Identification of novel deregulated RNA metabolism-related genes in non-small cell lung cancer

Lung cancer is a leading cause of cancer death worldwide. Several alterations in RNA metabolism have been found in lung cancer cells; this suggests that RNA metabolism-related molecules are involved in the development of this pathology. In this study, we searched for RNA metabolism-related genes that exhibit different expression levels between normal and tumor lung tissues. We identified eight genes differentially expressed in lung adenocarcinoma microarray datasets. Of these, seven were up-regulated whereas one was down-regulated. Interestingly, most of these genes had not previously been associated with lung cancer. These genes play diverse roles in mRNA metabolism: three are associated with the spliceosome (ASCL3L1, SNRPB and SNRPE), whereas others participate in RNA-related processes such as translation (MARS and MRPL3), mRNA stability (PCBPC1), mRNA transport (RAE), or mRNA editing (ADAR2, also known as ADARB1). Moreover, we found a high incidence of loss of heterozygosity at chromosome 21q22.3, where the ADAR2 locus is located, in NSCLC cell lines and primary tissues, suggesting that the downregulation of ADAR2 in lung cancer is associated with specific genetic losses. Finally, in a series of adenocarcinoma patients, the expression of five of the deregulated genes (ADAR2, MARS, RAE, SNRPB and SNRPE) correlated with prognosis. Taken together, these results support the hypothesis that changes in RNA metabolism are involved in the pathogenesis of lung cancer, and identify new potential targets for the treatment of this disease.     

Research on circadian clock genes in non-small-cell lung carcinoma

Circadian clock genes have become a hot topic in cancer research in recent years, and more and more studies are showing that clock genes are involved in regulating cell proliferation cycle and apoptosis of malignant tumors, neuroendocrine and immune function, and other processes. Lung cancer is a malignant tumor with increasing incidence worldwide. The pathogenesis of lung cancer is extremely complicated and includes genetic factors, living environment, and smoking, and the occurrence of lung cancer is related to the regulation of many oncogenes and tumor suppressor genes. But there are few studies on clock genes in lung cancer. Studies on clock genes may help to better understand the mechanism of lung cancer development for an improved treatment. The expressions of all 14 kinds of clock genes in adenocarcinoma (ADC) and squamous cell carcinoma (SCC), two main kinds of non-small-cell lung cancer (NSCLC), were studied based on integration and analysis of data from The Cancer Genome Atlas (TCGA) to show the association between clock gene expression and prognosis of cancer patients. Analysis of TCGA data indicated that overexpression of Cry2, BMAL1, and RORA with underexpression of Timeless and NPAS2 was associated with a favorable prognosis of ADC, and the expression of NPAS2 was associated with the time of patient survival. Additionally, the expression of Cry2 was related to TNM stage. In SCC, high expression of DEC1 was correlated with poor overall survival in patients and the expression of Timeless was associated with the time of patient survival. In NSCLC, circadian clock genes constitute cancer circadian rhythm by interacting with each other, showing that asynchrony with normal tissues, which collectively controlling the occurrence and development of NSCLC.     

Gene Expression Profiling in Human Lung Development: An Abundant Resource for Lung Adenocarcinoma Prognosis

A tumor can be viewed as a special “organ” that undergoes aberrant and poorly regulated organogenesis. Progress in cancer prognosis and therapy might be facilitated by re-examining distinctive processes that operate during normal development, to elucidate the intrinsic features of cancer that are significantly obscured by its heterogeneity. The global gene expression signatures of 44 human lung tissues at four development stages from Asian descent and 69 lung adenocarcinoma (ADC) tissue samples from ethnic Chinese patients were profiled using microarrays. All of the genes were classified into 27 distinct groups based on their expression patterns (named as PTN1 to PTN27) during the developmental process. In lung ADC, genes whose expression levels decreased steadily during lung development (genes in PTN1) generally had their expression reactivated, while those with uniformly increasing expression levels (genes in PTN27) had their expression suppressed. The genes in PTN1 contain many n-gene signatures that are of prognostic value for lung ADC. The prognostic relevance of a 12-gene demonstrator for patient survival was characterized in five cohorts of healthy and ADC patients [ADC_CICAMS (n = 69, p = 0.007), ADC_PNAS (n = 125, p = 0.0063), ADC_GSE13213 (n = 117, p = 0.0027), ADC_GSE8894 (n = 62, p = 0.01), and ADC_NCI (n = 282, p = 0.045)] and in four groups of stage I patients [ADC_CICAMS (n = 22, p = 0.017), ADC_PNAS (n = 76, p = 0.018), ADC_GSE13213 (n = 79, p = 0.02), and ADC_qPCR (n = 62, p = 0.006)]. In conclusion, by comparison of gene expression profiles during human lung developmental process and lung ADC progression, we revealed that the genes with a uniformly decreasing expression pattern during lung development are of enormous prognostic value for lung ADC.     

Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus)

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.     

Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes

Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.     

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non–small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80–100%.     

Molecular markers of various stages of nonsmall cell lung cancer development

Studies performed in the recent decade in the Laboratory of the Regulation of Cell and Virus Oncogenes associated structural and functional defects of several oncogenes and tumor suppressor genes with various stages of non-small-cell lung cancer. High risk of lung cancer was established for carriers of rare alleles of the Hras1 minisatellite, the hypermethylated p16INK4A promoter, and microsatellite defects in chromosome regions 3p12, 3p14.2, 3p22-24, 3p21, 3p25, 9p21, and 17p13. Analysis of the Hras1 minisatellite and microsatellites located in two minimal deletion overlap regions of 1p36 was shown to allow a more reliable prognosis in lung cancer. The results testified again that panels of molecular markers are useful for individual risk assessment, early and differential diagnosis, and prognosis in cancer.     

UGT1A6 Polymorphisms Modulated Lung Cancer Risk in a Chinese Population

Uridine diphosphoglucuronosyltransferases (UGTs) 1A6 is the only UGT1A isoform expressed in lung tissue. It is responsible for the detoxification of carcinogens such as benezo[a]pyrene from cigarette smoke. The purpose of this study was to evaluate the association of UGT1A6 polymorphisms and haplotypes with lung cancer risk and to evaluate the functional significance of UGT1A6 polymorphisms. Genomic DNA was isolated from leukocytes. Eight UGT1A6 polymorphisms were sequenced in a test set of 72 Chinese lung cancer patients and 62 healthy controls. Potential risk modifying alleles were validated in a separate set of 95 Chinese lung cancer patients and 100 healthy controls. UGT1A6 19T>G, 541A>G and 552A>C showed significant association with increased lung cancer risk, while UGT1A6 105C>T and IVS1+130G>T were significantly associated with reduced lung cancer risk. Multivariate logistic regression analysis demonstrated a significant association of lung cancer with UGT1A6 541A>G (OR: 3.582, 95% CI: 1.27–10.04, p = 0.015), 552A>C (OR: 5.364, 95% CI: 1.92–14.96, p = 0.001) and IVS1+130G>T (OR: 0.191, 95% CI: 0.09–0.36, p<0.001). Functional test demonstrated that UGT1A6 105C>T increased mRNA stability, providing a plausible explanation of its association with reduced lung cancer risk. Thus UGT1A6 polymorphisms may be used to identify people with increased risk of developing lung cancer.     

Systematic profiling of invasion-related gene signature predicts prognostic features of lung adenocarcinoma

Due to the high heterogeneity of lung adenocarcinoma (LUAD), molecular subtype based on gene expression profiles is of great significance for diagnosis and prognosis prediction in patients with LUAD. Invasion-related genes were obtained from the CancerSEA database, and LUAD expression profiles were downloaded from The Cancer Genome Atlas. The ConsensusClusterPlus was used to obtain molecular subtypes based on invasion-related genes. The limma software package was used to identify differentially expressed genes (DEGs). A multi-gene risk model was constructed by Lasso-Cox analysis. A nomogram was also constructed based on risk scores and meaningful clinical features. 3 subtypes (C1, C2 and C3) based on the expression of 97 invasion-related genes were obtained. C3 had the worst prognosis. A total of 669 DEGs were identified among the subtypes. Pathway enrichment analysis results showed that the DEGs were mainly enriched in the cell cycle, DNA replication, the p53 signalling pathway and other tumour-related pathways. A 5-gene signature (KRT6A, MELTF, IRX5, MS4A1 and CRTAC1) was identified by using Lasso-Cox analysis. The training, validation and external independent cohorts proved that the model was robust and had better prediction ability than other lung cancer models. The gene expression results showed that the expression levels of MS4A1 and KRT6A in tumour tissues were higher than in normal tissues, while CRTAC1 expression in tumour tissues was lower than in normal tissues. The 5-gene signature prognostic stratification system based on invasion-related genes could be used to assess prognostic risk in patients with LUAD.     

A Novel Five Gene Signature Derived from Stem-Like Side Population Cells Predicts Overall and Recurrence-Free Survival in NSCLC

Gene expression profiling has been used to characterize prognosis in various cancers. Earlier studies had shown that side population cells isolated from Non-Small Cell Lung Cancer (NSCLC) cell lines exhibit cancer stem cell properties. In this study we apply a systems biology approach to gene expression profiling data from cancer stem like cells isolated from lung cancer cell lines to identify novel gene signatures that could predict prognosis. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stem like properties. Differentially expressed genes that were over or under-expressed at least two fold commonly in all 4 cell lines were identified. We found 354 were upregulated and 126 were downregulated in SP cells compared to MP cells; of these, 89 up and 62 downregulated genes (average 2 fold changes) were used for Principle Component Analysis (PCA) and MetaCore™ pathway analysis. The pathway analysis demonstrated representation of 4 up regulated genes (TOP2A, AURKB, BRRN1, CDK1) in chromosome condensation pathway and 1 down regulated gene FUS in chromosomal translocation. Microarray data was validated using qRT-PCR on the 5 selected genes and all showed robust correlation between microarray and qRT-PCR. Further, we analyzed two independent gene expression datasets that included 360 lung adenocarcinoma patients from NCI Director''s Challenge Set for overall survival and 63 samples from Sungkyunkwan University (SKKU) for recurrence free survival. Kaplan-Meier and log-rank test analysis predicted poor survival of patients in both data sets. Our results suggest that genes involved in chromosome condensation are likely related with stem-like properties and might predict survival in lung adenocarcinoma. Our findings highlight a gene signature for effective identification of lung adenocarcinoma patients with poor prognosis and designing more aggressive therapies for such patients.     

Integrated analysis of gene expression and DNA methylation datasets identified key genes and a 6-gene prognostic signature for primary lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the main subtype of non-small cell lung cancer with a poor survival prognosis. In our study, gene expression, DNA methylation, and clinicopathological data of primary LUAD were utilized to identify potential prognostic markers for LUAD, which were recruited from The Cancer Genome Atlas (TCGA) database. Univariate regression analysis showed that there were 21 methylation-associated DEGs related to overall survival (OS), including 9 down- and 12 up-regulated genes. The 12 up-regulated genes with hypomethylation may be risky genes, whereas the other 9 down-regulated genes with hypermethylation might be protective genes. By using the Step-wise multivariate Cox analysis, a methylation-associated 6-gene (consisting of CCL20, F2, GNPNAT1, NT5E, B3GALT2, and VSIG2) prognostic signature was constructed and the risk score based on this gene signature classified patients into high- or low-risk groups. Patients of the high-risk group had shorter OS than those of the low-risk group in both the training and validation cohort. Multivariate Cox analysis and the stratified analysis revealed that the risk score was an independent prognostic factor for LUAD patients. The methylation-associated gene signature may serve as a prognostic factor for LUAD patients and the represent hypermethylated or hypomethylated genes might be potential targets for LUAD therapy.     

Identification of two molecular subtypes of dysregulated immune lncRNAs in ovarian cancer

Long non-coding RNA (lncRNA) has increasingly been identified as a key regulator in pathologies such as cancer. Multiple platforms were used for comprehensive analysis of ovarian cancer to identify molecular subgroups. However, lncRNA and its role in mapping the ovarian cancer subpopulation are still largely unknown. RNA-sequencing and clinical characteristics of ovarian cancer were acquired from The Cancer Genome Atlas database (TCGA). A total of 52 lncRNAs were identified as aberrant immune lncRNAs specific to ovarian cancer. We redefined two different molecular subtypes, C1(188) and C2(184 samples), in “iClusterPlus” R package, among which C2 grouped ovarian cancer samples have higher survival probability and longer median survival time (P <0.05) with activated IFN-gamma response, Wound Healing and Cytotoxic lymphocytes signal; 456 differentially expressed genes were acquired in C1 and C2 subtypes using limma (3.40.6) package, among which 419 were up-regulated and 37 were down-regulated, in TCGA dataset. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis revealed that these genes were actively involved in ECM-receptor interaction, PI3K-Akt signaling pathway interaction KEGG pathway. Compared with the existing immune subtype, the Cluster2 sample showed a substantial increase in the proportion of the existing C2 immune subtype, accounting for 81.37%, which was associated with good prognosis. Our C1 subtype contains only 56.49% of the existing immune C1 and C4, which also explains the poor prognosis of C1. Furthermore, 52 immune-related lncRNAs were used to divide the TCGA-endometrial cancer and cervical cancer samples into two categories, and C2 had a good prognosis. The differentially expressed genes were highly correlated with immune-cell-related pathways. Based on lncRNA, two molecular subtypes of ovarian cancer were identified and had significant prognostic differences and immunological characteristics.     

UHRF1 predicts poor prognosis by triggering cell cycle in lung adenocarcinoma

Accumulating evidence suggests that ubiquitin‐like with plant homeodomain and ring finger domains 1 (UHRF1) is overexpressed in non‐small cell lung cancer (NSCLC); however, the expression and function of UHRF1 in the subtype of NSCLC are still unclear. Here, we investigate the expression and prognosis traits of UHRF1 in large NSCLC cohorts and explore the molecular characters during UHRF1 up‐regulation. We find that UHRF1 is predominantly overexpressed in lung squamous cell carcinoma (SCC). Surprisingly, the up‐regulated UHRF1 is only associated with the overall survival of lung adenocarcinoma (ADC) and knockdown of UHRF1 dramatically attenuates ADC tumorigenesis. Mechanically, we identify a hub gene that includes a total of 55 UHRF1‐related genes, which are tightly associated with cell cycle pathway and yield to the poor clinical outcome in ADC patients. What's more, we observe knockdown of UHRF1 only affects ADC cells cycle and induces cell apoptosis. These results suggest that up‐regulated UHRF1 only contributes to lung ADC survival by triggering cell cycle pathway, and it may be a prognostic biomarker for lung ADC patients.