Brief Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 14, 2012; 18(6): 570-575
Published online Feb 14, 2012. doi: 10.3748/wjg.v18.i6.570
Identification of differential gene expressions in colorectal cancer and polyp by cDNA microarray
Yi-Chen Dai, Xiao-San Zhu, Qing-Zhen Nan, Zhang-Xin Chen, Jun-Pei Xie, Yu-Ka Fu, Yuan-Yuan Lin, Qing-Na Lian, Qiao-Fang Sang, Xiao-Juan Zhan
Yi-Chen Dai, Xiao-San Zhu, Zhang-Xin Chen, Jun-Pei Xie, Yu-Ka Fu, Yuan-Yuan Lin, Qing-Na Lian, Qiao-Fang Sang, Xiao-Juan Zhan, Department of Gastroenterology, Chenggong Hospital, Xiamen University, Xiamen 361003, Fujian Province, China
Yi-Chen Dai, Xiao-San Zhu, Zhang-Xin Chen, Jun-Pei Xie, Yu-Ka Fu, Yuan-Yuan Lin, Qing-Na Lian, Qiao-Fang Sang, Xiao-Juan Zhan, Department of Gastroenterology, The 174th Hospital of the Chinese PLA, Wenyuan Road, Xiamen 361003, Fujian Province, China
Qing-Zhen Nan, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China
Author contributions: Zhu XS and Lin YY performed the majority of experiments; Nan QZ, Zhan XJ and Chen ZX provided vital reagents and analytical tools and were also involved in editing the manuscript; Xie JP, Fu YK, Lian QN and Sang QF coordinated and provided the collection of all the human material in addition to providing financial support for this work; Dai YC and Zhu XS designed the study and wrote the manuscript.
Supported by Grant from Medical Technology Innovation Project of Nanjing Military, No. 09MA066
Correspondence to: Yi-Chen Dai, Professor, Department of Gastroenterology, The 174th Hospital of the Chinese PLA, Wenyuan Road, Xiamen 361003, Fujian Province, China. dyichen@sina.com
Telephone: +86-592-6335702 Fax: +86-592-6335702
Received: May 31, 2011
Revised: July 17, 2011
Accepted: August 15, 2011
Published online: February 14, 2012

Abstract

AIM: To screen the differential expressed genes in colorectal cancer and polyp tissue samples.

METHODS: Tissue specimens containing 16 cases of colorectal adenocarcinoma and colorectal polyp vs normal mucosae were collected and subjected to cDNA microarray and bioinformatical analyses. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm some of the cDNA microarray data.

RESULTS: The experimental data showed that eight genes were differentially expressed, most of which were upregulated in adenomatous polyp lesions. Forty-six genes expressions were altered in colorectal cancers, of which 29 were upregulated and 17 downregulated, as compared to the normal mucosae. In addition, 18 genes were similarly altered in both adenomatous polyps and colorectal cancer. qRT-PCR analyses confirmed the cDNA microarray data for four of those 18 genes: MTA1, PDCD4, TSC1 and PDGFRA.

CONCLUSION: These differentially expressed genes likely represent biomarkers for early detection of colorectal cancer and may be potential therapeutic targets after confirmed by further studies.

Key Words: Colorectal polyp, Colorectal cancer, cDNA microarray, Quantitative reverse transcription-polymerase chain reaction



INTRODUCTION

Colorectal polyp (CRP) is considered as a premalignant lesion for development of colorectal cancer (CRC)[1]. Although the mechanism underlying colorectal cancer development remains to be defined, a series of genetic and epigenetic events are thought to play important roles in colorectal carcinogenesis, including oncogene activation and tumor suppressor gene inactivation[2,3].

By attaining a detailed understanding of the altered gene expression profile of colorectal cancer novel strategies may be developed for earlier detection and more effective prevention and treatment, thereby reducing colorectal cancer incidence and increasing survival rates.

In this study, we performed a cDNA microarray analysis to profile differential gene expressions in tissue specimens of polyps and colorectal carcinoma and compared the expression profiles to that in corresponding normal tissues. We chose the genes with marked differential expressions for verification by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These data provide insightful information into the genetic mechanisms of colorectal cancer and identify genes that may be useful as biomarkers for early disease detection.

MATERIALS AND METHODS
Ethics

This study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and approved by the Medical Ethics Committee of Fujian Province, China. All patients read and signed an informed consent form prior to surgery and sample collection.

Patient tissue

A total of 16 patients with colorectal adenocarcinoma and adenomatous polyp lesions were collected from The 174th Hospital of the Chinese PLA between May 2006 and December 2010. Diagnosis of these patients was confirmed by surgical pathology. None of the patients received any pre-surgical chemo- or radiation-therapy. All tissue specimens were immediately taken from the operation room upon excision from the patient, snap frozen in liquid nitrogen, and stored at -80 °C until use. The tissue specimens from these patients were divided into two groups: adenomatous polyp lesions vs proximal non-cancerous colorectal mucosae (Group A) or colorectal cancer vs proximal non-cancerous colorectal mucosae (Group B). The clinicopathological characteristics of these patients are summarized Table 1.

Table 1 Clinical characteristics of patients with colorectal cancer or polyps.
CaseSexAgeSize in cm/nDifferentiationDepthDukes
CRCCRPCRCPolyps
1F411.7/20.6/2HighSB1
2M370.9/11.2/3PoorSsC1
3M682.9/20.7/1PoorSmD1
4F272.8/11.4/1PoorSsC2
5M591.8/10.5/3ModerateSC2
6F522.9/11.9/4PoorSsB2
7M713.3/20.4/1HighSmB1
8M462.7/13.1/1ModerateSsC1
9M280.9/12.8/2PoorSmB1
10F362.6/10.4/1PoorSsC2
11F302.4/12.8/2ModerateSC1
12M463.1/11.3/2PoorSmB1
13M621.5/12.0/3PoorSsC2
14F240.8/12.4/2ModerateSmC2
15M702.5/14.3/1PoorSB2
16M433.2/23.7/1HighSsB2
RNA isolation and cDNA microarray analysis

Total cellular RNA was extracted from the tissue samples by using the Trizol reagent (Sigma-Aldrich Inc., Germany)[2,4]. mRNA isolation was then carried out with Qiagen Oligotex beads, (Valencia, CA, United States) according to the manufacturer’s instructions. The final concentration of mRNA was measured by spectrophotometer.

Next, the mRNAs from colorectal cancer or adenomatous polyp lesions were reverse transcribed into cDNA by means of Cy5-dUTP labeling, while the mRNAs from the normal mucosae were processed with Cy3-dUTP labeling by following the manufacturer’s protocols (NEN Company, Boston, MA, United States). The labeled probes were then hybridized to the cDNA microarray (Chipscreen, Shenzhen, China), which contained 8064 human genes.

Microarray scanning and data analysis

Hybridized cDNA microarrays were scanned using a Gene PIX 4000 microarray fluorescence scanner (Axon Instruments, Foster City, CA, United States). Accompanying bioinformatical software was used convert the output images to data form and perform analysis. Ratios of Cy5: Cy3 were normalized to the median ratio value of all the microarray spots detected. Spots with intensities in both channels that were 0.5 to 2.0-fold higher than the local background were excluded from further analysis. SPSS v13.0 statistical software (Chicago, IL, United States) was used to carry out Student’s t-test statistical analysis to determine significant intergroup differences of gene expression. P-values < 0.05 were considered statistically significant.

Quantitative reverse transcription-polymerase chain reaction

Differentially expressed genes between the adenomatous polyp lesions and colorectal cancer samples detected by the cDNA microarray, as compared to non-cancerous tissues, were verified by using qRT-PCR. The primers for these mRNAs used for qRT-PCR is listed in Table 2.

Table 2 Primers used for quantitative reverse transcription polymerase chain reaction analysis.
Gene tagSequencesTm (°C)CycleProduct (bp)
MTA15’-AGCCGTGCTTCGGTATCTT-3’5730580
5’-CCCGTTGTGCTGCTCGTA-3’
PDCD45′-GCTGAATTCGGATGGATGTAGAAAATGAGCAGA-3’5427470
5’-CTGCTCGAGTCAGTAGCTCTCTGGTTTAAGA-3’
TSC15′-ATCGCCTTTATGGAATGT-3’4929510
5’-GCTTGTGGTGGTTCAGTT-3’
PDGFRA5’-ACCATAAGGCTCTTACTCT-3’4531490
5’-TTCTGGCACTTACCTACA-3’
RESULTS
Qualification of the isolated total RNA and mRNA from the tissue samples

Quality of the isolated total RNA and mRNA from the tissue samples was found to have high correlation coefficients (Figure 1). The fluorescence signal was consistent with the expectations and standards (Figure 2); for example, the good 28S to 18S RNA subunit ratio in these samples indicated that there was no significant degradation (data not shown).

Figure 1
Figure 1 Correlation of CY5/CY3 hybridization signal intensity of all gene spots in Group A (polyps vs normal) and B (colorectal cancer vs normal) (R = 0. 8591 and 0.8335, respectively). For visualization of these gene distributions, dispersion plots containing the log2 (normal tissues) and log2 (polyps or colorectal cancer) values were constructed.
Figure 2
Figure 2 Microarray scanned data from Group A and B.
Identification of differential gene expression profile between colorectal cancer and adenomatous polyp lesions

cDNA microarray analysis of these tissue mRNAs revealed that eight genes were differentially expressed between adenomatous polyp lesions and the normal mucosae, and most of these were upregulated in the polyps (Table 3, P < 0.05). Meanwhile, 46 genes were differentially expressed between colorectal cancer and normal tissues, of which 29 were up-regulated in the cancer samples (Table 3, P < 0.05). A total of 18 genes were found to be similarly altered in both adenomatous polyp lesions and colorectal cancer samples (Table 4). qRT-PCR confirmed the observed patter for four of those 18 genes: MTA1, PDCD4, TSC1 and PDGFRA (Table 5).

Table 3 Identification of differential gene expression profile between colorectal cancer and adenomatous polyp lesions.
AccessionGene functionGene tagRatio
AY421086Programmed cell death 4PDCD46.41 ± 0.10
AA630800Interferon, γ-inducible protein 30IFI301.31 ± 0.18
AA400973Lipocalin 2 (oncogene 24p3)LCN23.16 ± 0.22
AI817942Zeta-chain associated protein kinase (70 kD)ZAP703.29 ± 0.31
AA447515Mad4 homologMAD42.35 ± 0.20
W47350Retinoic acid receptor responder 3RARRES30.18 ± 0.13
AA436401TU3A proteinTU3A5.40 ± 0.27
AI650283Serum/glucocorticoid regulated kinase 2SGK21.29 ± 0.13
NM003542H4 histone family, member GH4FG7.04 ± 0.17
NM205510Fibroblast growth factor receptor 1FGFR12.94 ± 0.21
NM204434Cyclin-dependent kinase inhibitor 2ACDKN2A3.16 ± 0.28
NM005438FOS-like antigen-1FOSL11.93 ± 0.25
NM005439Myeloid leukemia factor 2MLF22.17 ± 0.24
BC08072v-raf murine sarcoma 3611 viral oncogene homolog 1ARAF10.98 ± 0.15
NM020531Chromosome 20open reading frame 3C20ORF32.23 ± 0.21
NM033158Hyaluronoglucosaminidase 2HYAL24.16 ± 0.28
NM001950E2F transcription factor 4,p107/p130-bindingE2F45.26 ± 0.28
BC059522Ribosomal protein S30FAU2.58 ± 0.13
NM008583Multiple endocrine neoplasia IMEN13.89 ± 0.11
NM011492Serine/threonine kinase 11STK115.12 ± 0.27
NM133862Fibrinogen, gamma polypeptideFGG3.0 4 ± 0.28
BC162533GRO2 oncogeneGRO21.39 ± 0.30
NM000612Insulin-like growth factor 2 receptorIGF25.06 ± 0.29
NM005343v-Ha-ras Harvey rat sarcoma viral oncogene homologHRAS4.74 ± 0.27
NM002634ProhibitinPHB2.98 ± 0.15
BC046375p53-induced proteinPIG116.67 ± 0.29
NM004448v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2ERBB23.15 ± 0.17
NM010658v-maf musculoaponeurotic fibrosarcoma oncogene familyMAFG5.61 ± 0.04
NM022012Mitogen-activated protein 3 kinase 11MAP3K113.32 ± 0.06
NM023983Melanoma adhesion moleculeMCAM7.38 ± 0.14
NM014567Breast cancer anti-estrogen resistance 1BCAR15.12 ± 0.23
NM000535Postmeiotic segregation increased 2PMS25.17 ± 0.25
NM183243Inosine monophosphate dehydrogenase 1IMPDH17.14 ± 0.10
NM005380Neuroblastoma, suppression of tumorigenicity 1NBL15.62 ± 0.16
NM002429Matrix metalloproteinase 19MMP193.90 ± 0.22
NM002466v-myb avian myeloblastosis viral oncogene homolog-like 2MYBL29.70 ± 0.21
NM022588Metastasis associated 1MTA110.41 ± 0.37
NM017045Retinoblastoma 1RB12.81 ± 0.14
NC006104SET translocationSET2.69 ± 0.11
NM002439Phosphatase and tensin homologPTEN3.94 ± 0.21
NM053455Fibrinogen-like 2 GTPase activatingFGL23.40 ± 0.27
NM005638ADP-ribosylation factor protein 1ARFGAP5.14 ± 0.25
NM032415Mucosa associated lymphoid tissue lymphoma translocation gene 1MALT14.84 ± 0.21
NM006283Transforming acidic coiled-coil containing protein 1TACC13.41 ± 0.13
NM00288v-ral simian leukemia viral oncogene homolog BRALB4.12 ± 0.18
NM003766Myosin-like BCL2-interacting proteinBECN15.05 ± 0.14
NG027821TRK-fused geneTFG4.33 ± 0.28
NM005805Cadherin 1,type 1,E-cadherin (epithelial)CDH14.11 ± 0.26
NM001982v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3ERBB35.82 ± 0.15
NM133250MutS (Escherichia coli) homolog 2MSH22.13 ± 0.21
AY805747Ras homolog gene family, member EARHE3.61 ± 0.22
NM002884RAP1A, member of RAS oncogene familyRAP1A5.18 ± 0.27
NM000368Tuberous sclerosis 1TSC16.02 ± 0.14
L36953Mothers against decapentaplegic homolog 4MADH44.93 ± 0.26
Table 4 Differentially expressed genes between polyps and colorectal cancer.
AccessionGene functionGene tagRatio
AB
AA191692StratifinSFN2.36 ± 0.253.86 ± 0.11a
H15456Calpain 1, (mu/I) large subunitCAPN18.17 ± 0.2010.25 ± 0.24a
AA043501v-maf musculoaponeurotic fibrosarcoma (avian) oncogene homologMAF1.36 ± 0.084.85 ± 0.27a
AA495936Microsomal glutathione S-transferase 1MGST1-2.14 ± 0.232.90 ± 0.16a
H23235Platelet-derived growth factor receptorPDGFRA-0.15 ± 0.314.81 ± 0.14a
AA430032Pituitary tumor-transforming 1PTTG4.91 ± 0.2311.46 ± 0.18a
N94468Jun B proto-oncogeneJUNB1.27 ± 0.2011.09 ± 0.14a
T61948FBJ murine osteosarcoma viral oncogene B homologFOSB1.37 ± 0.3110.21 ± 0.20a
AA460168Growth arrest and DNA damage inducible 34GADD341.07 ± 0.154.15 ± 0.20a
L36870MAP kinase kinase 4MKK44.01 ± 0.236.93 ± 0.24a
AA426216Malignant cell expression-enhanced geneLENG47.03 ± 0.168.81 ± 0.23a
AA486219SRp25 nuclear proteinLOC513292.09 ± 0.126.18 ± 0.23a
AA457705Immediate early response 3IER31.94 ± 0.259.17 ± 0.20a
AA485377v-fos FBJ murine osteosarcoma viral oncogene homologFOS4.03 ± 0.277.21 ± 0.05a
AA463204Pleiomorphic adenoma gene-like 1PLAGL17.11 ± 0.24-4.16 ± 0.06a
AA434373E74-like factor 3 (epithelial-specific )ELF31.09 ± 0.297.03 ± 0.15a
AI677994Fms-associated tyrosine kinase 3 ligandFLT3LG1.94 ± 0.324.35 ± 0.02a
AA464600v-myc avian myelocytomatosis viral oncogene homologMYC3.27 ± 0.178.01 ± 0.13a
Table 5 cDNA microarray data confirmed by quantitative reverse transcription-polymerase chain reaction.
Gene functionGene tagRatio
cDNA microarrayqRT-PCR
Metastasis associated 1MTA18.01 ± 0.476.72 ± 0.20a
Programmed cell death 4PDCD46.41 ± 0.105.35 ± 0.01a
Tuberous sclerosis 1TSC16.02 ± 0.144.83 ± 0.26a
Platelet-derived growth factor receptorPDGFRA-0.15 ± 0.310.03 ± 0.07a
DISCUSSION

Colorectal carcinoma is a major cause of cancer-related deaths in China[5,6]. Unfortunately, little is known about the gene expression profiles between colorectal cancer and adenomatous polyp lesions. Genes that are differentially expressed in adenomatous polyp lesions may represent useful biomarkers for risk of colorectal cancer development, while altered genes in cancerous tissues may be used as therapeutic targets for colorectal cancer treatment[7].

Adenomatous polyp is the premalignant lesion of colorectal cancer[8-12]. Therefore, we conducted the current study to profile the differential gene expressions between normal mucosae and adenomatous polyp lesions, between normal mucosae and colorectal cancer, and between adenomatous polyp lesions and colorectal cancer. We found that eight genes were differentially expressed in adenomatous polyp lesions, as compared to normal mucosae. In addition, 46 genes were differentially expressed in colorectal cancer, as compared to the normal mucosae; twenty-nine of which were up-regulated. A total of 18 genes were significantly upregulated in both colorectal cancer and adenomatous polyp lesions, further indicating that adenomatous polyp is a precancerous lesion. However, some genes were downregulated in adenomatous polyp lesions (such as MGST1 and PDGFRA) or upregulated in colorectal cancers only (PLAGL1). These genes encode proteins that are known to be involved in cell growth, apoptosis, and metastasis and are likely to contribute to colorectal carcinogenesis, as purported by previous studies[13,14].

The MGST1 gene is located in 12p13.1-13.2 and was previously considered to be a “housekeeping” gene[15]. However, it has been frequently observed as upregulated in various cancers. A recent in vitro study has implicated the role of MGST1 in development of multiple drug resistance during breast cancer chemotherapy with several cytostatic drugs (such as cisplatin)[16]. Polymorphisms in MGST1 have also been associated with colorectal cancer risk in Chinese[17]. In this study, we found that MGST1 mRNA levels were upregulated in colorectal cancer, as compared to those detected in normal mucosae (2.90 ± 0.16). Intriguingly, MGST1 was down-regulated in adenomatous polyps, as compared to the normal mucosae (2.14 ± 0.23), but further study is necessary to fully understand the implications of this finding.

PDGFRA gene mutation is commonly observed in tissues of gastrointestinal stromal tumors[18]. Mutated PDGFRA proteins demonstrate constitutively elevated tyrosine kinase activity and possess transforming ability, which can be reversed through PDGFR blockade[19]. Thus, mutants of PDGFRA protein behave as oncogenes, as has been demonstrated in glioma samples[20]. Here, we observed high expression of PDGFRA in colorectal cancers, as compared to that in normal tissues (4.81 ± 0.14). This observation suggests that PDGFRA may contribute to cancer development or maintenance of the tumor phenotype, possibly by supporting properties of tumor cell growth and invasiveness. However, to the reason why PDGFRA was down-regulated in adenomatous polyps remains unclear.

Finally, PLAGL1, a tumor suppressor gene, is localized on the chromosome 6q24-25 and is the target of several types of chromosomal rearrangement, including one identified in pleomorphic adenomas and malignant tumors. PLAGL1 is ubiquitously expressed in many human tissues where it regulates normal physiological functions; however, it has also been demonstrated to functionally contribute to complex pathologies such as cancer[21-23]. Our current study showed that PLAGL1 mRNA was down-regulated in colorectal cancer, as compared to adenomatous polyps, suggesting that PLAGL1 protein may also play a role in suppressing colorectal cancer development.

The functional roles for each of these genes in colorectal tumorigenesis remain to be verified. Nonetheless, our data provide insightful information into their potential roles in this complex and diverse disease[24]. Future studies will aim to verify these differentially expressed genes as biomarkers for early detection and/or therapeutic targets for treatment of colorectal cancers.

COMMENTS
Background

The mechanism of colorectal carcinogenesis remains to be defined and this study aims to obtain the gene expression profiles between colorectal cancer and adenomatous polyp lesions.

Research frontiers

Many researchs on genetic and epigenetic events which are thought to play important roles in colorectal carcinogenesis, such as oncogene activation and tumor suppressor gene inactivation.

Innovations and breakthroughs

The study firstly screened the differential expressed genes in colorectal adenocarcinoma and colorectal polyp vs normal mucosae.

Applications

These differentially expressed genes maybe as biomarkers for early detection and/or therapeutic targets for treatment of colorectal cancers.

Peer review

Study was well designed and performed methodologically.

Footnotes

Peer reviewers: Jean Wang, Professor, Washington University, 660 S. Euclid Ave, Campus Box 8124, St. Louis, United States; Zoran Krivokapic, Professor, MD, FRCS, Institute for Digestive Disease, First Surgical Clinic, Clinical Center of Serbia, 6, Dr Koste Todorovica, Belgrade, 11 000, Serbia

S- Editor Lv S L- Editor Webster JR E- Editor Xiong L

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