3 mAb could be used in histopathological diagnosis and cytopathological testing to detect early-stage NSCLC. We also studied the mechanisms of ERGIC3 regulation in vitro and in vivo by means of bioinformatics analysis, luciferase reporter assay, miRNA expression profiling and miRNA transfection. Results showed that miR-203a downregulation induced ERGIC3 overexpression in NSCLC cells.

Driver Gene Mutations of Non-Small-Cell Lung Cancer are Rare in Primary Carcinoids of the Lung: NGS Study by Ion Torrent

Lung carcinoids are rare neuroendocrine tumors of the lung. Very little is known about the genetic background of these tumors. We applied Ion Torrent Ampliseq next-generation technology to study hotspot mutations of 22 lung cancer-related genes from typical and atypical lung carcinoid tumors. DNA isolated from 25 formalin-fixed, paraffin-embedded carcinoid tumors were amplified to prepare barcoded libraries covering 507 mutations included in 90 amplicons. The libraries were pooled, purified, enriched, and sequenced on ion personal genome machine. The sequences were aligned and checked for known and novel variations using Torrent Suite Software v.4.0.2. One out of 25 patients had mutations in the targeted regions sequenced. This patient had mutations in BRAF, SMAD4, PIK3CA, and KRAS. All these mutations were confirmed as somatic and are previously known mutations. In summary, mutations in genes commonly mutated in non-small-cell lung cancer are not common in lung carcinoids.

Gene expression profiles on predicting protein interaction network and exploring of new treatments for lung cancer.

In the present study, we aimed to explore disease-associated genes and their functions in lung cancer. We downloaded the gene expression profile GSE4115 from Gene Expression Omnibus (GEO) database. Total 97 lung cancer and 90 adjacent non-tumor lung tissue (normal) samples were applied to identify the differentially expressed genes (DEGs) by paired t test and variance analysis in spectral angle mapper (SAM) package in R. Gene Ontology (GO) functional enrichment analysis of DEGs were performed with Database for Annotation Visualization and Integrated Discovery, followed by construction of protein-protein interaction (PPI) network from Human Protein Reference Database (HPRD). Finally, network modules were analyzed by the MCODE algorithm to detect protein complexes in the PPI network. Total 3,102 genes were identified as DEGs at FDR < 0.05, including 1,146 down-regulated and 1,956 up-regulated DEGs. GO functional enrichment analysis revealed that up-regulated DEGs mainly participated in cell cycle and intracellular related functions, and down-regulated DEGs might influence cell functions. There were 39,240 pairs of PPIs in human obtained from HPRD databases, 3,102 DEGs were mapped to this PPI network, in which 2,429 pairs of PPIs and 1,342 genes were identified. With MCODE algorithm, 48 modules were selected, including five corresponding modules and three modules with differences in gene expressing profiles. In addition, three DGEs, FXR2, ARFGAP1 and ELAVL1 were discovered as potential lung cancer related genes. The discovery of featured genes which were probably related to lung cancer, has a great significance on studying mechanism, distinguishing normal and cancer tissues, and exploring new treatments for lung cancer.

Inferring single nucleotide polymorphisms in microRNA binding sites of lung cancer-related inflammatory genes.

Single nucleotide polymorphisms located at microRNA (miRNA)-binding sites are likely to affect the expression of miRNA targets and may contribute to the susceptibility of humans to common diseases. Here 335 candidate lung cancer-related inflammatory genes were selected according to the existing literature and database. We identified putative miRNA-binding sites of 149 genes by specialised algorithms and screened SNPs in the 3'UTRs of these genes. By calculating binding free energy, we sorted 269 SNPs on the basis of the possibility of prediction. The proposed approach could help to easy the identification of functionally relevant SNPs and minimize the workflow and the costs.

Exploratory study on the correlation between 14 lung cancer-related gene expression and specific clinical characteristics of NSCLC patients

Personalized medicine has become essential in the treatment of lung cancer. However, the lung cancer-related gene expression profiles in non-small cell lung cancer (NSCLC) patients have not been elucidated. In this study, the correlation between gene expression profiles and clinicopathological characteristics was investigated in NSCLC patients. A total of 95 patients were enrolled in this study. The mRNA expression levels of 14 genes were assessed by multiplex branched DNA liquidchip (MBL) technology and data on 9 clinicopathological characteristics of patients were collected simultaneously. The correlation between gene expression and clinicopathological characteristics was investigated. Out of the 9 clinicopathological parameters, 6 were associated with several of the 14 genes analyzed. Patient gender was associated with TYMS and TOP2A. Clinical stage was associated with VEGFR2, KIT and HER2. There was weak correlation between primary tumor size of ≤3 cm and the expression level of KIT. The mRNA expression levels of VEGFR2 and HER2 correlated with distant metastasis. BRCA1, TYMS, TOP2A and HER2 were associated with histological type. Smoking correlated with higher expression levels of BRCA1, TYMS and TOP2A and lower expression levels of PDGFRβ. The results were suggestive of correlation between the clinicopathological parameters of the NSCLC patients and the mRNA expression levels of certain lung cancer-related genes, including BRCA1, TYMS, TOP2A, PDGFRβ, VEGFR2, KIT and HER2.

Advancement and the value of targeted gene related to lung cancer

The occurrence and development of lung cancer are closely related to gene expression and mutations.Recent studies of lung cancer related genes show that the expression of COPS3 gene may play a key role in the lung cancer cell proliferation.Echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase fusion gene and epidermal growth factor receptor mutations coexist.GA733 gene is closely related to lung cancer survival.Fibroblast growth factor receptor 1,high mobility protein family,members of the scavenger receptor class 5 and epidermal growth factor receptor provide new ideas for targeted therapy of lung cancer.For the therapy of lung cancer,multiple gene therapy and gene therapy combined with conventional treatment is required. Key words: Lung neoplasms; Gene expression; Gene therapy

Abstract 332: Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice.

Abstract Lung cancer is the leading cause of cancer death among malignant tumors. Lymphogenic and distant metastases were found approximately 70% at the diagnosis, and most of recurrence after surgery occurs as distant metastasis. Therefore, it is important to establish the new strategy for control of metastatic lung cancers. Since metastasis consists of several steps such as loss of cell adhesion, intravasation, survival in circulation, exit into new tissues, and colonization in a distant site, a suitable animal model is indispensable for investigation of metastasis. In addition, recent investigations revealed that biological and molecular characteristics of cancer cells were different between primary and metastatic lesions, as well as among each metastatic site. To establish the animal model reflecting human metastatic lung cancers, we used NOD/Scid/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and DC function, and complement activation as well as T and B cell deficiencies. This animal model enabled us to engraft various human cells more easily than previous immunodeficiency mouse models. At first, twenty kinds of human lung cancer cell lines were screened by expression pattern of E-cadherin and vimentin using western blot analysis, and two cell lines with high expression of vimentin and low expression of E-cadherin were selected. The cells (1×107/mouse) were injected into the dorsal flanks, and one cell line, H1975 showed lymphogenic metastasis to axillary and abdominal lymph nodes, resulting dissemination to pleura and diaphragm with ascites. Since H1975 cells carry epidermal growth factor receptor (EGFR) mutation, L858R and T790M, we can monitor the existence of cancer cells using a sensitive detection system, mutation-biased PCR and quenched probe system (MBP-QP), which were established in our laboratory. We have also tried to establish the animal model which lung cancer cells are inoculated into lung to mimic metastasis of human lung cancers. Using these model systems, we have been performing comprehensive analysis including mRNA expression, the mutation status of lung cancer related genes in each metastatic site, and compared the phosphorylation status of 38 kinds of molecules. Our goal of this study is to establish the animal model to reproduce phenotypes of metastasis of human lung cancers for screening of anti-metastatic agents. Citation Format: Naoko Aragane, Akemi Sato, Naomi Kobayashi, Masaru Ide, Eisaburo Sueoka, Seiji Okada, Shinya Kimura. Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2013-332

Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3as a novel lung cancer-related gene

BackgroundTo understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes.MethodsTwo cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR) and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression.ResultsTwo libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL) and the reverse-subtracted library (RSL) contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P < 0.05). The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells, and expression of ERGIC3 was correlated with the differentiated degree and histological type of lung cancer. The up-regulation of ERGIC3 could promote cellular migration and proliferation in vitro.ConclusionsThe two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer-related gene. ERGIC3 may play an active role in the development and progression of lung cancer.

Exome sequencing identifies MXRA5 as a novel cancer gene frequently mutated in non–small cell lung carcinoma from Chinese patients

Lung cancer has become the top killer among malignant tumors in China and is significantly associated with somatic genetic alterations. We performed exome sequencing of 14 non-small cell lung carcinomas (NSCLCs) with matched adjacent normal lung tissues extracted from Chinese patients. In addition to the lung cancer-related genes (TP53, EGFR, KRAS, PIK3CA, and ROS1), this study revealed "novel" genes not previously implicated in NSCLC. Especially, matrix-remodeling associated 5 was the second most frequently mutated gene in NSCLC (first is TP53). Subsequent Sanger sequencing of matrix-remodeling associated 5 in an additional sample set consisting of 52 paired tumor-normal DNA samples revealed that 15% of Chinese NSCLCs contained somatic mutations in matrix-remodeling associated 5. These findings, together with the results from pathway analysis, strongly indicate that altered extracellular matrix-remodeling may be involved in the etiology of NSCLC.

Abstract 4075: Aerosol azacytidine inhibits the orthotopic lung cancer in mice by its demethylation function but not cytotoxic effect

Abstract Introduction: Silencing of key tumor suppressor genes by promoter hypermethylation plays an important role in lung cancer carcinogenesis. Reversing the hypermethylation in the early lung cancer cells adjacent to the airway surface may have a potential to inhibit lung cancer development. This study was to explore the therapeutic potential of aerosol administered demethylating agent azacytidine (Aza) for the treatment of bronchial epithelial premalignancy or lung cancer. Methods: The demethylation and gene reactivation functions of Aza administered by aerosol were detected by qPCR methylation array and western blotting assay in the tumor tissues from the orthotopic human non-small cell lung cancer (NSCLC) xenograft models in nude mice. The therapeutic efficacy and toxicity of the aerosol Aza were also evaluate in the same lung cancer models. An intravenous administered Aza with clinical equivalent dose and schedule was used as a control. Results: The aerosol Aza is an appropriate pharmaceutical inhalation formulation with about 80% of droplets size distributing between 0.1 to 5 micron. This aerodynamic size range ensures the aerosol droplets depositing in the lower airway. In animal studies, aerosol administration of Aza significantly prolonged the survival of nude mice with intratracheally inoculated human NSCLC tumors. The %-increased life span was 5- to 10-fold higher than that of a systemic treatment of Aza at a clinical equivalent dose. The aerosol treatment did not cause any detectable lung toxicity or myelosuppression (systemic toxicity of Aza), since the treatment dose was &amp;gt;100-fold lower than the toxic dose. The lung tumors were resected after the aerosol Aza treatment and the methylation levels of the 24 promoters that drive the lung cancer related tumor suppressor genes (TSG) were detected. We found that aerosol Aza significantly reduced the methylation level in 9 of these 24 promoters. The dementhylating aerosol also resulted in the gene reactivation in the protein level of several tested genes. Conclusions: Aerosol administration of Aza has significant therapeutic advantages over the systemic Aza. It can be given at a non-cytotoxic dose, thus its main therapeutic efficacy is the demethylation function of Aza and the TSG reactivation. In our case, the therapeutic index of aerosol Aza is &amp;gt;800-fold higher than that of IV Aza. The clinical trial of aerosol Aza is worth to be conducted. Acknowledgement: Supported by a NIH (NCI) grant 5R01CA154755-02 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4075. doi:1538-7445.AM2012-4075

Abstract 5134: The identification and characterization of genetic variations in lung adenocarcinomas in a Japanese population

Abstract Lung adenocarcinoma is a major histological subtype of non-small cell lung cancer. Previous studies have revealed substantial clinical and molecular differences between the Asian and Caucasian populations. Kinase inhibitor-sensitive EGFR mutations are frequently detected in Asian, female and non-smoker population. These findings suggest unique genetic and/or environmental background of carcinogenesis of this tumor. In the present study, we generated and analyzed a dataset of single-nucleotide variants (SNVs) and small insertions and deletions (indels) in the entire exon regions (exomes) in Japanese lung adenocarcinomas. We chose consecutive 97 of surgically resected T2 cases registered to the database containing clinicopathological and lifestyle information established at National Cancer Center Hospital East. High integrity genomic DNA was extracted from archived methanol fixed paraffin embedded tissue samples. Whole exons were captured using Agilent SureSelect system and we generated 76-base paired-end sequences with an Illumina GAIIx platform. Approximately 30 million mapped sequences were obtained for each sample, providing 75x coverage of the target regions and 93% of the target regions also had 5x coverage. Using the sequence data, we identified SNVs and indels by BWA, SAMtools and GATK. EGFR mutations were observed among 55 cases and Sanger sequencing validated them. As well, known lung cancer-related gene mutations such as KRAS and TP53 were detected in exome data. Detailed profiling of somatic mutations provided wide variety of heterogeneity of known and novel gene mutations with both EGFR-mutant and -wildtype background. These heterogenity might define the unique signature of an individual tumor and be waited for further classification suitable for understanding genetic mechanism of carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5134. doi:1538-7445.AM2012-5134

Similar DNA methylation pattern in lung tumours from smokers and never-smokers with second-hand tobacco smoke exposure

Tobacco smoke causes lung cancer in smokers and in never-smokers exposed to second-hand tobacco smoke (SHS). Nonetheless, molecular mechanisms of lung cancer in SHS-exposed never-smokers are still elusive. We studied lung cancers from current smokers (n = 109), former smokers (n = 56) and never-smokers (n = 47) for promoter hypermethylation of five tumour suppressor genes--p16, RARB, RASSF1, MGMT and DAPK1--using methylation-specific polymerase chain reaction. Lung tumours from ever-smokers suggested an increased risk of p16 hypermethylation as compared to never-smokers (P = 0.073), with former smokers having the highest frequency of p16 hypermethylation (P = 0.044 versus current smokers and P = 0.009 versus never-smokers). In the never-smoking group, p16 hypermethylation was seen in lung tumours from SHS-exposed individuals (4/33; 12%) but in none of the non-exposed individuals (0/9). The overall occurrence of hypermethylation (measured both as methylation index and as number of genes affected) was similar in those ever exposed to tobacco smoke (smokers, SHS-exposed never-smokers) and differed from non-exposed never-smokers. In multivariate analysis, p16 hypermethylation was more prevalent in lung tumours from male than female patients (P = 0.018) and in squamous cell carcinomas than in adenocarcinomas (P = 0.025). Occurrence of TP53 mutation in the tumour was associated with hypermethylation of at least one gene (P = 0.027). In all, our data suggest that promoter hypermethylation pattern in SHS-exposed never-smokers resembles that observed in smokers. Association between TP53 mutation, a hallmark of smokers' lung cancer, and methylation of one or more of the lung cancer-related genes studied, provides further evidence for common tobacco smoke-related origin for both types of molecular alterations.

Microarray Analysis reveals that Dietary Retinoic Acid Suppresses Cancer-Related Gene Expression of the Lungs of Cigarette Smoke Exposed Rats

Previously we found that cigarette smoke depleted vitamin A in the lungs and induced tracheal precancerous lesions. To understand the molecular consequences underlying cigarette smoke-induced vitamin A depletion and its associated lung cancer risk, this study investigated the lung cancer-related genes in cigarette smoke-exposed rats with or without dietary retinoic acid, the active metabolite of vitamin A. Twenty-four male weanling rats were fed either a control or a retinoic acid supplemented diet. Half of each group was exposed to 40 commercial cigarettes/ d, 5 d/ wk. After 4 weeks, the rats were sacrificed and their lungs were immediately frozen. Total RNA was extracted and purified, from which cDNA was synthesized and labeled for gene expression analysis. Expressions of 120 genes were measured via a customized microarray. In lungs exposed to cigarette smoke, most of the genes involved in cell division, transcription and cell adhesion were up-regulated. The dietary retinoic acid treatment of the cigarette smoke-exposed lungs was found to down-regulate most of the genes involved in similar functions. In addition, retinoic acid down-regulated four genes, Egr1, Fos, Icam1 and Mmp9, all of which were up-regulated by cigarette smoke. These findings suggest possible molecular mechanisms of cigarette smoke induced-lung cancer and define potential targets of retinoic acid anticarcinogenic actions.

Comparative Analysis of Human Protein-Coding and Noncoding RNAs between Brain and 10 Mixed Cell Lines by RNA-Seq

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome.

Toxicogenomic comparison of multi-wall carbon nanotubes (MWCNTs) and asbestos

Carbon nanotubes (CNTs) have specific properties, including electrical and thermal conductivity, great strength, and rigidity, that allow them to be used in many fields. However, this increasing contact with humans and the environment is also raising health and safety concerns. Thus, research on the safety of CNTs has attracted much interest, including a comparison of the toxic effects of asbestos and carbon nanotubes, due to their physical similarity of a high aspect ratio (length/diameter). Nonetheless, there has not yet been a toxicogenomic comparison. Therefore, to examine toxicogenomic effects, the 50% growth inhibition (GI(50)) concentration was determined for multi-wall carbon nanotubes (MWCNTs) and asbestos (crocidolite) and found to be approximately 0.0135 and 0.066%, respectively, in the case of 24-h treatment of normal human bronchial epithelia (NHBE) cells. Using these GI(50) concentrations, NHBE cells were then treated with MWCNTs and asbestos for 6 and 24 h, followed by a DNA microarray analysis. Among 31,647 genes, 1,201 and 1,252 were up-regulated by both asbestos and MWCNTs after 6 and 24 h of exposure, respectively. Meanwhile, 1,977 and 1,542 genes were down-regulated by both asbestos and MWNCTs after 6 and 24 h of exposure, respectively. In particular, the asbestos and MWCNTs both induced an over twofold up- and down-regulated expression of 12 mesothelioma-related genes and 22 lung cancer-related genes when compared with the negative control. Plus, the genes induced by the MWCNT exposure were expressed in the brain, lungs, epithelium, liver, and colon.

Construction and identification of a recombinant lentiviral vector of RNA interference of lung cancer related gene SLC35F2

Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference (RNAi) and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 (SLC-siRNA) was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction (PCR) and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group ( 14.88% vs 3.16% ,P ＜0.05 ).Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established. Key words: Long carcinoma; RNA interference; Lentiviral vector; Cell proliferation

Wood Smoke Exposure and Gene Promoter Methylation Are Associated with Increased Risk for COPD in Smokers

Wood smoke-associated chronic obstructive pulmonary disease (COPD) is common in women in developing countries but has not been adequately described in developed countries. Our objective was to determine whether wood smoke exposure was a risk factor for COPD in a population of smokers in the United States and whether aberrant gene promoter methylation in sputum may modify this association. For this cross-sectional study, 1,827 subjects were drawn from the Lovelace Smokers' Cohort, a predominantly female cohort of smokers. Wood smoke exposure was self-reported. Postbronchodilator spirometry was obtained, and COPD outcomes studied included percent predicted FEV₁, airflow obstruction, and chronic bronchitis. Effect modification of wood smoke exposure with current cigarette smoke, ethnicity, sex, and promoter methylation of lung cancer-related genes in sputum on COPD outcomes were separately explored. Multivariable logistic and poisson regression models were used for binary and rate-based outcomes, respectively. Self-reported wood smoke exposure was independently associated with a lower percent predicted FEV₁ (point estimate [± SE] -0.03 ± 0.01) and a higher prevalence of airflow obstruction and chronic bronchitis (odds ratio, 1.96; 95% confidence interval, 1.52-2.52 and 1.64 (95% confidence interval, 1.31-2.06, respectively). These associations were stronger among current cigarette smokers, non-Hispanic whites, and men. Wood smoke exposure interacted in a multiplicative manner with aberrant promoter methylation of the p16 or GATA4 genes on lower percent predicted FEV₁. These studies identify a novel link between wood smoke exposure and gene promoter methylation that synergistically increases the risk for reduced lung function in cigarette smokers.