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Identification and validation of genes involved in cervical tumourigenesis.

ABSTRACT: BACKGROUND: Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer. MATERIALS AND METHODS: A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay. RESULTS: Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells. CONCLUSIONS: Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.

SUBMITTER: Rajkumar T

PROVIDER: S-EPMC3050856 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Identification and validation of genes involved in cervical tumourigenesis.

Rajkumar Thangarajan T Sabitha Kesavan K Vijayalakshmi Neelakantan N Shirley Sundersingh S Bose Mayil Vahanan MV Gopal Gopisetty G Selvaluxmy Ganesharaja G

BMC cancer 20110222

<h4>Background</h4>Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer.<h4>Materials and methods</h4>A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the signi ...[more]

PMID: 21338529

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Project description:BACKGROUND:Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets. MATERIALS AND METHODS:We used 24 gastric cancers, 20 Paired normal (PN) and 5 apparently normal gastric tissues obtained from patients with non-gastric cancers (Apparently normal - AN) for the microarray study followed by validation of the significant genes (n = 63) by relative quantitation using Taqman Low Density Array Real Time PCR. We then used a custom made Quantibody protein array to validate the expression of 15 proteins in gastric tissues (4 AN, 9 PN and 9 gastric cancers). The same array format was used to study the plasma levels of these proteins in 58 patients with gastric cancers and 18 from patients with normal/non-malignant gastric conditions. RESULTS:Seventeen genes (ASPN, CCL15/MIP-1?, MMP3, SPON2, PRSS2, CCL3, TMEPAI/PMEPAI, SIX3, MFNG, SOSTDC1, SGNE1, SST, IGHA1, AKR1B10, FCGBP, ATP4B, NCAPH2) were shown to be differentially expressed between the tumours and the paired normal, for the first time. EpCAM (p = 0.0001), IL8 (p = 0.0003), CCL4/MIP-1? (p = 0.0026), CCL20/MIP-3? (p = 0.039) and TIMP1 (p = 0.0017) tissue protein levels were significantly different (Mann Whitney U test) between tumours versus AN & PN. In addition, median plasma levels of IL8, CXCL9/MIG, CCL3/MIP-1?, CCL20/MIP-3?, PDGFR-B and TIMP1 proteins were significantly different between the non-malignant group and the gastric cancer group. The post-surgical levels of EpCAM, IGFBP3, IL8, CXCL10/IP10, CXCL9/MIG, CCL3/MIP-1?, CCL20/MIP-3?, SPP1/OPN and PDGFR-B showed a uniform drop in all the samples studied. CONCLUSIONS:Our study has identified several genes differentially expressed in gastric cancers, some for the first time. Some of these have been confirmed at the protein level, as well. Some of these proteins will need to be evaluated further for their potential as diagnostic biomarkers in gastric cancers and some could be useful as follow-up markers in gastric cancer.

Project description:Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n = 74) and ABrix (P<0.05, n = 32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.

Project description:BackgroundCervical cancer belongs to one of the most common female cancers; yet, the exact underlying mechanisms are still elusive. Recently, microarray and sequencing technologies have been widely used for screening biomarkers and molecular mechanism discovery in cancer studies. In this study, we aimed to analyse the microarray datasets using comprehensive bioinformatics tools and identified novel biomarkers associated with the prognosis of patients with cervical cancer.MethodsThe differentially expressed genes (DEGs) from Gene Expression Omnibus (GEO) datasets including GSE138080, GSE113942 and GSE63514 were analysed using GEO2R tool. The functional enrichment analysis was performed using g:Profiler tool. The protein-protein interaction (PPI) network construction and hub genes identification were performed using the STRING database and Cytoscape software, respectively. The hub genes were subjected to expression and survival analysis in the cervical cancer. The EdU incorporation and Cell Counting Kit-8 assays were performed to evaluate the effects of hub gene knockdown on the proliferation of cervical cancer cells.ResultsA total of 89 overlapping DEGs (63 up-regulated and 26 down-regulated genes) were identified in the microarray datasets. The functional enrichment analysis indicated that the overlapping DEGs were mainly associated with "DNA replication" and "cell cycle". Furthermore, the PPI network analysis revealed that the network contains 87 nodes and 309 edges. Sub-module analysis using the Molecular Complex Detection tool identified 21 hub genes from the PPI network. The expression levels of the 21 hub genes were all up-regulated in the cervical cancer tissues when compared to normal cervical tissues as analysed by GEPIA tool. The survival analysis showed that the low expression of cell division cycle 45 (CDC45), GINS complex subunit 2 (GINS2), minichromosome maintenance complex component 2 (MCM2) and proliferating cell nuclear antigen (PCNA) was significantly correlated with the shorter overall survival of patients with cervical cancer. Moreover, the protein expression levels of GINS2, MCM2 and PCNA, but not CDC45, were significantly up-regulated in the cervical cancer tissues when compared to normal cervical tissues. Finally, knockdown of MCM2 significantly suppressed the proliferation of HeLa and SiHa cells.ConclusionIn conclusion, we screened a total of 89 overlapping DEGs from the GEO datasets, and further analysis identified four hub genes (CDC45, GINS2, MCM2 and PCNA) that were likely associated with the prognosis of patients with cervical cancer. MCM2 knockdown repressed the cervical cancer cell proliferation. The current findings may provide novel insights into understanding the pathophysiology of cervical cancer and develop therapeutic targets for patients with cervical cancer.

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Identification and validation of genes involved in cervical tumourigenesis.

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Identification and validation of genes involved in cervical tumourigenesis.

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