Roslan Harun

Integrated analysis of copy number variation and genome-wide expression profiling in colorectal cancer tissues.

Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV) and gene expression in colorectal cancer (CRC) samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

Identification of diagnostic markers in colorectal cancer via integrative epigenomics and genomics data

Apart from genetic mutations, epigenetic alteration is a common phenomenon that contributes to neoplastic transformation in colorectal cancer. Transcriptional silencing of tumor-suppressor genes without changes in the DNA sequence is explained by the existence of promoter hypermethylation. To test this hypothesis, we integrated the epigenome and transcriptome data from a similar set of colorectal tissue samples. Methylation profiling was performed using the Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent normal epithelial cells. Fifteen of the 55 paired tissues were used for gene expression profiling using the Affymetrix GeneChip Human Gene 1.0 ST array. Validation was carried out on 150 colorectal tissues using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique. PCA and supervised hierarchical clustering in the two microarray datasets showed good separation between cancer and normal samples. Significant genes from the two analyses were obtained based on a ≥2-fold change and a false discovery rate (FDR) P-value of <0.05. We identified 1,081 differentially hypermethylated CpG sites and 36 hypomethylated CpG sites. We also found 709 upregulated and 699 downregulated genes from the gene expression profiling. A comparison of the two datasets revealed 32 overlapping genes with 27 being hypermethylated with downregulated expression and 4 hypermethylated with upregulated expression. One gene was found to be hypomethylated and downregulated. The most enriched molecular pathway identified was cell adhesion molecules that involved 4 overlapped genes, JAM2, NCAM1, ITGA8 and CNTN1. In the present study, we successfully identified a group of genes that showed methylation and gene expression changes in well-defined colorectal cancer tissues with high purity. The integrated analysis gives additional insight regarding the regulation of colorectal cancer-associated genes and their underlying mechanisms that contribute to colorectal carcinogenesis.

Tocotrienol-Rich Fraction, [6]-Gingerol and Epigallocatechin Gallate Inhibit Proliferation and Induce Apoptosis of Glioma Cancer Cells

Plant bioactives [6]-gingerol (GING), epigallocatechin gallate (EGCG) and asiaticoside (AS) and vitamin E, such as tocotrienol-rich fraction (TRF), have been reported to possess anticancer activity. In this study, we investigated the apoptotic properties of these bioactive compounds alone or in combination on glioma cancer cells. TRF, GING, EGCG and AS were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) in culture by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay. With the exception of AS, combinations of two compounds were tested, and the interactions of each combination were evaluated by the combination index (CI) using an isobologram. Different grades of glioma cancer cells showed different cytotoxic responses to the compounds, where in 1321N1 and LN18 cells, the combination of EGCG + GING exhibited a synergistic effect with CI = 0.77 and CI = 0.55, respectively. In contrast, all combinations tested (TRF + GING, TRF + EGCG and EGCG + GING) were found to be antagonistic on SW1783 with CI values of 1.29, 1.39 and 1.39, respectively. Combined EGCG + GING induced apoptosis in both 1321N1 and LN18 cells, as evidenced by Annexin-V FITC/PI staining and increased active caspase-3. Our current data suggests that the combination of EGCG + GING synergistically induced apoptosis and inhibits the proliferation 1321N1 and LN18 cells, but not SW1783 cells, which may be due to their different genetic profiles.

MicroRNAs and Lymph Node Metastasis in Papillary Thyroid Cancers.

Lymph node metastasis (LNM) in papillary thyroid cancer (PTC) has been shown to be associated with increased risk of locoregional recurrence, poor prognosis and decreased survival, especially in older patients. Hence, there is a need for a reliable biomarker for the prediction of LNM in this cancer. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene translation or degradation and play key roles in numerous cellular functions including cell-cycle regulation, differentiation, apoptosis, invasion and migration. Various studies have demonstrated deregulation of miRNA levels in many diseases including cancers. While a large number of miRNAs have been identified from PTCs using various means, association of miRNAs with LNM in such cases is still controversial. Furthermore, studies linking most of the identified miRNAs to the mechanism of LNM have not been well documented. The aim of this review is to update readers on the current knowledge of miRNAs in relation to LNM in PTC.

Senescence-related changes in gene expression of peripheral blood mononuclear cells from octo/nonagenarians compared to their offspring.

Mechanisms determining both functional rate of decline and the time of onset in aging remain elusive. Studies of the aging process especially those involving the comparison of long-lived individuals and young controls are fairly limited. Therefore, this research aims to determine the differential gene expression profile in related individuals from villages in Pahang, Malaysia. Genome-wide microarray analysis of 18 samples of peripheral blood mononuclear cells (PBMCs) from two groups: octo/nonagenarians (80–99 years old) and their offspring (50.2  ±  4.0 years old) revealed that 477 transcripts were age-induced and 335 transcripts were age-repressed with fold changes ≥1.2 in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression were associated with increased capacity for apoptosis (BAK1), cell cycle regulation (CDKN1B), metabolic process (LRPAP1), insulin action (IGF2R), and increased immune and inflammatory response (IL27RA), whereas response to stress (HSPA8), damage stimulus (XRCC6), and chromatin remodelling (TINF2) pathways were downregulated in octo/nonagenarians. These results suggested that systemic telomere maintenance, metabolism, cell signalling, and redox regulation may be important for individuals to maintain their healthy state with advancing age and that these processes play an important role in the determination of the healthy life-span.

γ-Tocotrienol and 6-Gingerol in Combination Synergistically Induce Cytotoxicity and Apoptosis in HT-29 and SW837 Human Colorectal Cancer Cells.

Numerous bioactive compounds have cytotoxic properties towards cancer cells. However, most studies have used single compounds when bioactives may target different pathways and exert greater cytotoxic effects when used in combination. Therefore, the objective of this study was to determine the anti-proliferative effect of γ-tocotrienol (γ-T3) and 6-gingerol (6G) in combination by evaluating apoptosis and active caspase-3 in HT-29 and SW837 colorectal cancer cells. MTS assays were performed to determine the anti-proliferative and cytotoxicity effect of γ-T3 (0–150 µg/mL) and 6G (0–300 µg/mL) on the cells. The half maximal inhibitory concentration (IC50) value of 6G+ γ-T3 for HT-29 was 105 + 67 µg/mL and for SW837 it was 70 + 20 µg/mL. Apoptosis, active caspase-3 and annexin V FITC assays were performed after 24 h of treatment using flow cytometry. These bioactives in combination showed synergistic effect on HT-29 (CI: 0.89 ± 0.02,) and SW837 (CI: 0.79 ± 0.10) apoptosis was increased by 21.2% in HT-29 and 55.4% in SW837 (p < 0.05) after 24 h treatment, while normal hepatic WRL-68 cells were unaffected. Increased apoptosis by the combined treatments was also observed morphologically, with effects like cell shrinkage and pyknosis. In conclusion, although further studies need to be done, γ-T3 and 6G when used in combination act synergistically increasing cytotoxicity and apoptosis in cancer cells.

NFIX as a master regulator for lung cancer progression

About 40% of lung cancer cases globally are diagnosed at the advanced stage. Lung cancer has a high mortality and overall survival in stage I disease is only 70%. This study was aimed at finding a candidate of transcription regulator that initiates the mechanism for metastasis by integrating computational and functional studies. The genes involved in lung cancer were retrieved using in silico software. 10 kb promoter sequences upstream were scanned for the master regulator. Transient transfection of shRNA NFIXs were conducted against A549 and NCI-H1299 cell lines. qRT-PCR and functional assays for cell proliferation, migration and invasion were carried out to validate the involvement of NFIX in metastasis. Genome-wide gene expression microarray using a HumanHT-12v4.0 Expression BeadChip Kit was performed to identify differentially expressed genes and construct a new regulatory network. The in silico analysis identified NFIX as a master regulator and is strongly associated with 17 genes involved in the migration and invasion pathways including IL6ST, TIMP1 and ITGB1. Silencing of NFIX showed reduced expression of IL6ST, TIMP1 and ITGB1 as well as the cellular proliferation, migration and invasion processes. The data was integrated with the in silico analyses to find the differentially expressed genes. Microarray analysis showed that 18 genes were expressed differentially in both cell lines after statistical analyses integration between t-test, LIMMA and ANOVA with Benjamini-Hochberg adjustment at p-value < 0.05. A transcriptional regulatory network was created using all 18 genes, the existing regulated genes including the new genes PTCH1, NFAT5 and GGCX that were found highly associated with NFIX, the master regulator of metastasis. This study suggests that NFIX is a promising target for therapeutic intervention that is expected to inhibit metastatic recurrence and improve survival rate.

Exploring breast carcinogenesis through integrative genomics and epigenomics analyses

There have been many DNA methylation studies on breast cancer which showed various methylation patterns involving tumour suppressor genes and oncogenes but only a few of those studies link the methylation data with gene expression. More data are required especially from the Asian region and to analyse how the epigenome data correlate with the transcriptome. DNA methylation profiling was carried out on 76 fresh frozen primary breast tumour tissues and 25 adjacent non-cancerous breast tissues using the Illumina Infinium(®) HumanMethylation27 BeadChip. Validation of methylation results was performed on 7 genes using either MS-MLPA or MS-qPCR. Gene expression profiling was done on 15 breast tumours and 5 adjacent non-cancerous breast tissues using the Affymetrix GeneChip(®) Human Gene 1.0 ST array. The overlapping genes between DNA methylation and gene expression datasets were further mapped to the KEGG database to identify the molecular pathways that linked these genes together. Supervised hierarchical cluster analysis revealed 1,389 hypermethylated CpG sites and 22 hypomethylated CpG sites in cancer compared to the normal samples. Gene expression microarray analysis using a fold-change of at least 1.5 and a false discovery rate (FDR) at p>0.05 identified 404 upregulated and 463 downregulated genes in cancer samples. Integration of both datasets identified 51 genes with hypermethylation with low expression (negative association) and 13 genes with hypermethylation with high expression (positive association). Most of the overlapping genes belong to the focal adhesion and extracellular matrix-receptor interaction that play important roles in breast carcinogenesis. The present study displayed the value of using multiple datasets in the same set of tissues and how the integrative analysis can create a list of well-focused genes as well as to show the correlation between epigenetic changes and gene expression. These gene signatures can help us understand the epigenetic regulation of gene expression and could be potential targets for therapeutic intervention in the future.

Gene expression profiles predict survival of patients with advanced non-small cell lung cancers

A large variation in prognosis is observed despite the use of clinical prognostic factors in patients with advanced non-small cell lung cancer (NSCLC). It is likely that this variation is due to the different biological properties of the tumour cells. In this work we aimed to identify gene signature that could predict survival in advanced NSCLC. Total RNA was extracted from five 5 μm-thick sections of the FFPE using the High Pure RNA Paraffin Kit (Roche). RNA amplification was performed using WT-Ovation™ FFPE RNA Amplification System V2 (NuGen). The amplified cDNA was then labelled and hybridised onto Illumina HumanRef-8 v3.0 Expression BeadChips. Microarray data analysis was subsequently performed using Genespring GX version 9.0. Out of 75 FFPE samples, only 32 had sufficient RNA quality and quantity for microarray gene expression analysis. Patients were grouped into long and short survival groups based on the time to cancer-related death. After normalization and filtration, 19,002 genes were selected for differential gene expression analysis. A total of 440 genes differed significantly between the long and short survival groups (ANOVA, p < 0.05, with Benjamini and Hochberg False Discovery Rate multiple testing correction). Unsupervised Hierarchial Clustering with Pearson correlation and average linkage identified two broad clusters of patients corresponding to the long and short survival. Thirteen genes were selected based on the TTest, 2-fold expression changes, principal components analysis and univariate Cox regression analysis and risk scores were calculated for each patient. These gene signatures were independent predictors of survival. The model was validated with a published microarray data from 130 patients with NSCLC. Using Gene Set Analysis (GSA), we found certain biological processes including metastasis and chemotherapy resistance were up-regulated in the short survival group while TID pathway and MAPKKK cascade were enriched in the long survival group. As the conclusion, there is several distinct gene expression profiles associated with survival of patients with advanced stage NSCLC. Survival outcomes in advanced NSCLC could be predicted based on a 13-gene signature.

Mitochondrial DNA Mutations in Grade II and III Glioma Cell Lines Are Associated with Significant Mitochondrial Dysfunction and Higher Oxidative Stress

The role of mitochondria in tumorigenesis has regained much attention as it could dysregulate cellular energetics, oxidative stress and apoptosis. However, the role of mitochondria in different grade gliomasis still unknown. This study aimed to identify mitochondrial DNA (mtDNA) sequence variations that could possibly affect the mitochondrial functions and also the oxidative stress status. Three different grades of human glioma cell lines and a normal human astrocyte cell line were cultured in-vitro and tested for oxidative stress biomarkers. Relative oxidative stress level, mitochondria activity, and mitochondrial mass were determined by live cell imaging with confocal laser scanning microscope using CM-H2DCFDA, MitoTracker Green, and MitoTracker Orange stains. The entire mitochondrial genome was sequenced using the AffymetrixGeneChip Human Mitochondrial Resequencing Array 2.0. The mitochondrial sequence variations were subjected to phylogenetic haplogroup assessment and pathogenicity of the mutations were predicted using pMUT and PolyPhen2. The Grade II astrocytoma cells showed increased oxidative stress wherea high level of 8-OHdG and oxidative stress indicator were observed. Simultaneously, Grade II and III glioma cells showed relatively poor mitochondria functions and increased number of mutations in the coding region of the mtDNA which could be due to high levels of oxidative stress in these cells. These non-synonymous mtDNA sequence variations were predicted to be pathogenic and could possibly lead to protein dysfunction, leading to oxidative phosphorylation (OXPHOS) impairment, mitochondria dysfunction and could create a vicious cycle of oxidative stress. The Grade IV cells had no missense mutation but preserved intact mitochondria and excellent antioxidant defense mechanisms thus ensuring better survival. In conclusion, Grade II and III glioma cells demonstrated coding region mtDNA mutations, leading to mitochondrial dysfunction and higher oxidative stress.