Microarray Method Research Articles

Expression of migration and invasion inhibitory protein (MIIP) in gastric adenocarcinoma and its clinicopathological significance

Objective To investigate the expression of migration and invasion inhibitory protein (MIIP) in gastric adenocarcinoma and its clinicopathological significance. Methods The expression of MIIP was detected in 90 samples of gastric adenocarcinoma and 90 samples of normal gastric tissues by tissue microarray and immunohistochemical method. The correlation of MIIP expression with clinicopathological parameters and prognosis of gastric adenocarcinoma patients was analyzed. Results The expression level of MIIP in gastric adenocarcinoma tissues was significantly lower than that in normal gastric tissues [30.0%(27/90)vs. 55.6%(50/90), χ2=12.006, P=0.001]. The down-regulated expression of MIIP in gastric adenocarcinoma tissues was significantly associated with larger tumor mass (χ2=4.116, P=0.042), higher T stage (χ2= 6.752, P=0.009 ), lymph nodes metastasis (χ2=7.234, P=0.007) and higher TNM stage (χ2=7.608, P=0.006). The 5-year overall survival rate was significantly higher in patients with high expression of MIIP than those with low expression (χ2=11.378, P=0.001). The tumor mass (OR=0.570, P=0.043), T stage (OR=0.213, P=0.036) and the expression level of MIIP (OR=1.967, P=0.042) were the independent predictors for prognosis of gastric adenocarcinoma. Conclusions The low expression level of MIIP is associated with poor prognosis in gastric adenocarcinomas. Key words: Gastric cancer; Adenocarcinoma; Prognosis; Migration and invasion inhibitory protein

Bioinformatics analysis of gene expression data for the identification of critical genes in breast invasive carcinoma.

Gene expression data were analyzed in order to identify critical genes in breast invasive carcinoma (BRCA). Data from 1,073 BRCA samples and 99 normal samples were analyzed, which were obtained from The Cancer Genome Atlas. Differentially expressed genes (DEGs) were identified using the significance analysis of microarrays method and a functional enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. Relevant microRNAs (miRNAs), transcription factors (TFs) and associated small molecule drugs were revealed by Fisher's exact test. Furthermore, protein-protein interaction (PPI) information was downloaded from the Human Protein Reference Database. Interactions with a Pearson's correlation coefficient >0.5 were identified and PPI networks were subsequently constructed. A survival analysis was also conducted according to the Kaplan-Meier method. Initially, the 1,073 BRCA samples were clustered into seven groups, and 5,394 DEGs that were identified in ≥4 groups were selected. These DEGs were involved in the cell cycle, ubiquitin-mediated proteolysis, oxidative phosphorylation and human immunodeficiency virus infection. In addition, TFs, including Sp1 transcription factor, DAN domain BMP antagonist family member 5, MYCN proto-oncogene, bHLH transcription factor and cAMP responsive element binding protein (CREB)1, were identified in the BRCA groups. Seven PPI networks were subsequently constructed and the top 10 hub genes were acquired, including RB transcriptional corepressor 1, inhibitor of nuclear factor (NF)-κB kinase subunit γ, NF-κB subunit 2, transporter 1, ATP binding cassette subfamily B member, CREB binding protein and proteasome subunit α3. A significant difference in survival was observed between the two combined groups (groups-2, −4 and −5 vs. groups-1, −3, −6 and −7). In conclusion, numerous critical genes were detected in BRCA, and relevant miRNAs, TFs and small molecule drugs were identified. These findings may advance understanding regarding the pathogenesis of BRCA.

Differences in echocardiography, blood pressure, stroke volume, maximal power and profile of genes related to cardiac hypertrophy in elite road cyclists.

Regular and moderate exercise is beneficial for improving the efficiency of the heart, but high-intensity physical activity may result in cardiac changes. This study focuses on the identification of the differences in echocardiography and blood variables before exercise, as well as the genes associated with cardiac hypertrophy at rest and in response to graded exercise test. The study group was made up of 28 road cyclists. Echocardiographic parameters and blood pressure were measured before exercise tests (N = 28). Blood samples were collected at rest, at maximal exercise intensity in a graded bicycle test and after 15 min of recovery; afterwards, blood morphology was estimated and RNA was isolated. Analysis of the expression profile of genes was performed for randomly selected road cyclists using the microarray method. Echocardiographic results and blood parameters divided cyclists into two groups: with and without left ventricular hypertrophy (N = 14). Differences in the structure and function of the left ventricle cyclists with a similar level of training were observed (p < 0.05). Diastolic blood pressure and resting heart rate were significantly lower in subjects with left ventricular hypertrophy (p < 0.05). The myosin light chain 9 and interleukin-6 signal transducer gene expression were differentially regulated in cyclists with left ventricular hypertrophy compared to athletes with normal heart dimensions in response to intensive exercise. We have found differences in echocardiography parameters, blood pressure, stroke volume and maximal power in the cyclists examined. These studies indicate the benefits of the recommended echocardiography measurements for professional endurance-athletes. The graded exercise altered the myosin light chain 9 and interleukin-6 signal transducer gene expression in the peripheral blood of road cyclists has also been found.

Cesarean Section, Formula Feeding, and Infant Antibiotic Exposure: Separate and Combined Impacts on Gut Microbial Changes in Later Infancy.

Established during infancy, our complex gut microbial community is shaped by medical interventions and societal preferences, such as cesarean section, formula feeding, and antibiotic use. We undertook this study to apply the significance analysis of microarrays (SAM) method to quantify changes in gut microbial composition during later infancy following the most common birth and postnatal exposures affecting infant gut microbial composition. Gut microbiota of 166 full-term infants in the Canadian Healthy Infant Longitudinal Development birth cohort were profiled using 16S high-throughput gene sequencing. Infants were placed into groups according to mutually exclusive combinations of birth mode (vaginal/cesarean birth), breastfeeding status (yes/no), and antibiotic use (yes/no) by 3 months of age. Based on repeated permutations of data and adjustment for the false discovery rate, the SAM statistic identified statistically significant changes in gut microbial abundance between 3 months and 1 year of age within each infant group. We observed well-known patterns of microbial phyla succession in later infancy (declining Proteobacteria; increasing Firmicutes and Bacteroidetes) following vaginal birth, breastfeeding, and no antibiotic exposure. Genus Lactobacillus, Roseburia, and Faecalibacterium species appeared in the top 10 increases to microbial abundance in these infants. Deviations from this pattern were evident among infants with other perinatal co-exposures; notably, the largest number of microbial species with unchanged abundance was seen in gut microbiota following early cessation of breastfeeding in infants. With and without antibiotic exposure, the absence of a breast milk diet by 3 months of age following vaginal birth yielded a higher proportion of unchanged abundance of Bacteroidaceae and Enterobacteriaceae in later infancy, and a higher ratio of unchanged Enterobacteriaceae to Alcaligenaceae microbiota. Gut microbiota of infants born vaginally and exclusively formula fed became less enriched with family Veillonellaceae and Clostridiaceae, showed unchanging levels of Ruminococcaceae, and exhibited a greater decline in the Rikenellaceae/Bacteroidaceae ratio compared to their breastfed, vaginally delivered counterparts. These changes were also evident in cesarean-delivered infants to a lesser extent. The clinical relevance of these trajectories of microbial change is that they culminate in taxon-specific abundances in the gut microbiota of later infancy, which we and others have observed to be associated with food sensitization.

Validation of a PCR coupled to a microarray method for detection of mycoplasma in vaccines

The revised section of the European, United States, and Japan Pharmacopeias on mycoplasma testing provided guidance for the set up and validation of a nucleic acid amplification technique (NAT) as an alternative method to agar culture and indicator cell culture compendial methods. The CytoInspect™ method, based on Polymerase Chain Reaction (PCR) coupled to microarray analysis, has been selected for detection and identification of mycoplasma in vaccines. To replace compendial methods, the alternative method must demonstrate equivalence in both limit of detection (LOD) and specificity compared with compendial methods.Here, we summarize the validation of the CytoInspect™ method according to current pharmacopeia requirements. Validation of the robustness, sensitivity (at least 10 colony forming units/ml) and specificity of the CytoInspect™ method are demonstrated. Likewise, a comparability study was performed to compare the LOD for CytoInspect™ compared with the previously validated LOD for compendial culture tests.

Comparative effect of imatinib and ponatinib on autophagy and miRNome in chronic myeloid leukemia

BCR-ABL tyrosine kinase inhibitors (TKIs) are selective therapies for the patients with Chronic Myeloid Leukemia (CML). Imatinib and ponatinib have remarkable long-term efficacy on a major molecular response. Although TKI related induction of cytotoxicity and apoptosis have been clearly investigated in molecular levels, their comparative effect on autophagy and miRNome are largely unknown. This study aimed to investigate the involvement of alterations of miRNA expressions in CML progression, and how imatinib and ponatinib affect this process, by comparing CML, imatinib-resistant CML and leukemia stem cells (LSC). Cytotoxicity analysis was conducted by WST-1, apoptosis was evaluated by AnnexinV, autophagy was analyzed by Tb/GFP TR-FRET LC3B assay and changes in miRNomes were evaluated with microarray method. Ponatinib showed higher cytotoxicity and apoptosis at far fewer concentrations than imatinib. Both imatinib and ponatinib was able to trigger autophagy in imatinib-resistant K562ima3 cell line but not in LSC. We pointed that imatinib and ponatinib caused significant miRNA profile alterations, especially in the expressions of miR-214-pre, miR-218, miR-19a-5p, miR-19b-1-5p, miR-27b-pre, miR-23b-pre, miR-320e, miR-200a-pre, miR-508-3p, miR-33-pre and miR-766. This study is the first comparative miRNome analysis of CML, resistant CML and LSCs following the imatinib or ponatinib treatment and may guide to identify new markers for diagnosis, follow-up of the disease and to develop novel therapeutic strategies if supported by preclinical studies.

Simultaneous detection of \u03b1-Lactoalbumin, \u03b2-Lactoglobulin and Lactoferrin in milk by Visualized Microarray

Backgroundα-Lactalbumin (a-LA), β-lactoglobulin (β-LG) and lactoferrin (LF) are of high nutritional value which have made ingredients of choice in the formulation of modern foods and beverages. There remains an urgent need to develop novel biosensing methods for quantification featuring reduced cost, improved sensitivity, selectivity and more rapid response, especially for simultaneous detection of multiple whey proteins.ResultsA novel visualized microarray method was developed for the determination of a-LA, β-LG and LF in milk samples without the need for complex or time-consuming pre-treatment steps. The measurement principle was based on the competitive immunological reaction and silver enhancement technique. In this case, a visible array dots as the detectable signals were further amplified and developed by the silver enhancement reagents. The microarray could be assayed by the microarray scanner. The detection limits (S/N = 3) were estimated to be 40 ng/mL (α-LA), 50 ng/mL (β-LG), 30 ng/mL (LF) (n = 6).ConclusionsThe method could be used to simultaneously analyze the whey protein contents of various raw milk samples and ultra-high temperature treated (UHT) milk samples including skimmed milk and high calcium milk. The analytical results were in good agreement with that of the high performance liquid chromatography. The presented visualized microarray has showed its advantages such as high-throughput, specificity, sensitivity and cost-effective for analysis of various milk samples.

HLA polymorphisms and risk of red blood cell alloimmunisation in polytransfused patients with sickle cell anaemia.

Red blood cell (RBC) alloimmunisation is an event that may occur due to factors such as numerous blood transfusions, age, gender and genetic factors such as human leukocyte antigen (HLA). The aim of the present study was to investigate the possibility of alloimmunisation to red blood cell group antigens associated with the HLA of individuals and to relate alloimmunisation to risk factors. A total of 172 polytransfused patients with sickle cell anaemia (SCA) (44 alloimmunised, 128 non-alloimmunised) participated in this study. Blood group genotyping was performed by the DNA microarray method and HLA genotyping by polymerase chain reaction-specific sequence of oligonucleotides. The number of transfusions received directly influenced the incidence of alloimmunisation, and the most common alloantibodies were against Rh (48·8%) and Kell (17%) systems. The HLA-C*06 and HLA-DQB1*03 variants were significantly higher in alloimmunised patients. The HLA-DRB1*04 and HLA-DRB1*11 were more often found in individuals who developed the alloantibodies anti-Fya and anti-K, respectively. This study suggests that polytransfused patients with SCA possessing the HLA-DQB1*03 and HLA-C*06 allele variants are more susceptible to alloimmunisation. In addition, HLA-DRB1*04 and HLA-DRB1*11 alleles were seen to be associated with the production of anti-Fya and anti-K antibodies, respectively.

Toll-like receptor 5 and 7 expression may impact prognosis of HPV-positive oropharyngeal squamous cell carcinoma patients.

A large subset of oropharyngeal squamous cell carcinomas (OPSCCs) is associated with HPV infection and has better outcome than non-viral-related tumors. Various malignancies also carry a role for TLRs, key activators of inflammation and innate immunity. We examined the expression of TLRs in OPSCC, and their association with HPV status and treatment outcome. TLR 5, 7, 9, and p16 were studied by immunohistochemistry and HPV status was detected with in situ hybridization in 202 tumors of consecutively treated OPSCC patients using tissue microarray method. The relations between TLR expression and HPV status, p16 expression, clinicopathological factors, and survival were analyzed. TLR 5, 7, and 9 expression patterns differed between HPV-positive and -negative tumors, and they were statistically significantly associated with history of smoking, heavy drinking, tumor site, grade, size (T), metastasis (N), and stage. Moreover, in HPV-positive tumors the expression of TLR 5 and 7 correlated with tumor recurrence. After adjustment, among HPV-positive OPSCC patients, high TLR 5 and low TLR 7 expression were associated with poor disease-specific survival. Our results indicate that TLR 5 and 7 may have a role in the prognostication of HPV-positive OPSCC, however, further studies are needed to clarify the comprehensive role of these TLRs in OPSCC.

Transcriptome analysis suggests a role for the differential expression of cerebral aquaporins and the MAPK signalling pathway in human temporal lobe epilepsy.

Epilepsies are common disorders of the central nervous system (CNS), affecting up to 2% of the global population. Pharmaco-resistance is a major clinical challenge affecting about 30% of temporal lobe epilepsy (TLE) patients. Water homeostasis has been shown crucial for regulation of neuronal excitability. The control of water movement is achieved through a family of small integral membrane channel proteins called aquaporins (AQPs). Despite the fact that changes in water homeostasis occur in sclerotic hippocampi of people with TLE, the expression of AQPs in the epileptic brain is not fully characterised. This study uses microarray and ELISA methods to analyse the mRNA and protein expression of the human cerebral AQPs in sclerotic hippocampi (TLE-HS) and adjacent neocortex tissue (TLE-NC) of TLE patients. The expression of AQP1 and AQP4 transcripts was significantly increased, while that of the AQP9 transcript was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while expression of AQP1 protein remained unchanged, and AQP9 was undetected. Microarray data analysis identified 3333 differentially regulated genes and suggested the involvement of the MAPK signalling pathway in TLE pathogenesis. Proteome array data validated the translational profile for 26 genes and within the MAPK pathway (e.g. p38, JNK) that were identified as differentially expressed from microarray analysis. ELISA data showed that p38 and JNK inhibitors decrease AQP4 protein levels in cultured human primary cortical astrocytes. Elucidating the mechanism of selective regulation of different AQPs and associated regulatory proteins may provide a new therapeutic approach to epilepsy treatment.

Identification of the anticancer effects of a novel proteasome inhibitor, ixazomib, on colorectal cancer using a combined method of microarray and bioinformatics analysis.

PurposeThe study aimed to explore the anticancer effects of a novel proteasome inhibitor, ixazomib, on colorectal cancer (CRC) using a combined method of microarray and bioinformatics analysis.Materials and methodsCell proliferation was tested by Cell Counting Kit-8 (CCK-8) assay for SW620 cells treated with different concentrations of ixazomib and different treatment times. The microarray analysis was conducted for six samples, including three samples of SW620 cells untreated with ixazomib and three samples of SW620 cells treated with ixazomib. The differentially expressed genes (DEGs) between untreated and treated samples were identified by the Linear Models for Microarray data (LIMMA) package in R language. The Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the DEGs using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and KEGG Orthology-Based Annotation System (KOBAS) online tool. The protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and module analysis was performed for the PPI network.ResultsIxazomib could inhibit the proliferation of SW620 cells in a dose-dependent and time-dependent manner. A total of 743 DEGs, including 203 upregulated DEGs such as HSPA6 and 540 downregulated DEGs such as APCDD1, were identified. Eighty-three GO terms were enriched for DEGs, which were mainly related to protein folding, apoptotic process, transcription factor activity, and proteasome. Thirty-seven KEGG pathways were perturbed, including pathway of apoptosis and cell cycle. Forty-six hub genes, such as TP53, JUN, and ITGA2, were screened out, and three modules with important functions were mined from the PPI network.ConclusionThe novel proteasome inhibitor ixazomib significantly inhibited the proliferation of human CRC SW620 cells. It exerted anticancer effects through targeting the expression of DEGs, such as HSPA6, APCDD1, TP53, and JUN, and affecting the signaling pathways including apoptosis and cell cycle pathway, which demonstrated the promising potential of ixazomib for CRC therapy.

Abstract 713: Proteomic early detection biomarkers for ER+ breast cancer to improve mammography screening

Abstract There are clear clinical and public health needs to improve the early detection of breast cancer in order to save lives. Breast cancer is the leading cause of cancer death in women worldwide and is the second leading cause of cancer mortality in women in the United States. Although mammography is widely used to screen for breast cancer, it suffers both from false negative and false positive readings resulting in later diagnoses or unnecessary follow-up (e.g., biopsies), respectively. Improvement of breast cancer early detection may be possible through the measurement of minimally invasive, easy to perform blood based biomarkers. Our studies were performed on pre-clinical plasma samples collected up to 12.5 months before breast cancer diagnosis from cases and matched controls obtained from the Women’s Health Initiative (WHI) Observational Study (a prospective cohort of 93,676 post-menopausal women). We utilized high-density antibody microarray methods for both discovery and preliminarily validation on distinct sample sets for early detection biomarkers of estrogen receptor positive (ER+) breast cancer (around 70% of all breast cancers are classified as ER+). Antibody microarray features (i.e., spots) identified as having ≥30% sensitivity at 80% specificity were considered to have met the cutoff criteria and identified as potential markers. A total of 129 features, corresponding to 101 proteins, were identified as potential markers in one WHI dataset (90 cases vs. 90 controls). Of the 129 features (101 proteins), 47 (34 proteins) validated by our cutoffs and demonstrated over or under-expression in cases compared to controls consistent with the first dataset in the second set of WHI samples (121 cases vs. 121 controls). Given this level of reproducibility, we believe these biomarkers could be used to improve early detection of ER+ breast cancer for these clinical scenarios: 1. Informing timing of a subsequent mammogram in women with a negative screening mammogram; 2. Informing continuation of mammographic screening among women 75-79 years; 3. Prioritizing women who should be screened with mammography in areas with limited resources. We are currently performing further validation of the protein markers in Early Detection Research Network (EDRN) specimen reference set from patients at the time of diagnosis. We plan to test our markers on a pre-diagnostic cohort from the Cardiovascular Health Study using a different, more clinically applicable platform. We used the same high-density antibody microarray platform to measure autoantibody-autoantigen complexes and sialyl Lewis-A and –X modifications in one of the WHI sample sets and are now testing these markers in the EDRN set for validation. Given the strength of our biomarker candidates, the high quality sets of biospecimens we used, the study designs employed, and the clearly delineated clinical applications proposed, we believe these markers could have near-term clinical impact. Citation Format: Carly B. Garrison, Tracey Marsh, Matthew Buas, Yuzheng Zhang, Margaret Pepe, Paul D. Lampe, Christopher I. Li. Proteomic early detection biomarkers for ER+ breast cancer to improve mammography screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 713. doi:10.1158/1538-7445.AM2017-713

Measuring ultra-low levels of nucleotide biomarkers using quartz crystal microbalance and SPR microarray imaging methods: A comparative analysis

Circulating serum nucleotide biomarkers are useful indicators for early diagnosis of cancer, respiratory illnesses, and other deadly diseases. In this work, we compared detection performances of a quartz crystal microbalance (QCM), which is a mass sensor, with that of a surface plasmon resonance (SPR) microarray for an oligonucleotide mimic of a microRNA-21 biomarker. A surface immobilized capture oligonucleotide probe was used to hybridize with the target oligonucleotide (i.e., the microRNA-21 mimic) to facilitate selective detection. To obtain ultra-low femtomolar (fM) detection sensitivity, gold nanoparticles (50nm) were conjugated with the target oligonucleotide. We achieved detection limits of 28 and 47 fM for the target oligonucleotide by the QCM and SPRi microarray, respectively. We also conducted sample recovery studies and performed matrix effect analysis. Although the QCM had a lower detection limit, the microarray approach offered better throughput for analysis of up to 16 samples. We confirmed that the designed assay was selective for the target oligonucleotide and did not show signals for the control oligonucleotide with five mismatch sites relative to the target sequence. Combination of the QCM and microarray methods that utilize the same assay chemistry on gold are useful for overcoming clinical sample matrix effects and achieving ultra-low detection of small nucleotide biomarkers with quantitative insights.

Genomic profiling of long non-coding RNA and mRNA expression associated with acquired temozolomide resistance in glioblastoma cells.

Temozolomide (TMZ) is an alkylating chemotherapeutic agent widely used in anti-glioma treatment. However, acquired TMZ resistance represents a major clinical challenge that leads to tumor relapse or progress. This study investigated the genomic profiles including long non-coding RNA (lncRNA) and mRNA expression associated with acquired TMZ resistance in glioblastoma (GBM) cells in vitro. The TMZ-resistant (TR) of GBM sub-cell lines were established through repetitive exposure to increasing TMZ concentrations in vitro. The differentially expressed lncRNAs and mRNAs between the parental U87 and U87TR cells were detected by human lncRNA microarray method. In this study, we identified 2,692 distinct lncRNAs demonstrating >2-fold differential expression with 1,383 lncRNAs upregulated and 1,309 lncRNAs downregulated. Moreover, 4,886 differential mRNAs displayed 2,933 mRNAs upregulated and 1,953 mRNAs downregulated. Further lncRNA classification and subgroup analysis revealed the potential functions of the lncRNA-mRNA relationship associated with the acquired TMZ resistance. Gene ontology and pathway analysis on mRNAs showed significant biological regulatory genes and pathways involved in acquired TMZ resistance. Moreover, we found the ECM-receptor interaction pathway was significantly downregulated and ECM related collagen I, fibronectin, laminin and CD44 were closely associated with the TR phenotype in vitro. Our findings indicate that the dysregulated lncRNAs and mRNAs identified in this work may provide novel targets for overcoming acquired TMZ resistance in GBM chemotherapy.

Long non-coding RNA expression profile in cervical cancer tissues.

Cervical cancer (CC), one of the most common types of cancer of the female population, presents an enormous challenge in diagnosis and treatment. Long non-coding (lnc)RNAs, non-coding (nc)RNAs with length >200 nucleotides, have been identified to be associated with multiple types of cancer, including CC. This class of nc transcripts serves an important role in tumor suppression and oncogenic signaling pathways. In the present study, the microarray method was used to obtain the expression profile of lncRNAs and protein-coding mRNAs and to compare the expression of lncRNAs between CC tissues and corresponding adjacent non-cancerous tissues in order to screen potential lncRNAs for associations with CC. Overall, 3356 lncRNAs with significantly different expression pattern in CC tissues compared with adjacent non-cancerous tissues were identified, while 1,857 of them were upregulated. These differentially expressed lncRNAs were additionally classified into 5 subgroups. Reverse transcription quantitative polymerase chain reactions were performed to validate the expression pattern of 5 random selected lncRNAs, and 2lncRNAs were identified to have significantly different expression in CC samples compared with adjacent non-cancerous tissues. This finding suggests that those lncRNAs with different expression may serve important roles in the development of CC, and the expression data may provide information for additional study on the involvement of lncRNAs in CC.

Whole-genome expression analysis of synovial fibroblasts in rheumatoid arthritis

Objective To identify the crucial gene implicated in rheumatoid arthritis (RA) pathogenesis by comparing microarray-based gene expression profiles of synovial fibroblast in arthritis patients and that in control. Methods The public da-tasets were obtained from NCBI GEO and EBI ArrayExpress. The qualified microarray-based gene expression profiles were inte-grated and normalized using the method implemented in GeneSpring software. Furthermore, the differentially expressed genes (DEGs) were identified using significance analysis of microarrays (SAM) method. The online tool DAVID and STRING were ap-plied to conduct the enrichment analysis and gene product interaction analysis respectively. Results There were two datasets that were qualified and analyzed in the present study. A total of 336 significant DEGs were identified by comparing the whole-ge-nome gene expression profiles from synovial fibroblast of RA patients and control group. Among these DEGs, 261 were significant-ly downregulated and 75 upregulated. About 13.6% of the downregulated genes were associated with extracellular matrix degrada-tion. The COL9A3 and COL4A5, indispensable component of hyaline cartilage and basement membrane respectively, were signifi-cantly downregulated, as well as genes in WNT family, including WNT2, WNT11, and WNT16. In contrast, matrix metalloprotein-ase 13 (MMP13) was found to be significantly upregulated in RA patients. MMP13 is a matrix metallopeptidase that degrade extra-cellular matrix and hyaline cartilage, and it could possibly interact with other proteins to regulate morphogenesis. Conclusion Molecular mechanisms underlying RA pathogenesis were investigated by analyzing the public datasets. A few genes that associat-ed with extracellular matrix degradation, construction and regulation, including MMP13, WNT2, WNT11, WNT16, COL9A3 and COL4A5, could be regarded as therapeutic targets in RA treatment. Key words: Arthritis, rheumatoid; Knee joint; Synovial Membrane; Fibroblasts; Gene expression profiling

CDKL1 promotes tumor proliferation and invasion in colorectal cancer.

BackgroundCDKL1 is a member of the cell division cycle 2 (CDC2)-related serine threonine protein kinase family and is overexpressed in malignant tumors such as melanoma, breast cancer, and gastric cancer.ObjectiveThis study aimed to evaluate whether CDKL1 can serve as a potential molecular target for colorectal cancer therapy.Materials and methodsExpression of CDKL1 in colorectal cancer tissues and cell lines was measured by immunohistochemistry and Western blot, respectively. To investigate the role of CDKL1 in colorectal cancer, CDKL1-small hairpin RNA-expressing lentivirus was constructed and infected into HCT116 and Caco2 cells. The effects of RNA interference (RNAi)-mediated CDKL1 downregulation on cell proliferation and invasion were assessed by CCK-8, colony formation, transwell, and tumorigenicity assays in nude mice. The effects of CDKL1 downregulation on cell cycle and apoptosis were analyzed by flow cytometry. Furthermore, microarray method and data analysis elucidated the molecular mechanisms underlying the phenomenon.ResultsCDKL1 protein was overexpressed in colorectal cancer tissues compared with paired normal tissues. Knockdown of CDKL1 in HCT116 and Caco2 significantly inhibited cell growth, colony formation ability, tumor invasion, and G1–S phase transition of the cell cycle. The knockdown of CDKL1 stimulated the upregulation of p15 and retinoblastoma protein.ConclusionCDKL1 plays a vital role in tumor proliferation and invasion in colorectal cancer in vitro and in vivo and, thus, may be considered as a valuable target for therapeutic intervention.

Clarifying the molecular mechanism associated with carfilzomib resistance in human multiple myeloma using microarray gene expression profile and genetic interaction network.

Carfilzomib is a Food and Drug Administration-approved selective proteasome inhibitor for patients with multiple myeloma (MM). However, recent studies indicate that MM cells still develop resistance to carfilzomib, and the molecular mechanisms associated with carfilzomib resistance have not been studied in detail. In this study, to better understand its potential resistant effect and its underlying mechanisms in MM, microarray gene expression profile associated with carfilzomib-resistant KMS-11 and its parental cell line was downloaded from Gene Expression Omnibus database. Raw fluorescent signals were normalized and differently expressed genes were identified using Significance Analysis of Microarrays method. Genetic interaction network was expanded using String, a biomolecular interaction network JAVA platform. Meanwhile, molecular function, biological process and signaling pathway enrichment analysis were performed based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Totally, 27 upregulated and 36 downregulated genes were identified and a genetic interaction network associated with the resistant effect was expanded basing on String, which consisted of 100 nodes and 249 edges. In addition, signaling pathway enrichment analysis indicated that cytokine–cytokine receptor interaction, autophagy, ErbB signaling pathway, microRNAs in cancer and fatty acid metabolism pathways were aberrant in carfilzomib-resistant KMS-11 cells. Thus, in this study, we demonstrated that carfilzomib potentially conferred drug resistance to KMS-11 cells by cytokine–cytokine receptor interaction, autophagy, ErbB signaling pathway, microRNAs in cancer and fatty acid metabolism pathways, which may provide some potential molecular therapeutic targets for drug combination therapy against carfilzomib resistance.

Identification of characteristic gene modules of osteosarcoma using bioinformatics analysis indicates the possible molecular pathogenesis.

The aim of the present study was to investigate the possible pathogenesis of osteosarcoma using bioinformatics analysis to examine gene-gene interactions. A total of three datasets associated with osteosarcoma were downloaded from the Gene Expression Omnibus. The differentially expressed genes (DEGs) were identified using the significance analysis of microarrays method, which then were subjected to the Human Protein Reference Database to identify the protein-protein interaction (PPI) pairs and to construct a PPI network of the DEGs. Subsequent multilevel community analysis was applied to mine the modules in the network, followed by screening of the differential expression module using the GlobalAncova package. The genes in the differential expression modules were verified in the valid datasets. The verified genes underwent functional and pathway enrichment analysis. A total of 616 DEGs were selected to construct the PPI network, which included 5,808 osteosarcoma-specific interaction pairs and 8,012 normal-specific pairs. Tumor protein p53 (TP53), mitogen-activated protein kinase 1 (MAPK1) and estrogen receptor 1 (ESR1) were identified the most important osteosarcoma-associated genes, with the highest levels of topological properties. Neurogenic locus notch homolog protein 3 (NOTCH3) and caspase 1 (CASP1) were identified as the osteosarcoma-specific interaction pairs. Among all 23 mined modules, three were identified as differential expression modules, which were verified in the other two datasets. The genes in these modules were predominantly enriched in the FGFR, MAPK and Notch signaling pathways. Therefore, TP53, MAPK1, ESR1, NOTCH3 and CASP1 may be important in the development of osteosarcoma, and provides valuable clues to investigate the pathogenesis of osteosarcoma using the three differential expression modules.

Risk analysis of colorectal cancer incidence by gene expression analysis.

BackgroundColorectal cancer (CRC) is one of the leading cancers worldwide. Several studies have performed microarray data analyses for cancer classification and prognostic analyses. Microarray assays also enable the identification of gene signatures for molecular characterization and treatment prediction.ObjectiveMicroarray gene expression data from the online Gene Expression Omnibus (GEO) database were used to to distinguish colorectal cancer from normal colon tissue samples.MethodsWe collected microarray data from the GEO database to establish colorectal cancer microarray gene expression datasets for a combined analysis. Using the Prediction Analysis for Microarrays (PAM) method and the GSEA MSigDB resource, we analyzed the 14,698 genes that were identified through an examination of their expression values between normal and tumor tissues.ResultsTen genes (ABCG2, AQP8, SPIB, CA7, CLDN8, SCNN1B, SLC30A10, CD177, PADI2, and TGFBI) were found to be good indicators of the candidate genes that correlate with CRC. From these selected genes, an average of six significant genes were obtained using the PAM method, with an accuracy rate of 95%. The results demonstrate the potential of utilizing a model with the PAM method for data mining. After a detailed review of the published reports, the results confirmed that the screened candidate genes are good indicators for cancer risk analysis using the PAM method.ConclusionsSix genes were selected with 95% accuracy to effectively classify normal and colorectal cancer tissues. We hope that these results will provide the basis for new research projects in clinical practice that aim to rapidly assess colorectal cancer risk using microarray gene expression analysis.