Research Report
Association between phospholipase C epsilon gene (PLCE1) polymorphism and colorectal cancer risk in a Chinese population
Journal of International Medical Research 2014, Vol. 42(2) 270–281 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513492484 imr.sagepub.com
Qi Wang1, Ping Chen1, Dafang Chen1, Fanlong Liu2 and Weihuo Pan3
Abstract Objective: To investigate the association between a single nucleotide polymorphism rs2274223 (adenine [A] to guanine [G]) in the phospholipase C epsilon 1 (PLCE1) gene and susceptibility to colorectal cancer (CRC). Methods: The PLCE1 rs2274223 polymorphism was genotyped in patients with CRC and age- and sex-matched cancer-free control subjects from a Chinese population in a case–control study. PLCE1 mRNA levels in pair-matched tumour and adjacent noncancerous tissue were evaluated by real-time quantitative reverse transcription–polymerase chain reaction. Results: A total of 417 patients with CRC and 416 control subjects were enrolled in the study. The AG and GG genotypes of PLCE1 rs2274223 were associated with a significantly increased risk of CRC (for AG þ GG versus AA: adjusted odds ratio 1.52, 95% confidence interval 1.15, 2.00). PLCE1 mRNA levels were significantly lower in tumours than in adjacent noncancerous tissue. Lower levels of PLCE1 mRNA were observed in both AG and GG genotype carriers compared with the AA genotype carriers. Conclusions: These results indicate that the PLCE1 rs2274223 A > G change might reduce gene expression and that the variant G genotype might contribute to the increased risk of CRC.
Keywords Phospholipase C epsilon 1, single nucleotide polymorphism, colorectal cancer, real-time quantitative reverse transcription–polymerase chain reaction 3
Department of Colorectal Surgery, Shaoxing People’s Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, Zhejiang Province, China
1 Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang Province, China 2 Department of Anus, Rectum and Colon Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
Corresponding author: Dr Weihuo Pan, Department of Colorectal Surgery, Shaoxing People’s Hospital, 568 Zhongxing North Road, Shaoxing 312000, Zhejiang Province, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without furtherDownloaded permission provided the original work is attributed ason specified on2015 the SAGE and Open Access page from imr.sagepub.com at GEORGIAN COURT UNIV March 11, (http://www.uk.sagepub.com/aboutus/openaccess.htm).
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Date received: 1 April 2013; accepted: 13 April 2013
Introduction Colorectal cancer (CRC) is one of the most aggressive cancers and causes approximately 1 million deaths worldwide each year.1 It is a frequent cause of cancer-related death in developed countries.1 With improvements in the standard of living and changes in lifestyle, food and the environment, the incidence of CRC is constantly increasing in China, where it is now the third most frequent malignancy.2 Although advances in surgical techniques, improved chemotherapy and early detection have decreased the mortality rate of CRC,1 there is still an urgent need for early diagnosis and effective cancer prevention because of the poor prognosis and lack of novel treatments for this disease. A number of theories have been put forward to explain the aetiology of CRC, such as the classical adenoma–carcinoma sequence model.3 Both environmental and genetic factors appear to contribute to the tumorigenesis of CRC.3 Environmental risk factors (such as an unbalanced diet, nutritional deficiencies, environmental exposure to carcinogens, and infectious agents such as viruses and bacteria) may also contribute to the carcinogenesis of CRC.4–8 Genetic polymorphisms in a variety of genes have been shown to be involved in the susceptibility to CRC, particularly for those genes involved in cell-cycle control,9 DNA repair,10 metabolism,11 insulin resistance, obesity and glucose level regulation,12 and inflammation.13 Phospholipase C epsilon 1 (PLCE1), encoded by the PLCE1 gene on chromosome 10q23, belongs to the phospholipase family of enzymes and catalyses the hydrolysis of polyphosphoinositides to generate the secondary messengers inositol 1,4,5triphosphate and diacylglycerol, thereby
contributing to intracellular signalling.14–16 PLCE1 protein initiates a cascade of intracellular responses that result in cell growth, differentiation and gene expression.14,16,17 PLCE1 has been reported to play a critical role in carcinogenesis and progression of a variety of human cancers including those of the oesophagus,18 stomach,19 skin,20 bladder,21 and head and neck.22 Two large-scale genome-wide association studies simultaneously showed that a new and notable lowpenetrance susceptibility single nucleotide polymorphism (SNP) rs2274223, located in exon 26 of the PLCE1 gene, was strongly associated with increased risk of oesophageal squamous cell carcinoma and gastric cancer in Chinese populations.23,24 Subsequent research confirmed that the SNP rs2274223 was a critical risk factor for the incidence of oesophageal squamous cell carcinoma and gastric cancer.19,25–27 There is also evidence that the PLCE1 SNP rs2274223 adenine (A) >guanine (G) change might reduce gene expression, and that variant G genotypes might contribute to the increased risk of oesophageal squamous cell carcinoma.18 PLCE1 mRNA levels were also shown to be significantly lower in gastric tumours compared with adjacent normal tissue, which might be associated with the SNP rs2274223.19 Research has demonstrated that PLCE1 gene expression was downregulated in sporadic CRC and that this gene exhibited a suppressive role on the incidence of CRC.28,29 However, no association between the potentially functional variants of the PLCE1 gene and susceptibility to CRC has been reported. The aim of the present study was to evaluate the possible correlation between the SNP rs2274223 in the PLCE1 gene and
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susceptibility to CRC in a Chinese population. The PLCE1 mRNA levels in pairmatched tissue samples were analysed and the results stratified according to different rs2274223 genotypes.
Patients and methods Study population This hospital-based case–control study enrolled consecutive Han Chinese patients with newly diagnosed and histopathologically confirmed CRC, receiving care at the Department of Anus, Rectum and Colon Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China between May 2007 and May 2012. In addition, 416 Han Chinese subjects from Zhejiang Province, who were healthy according to a physical examination, were recruited from several clinics at the First Affiliated Hospital, Zhejiang University School of Medicine between May 2007 and May 2012 with the selection criteria including no history of cancer. These cancer-free control subjects were matched to the patients based on age (5 years) and sex. At recruitment, each study participant was interviewed to gather demographic data (such as age and sex) and environmental exposure history, including smoking status (‘no’ for smoking included nonsmokers and former smokers) and alcohol consumption (‘no’ for alcohol consumption included former alcohol drinker and those who had never drunk alcohol). Tumours were graded according to the WHO Classification of Tumours of the Digestive System.30 This case–control study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (no. 20120118-01). Written informed consent was provided by all of the study participants.
Genotyping Blood samples (about 3 ml) were collected from the antecubital veins of CRC patients or healthy donors after an overnight fast during the first examination. Genomic DNA was extracted immediately from 200 ml of ethylenediaminetetra-acetic acid (20 g/l) anticoagulated venous blood using the QIAampÕ DNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) and stored at 80 C until analysis. The rs2274223 SNP was genotyped using the TaqManÕ SNP Genotyping Assay for the rs2274223 SNP, which contained the appropriate primers and probes (sequence information was not provided by the manufacturer; Applied Biosystems, Foster City, CA, USA). The polymerase chain reaction (PCR) was performed with 50 ng of genomic DNA, 0.2 mM of each primer, 0.1 mM of each probe, 200 mM of each deoxyribonucleotide, 3 mM MgCl2 and 1 U PlatinumÕ Taq DNA polymerase (Applied Biosystems). The PCR amplification was carried out with an initial denaturation at 95 C for 2 min, followed by 40 cycles of denaturation at 95 C for 15 s and annealing at 65 C for 30 s, using an Applied BiosystemsÕ 7900HT Fast Real-Time PCR System according to the manufacturer’s instructions. The ABI PRISMÕ 7900 Sequence Detection System software version 2.3 (Applied Biosystems) was used for data analysis. Approximately 5% of the samples were randomly selected for repeated genotyping for confirmation and the results were 100% concordant.
Quantitative real-time RT–PCR Noncancerous colorectal tissue was collected from an area of mucosa 15 cm away from the tumour when the tumour samples were collected. Tissue samples were divided into two parts: one was for histopathological examination and the other was transferred into a cryotube and immediately
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frozen in liquid nitrogen before further analysis. Total RNA was isolated from 50 mg of tumour tissue and pair-matched noncancerous tissue adjacent to the tumour using the RNeasyÕ Mini kit (QIAGEN) according to the manufacturer’s instructions. The quality and quantity of the RNA were assessed using a NanoDropTM 1000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA). A total of 36 CRC tumour samples and their corresponding noncancerous tissues were selected to quantify PLCE1 mRNA levels. These samples were selected at random from the total samples (12 with AA, 12 with AG, 12 with GG): we only identified the genotypes of blood samples whose genotypes we considered as the same as the genotypes in tumour or healthy tissues. Quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR) was used to measure the amount of PLCE1 mRNA in pair-matched tissue samples from patients with different PLCE1 genotypes. For each sample, 1 mg of total RNA was subjected to reverse transcription using the PrimeScriptTM RT Master Mix System (TaKaRa, Dalian, China). A SYBRÕ Green real-time PCR method was used to assess the PLCE1 mRNA levels in colon tissue. Primer sequences for RT–PCR for the PLCE1 gene were 50 -CCTGGG CATAAGCACTACCAAG-30 (forward) and 50 -GTCTTGAGGATCAGAACCACT CC-30 (reverse).19 Primer sequences for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were 50 -GCACCGTCAA GGCTGAGAAC-30 (forward) and 50 -ATG GTGGTGAAGACGCCAGT-30 (reverse). All the primers were obtained from Invitrogen (Shanghai, China). Real-time PCR was performed in a Bio-Rad CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using 1 ml of the resulting cDNA from the RT step, 10 ml of SYBRÕ Green PCR Master Mix (TaKaRa) and 4 pmol each of the forward
and reverse primers. The cycling programme involved preliminary denaturation at 95 C for 30 s, followed by 40 cycles of denaturation at 95 C for 15 s and annealing at 60 C for 30 s; amplification fluorescence was monitored at 60 C at the end of each cycle. Three replicates were performed for each sample and the quantitative real-time PCR products were analysed using Bio-Rad CFX ManagerTM software, version 3.0 (Bio-Rad) using the 2CT method.31
Statistical analyses All statistical analyses were performed using the SPSSÕ software package, version 16.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ . The Hardy–Weinberg equilibrium was tested by a goodness-of-fit 2-test to compare the observed genotype frequencies compared with the expected frequencies among the control subjects. Differences in the distributions of demographic characteristics, selected variables, and the genotypes of the PLCE1 rs2274223 SNP between patients with CRC and control subjects were evaluated using a 2-test-based Q-test and the Wilcoxon signed-rank test. Stratification analysis was performed to evaluate the potential association of genetic variants of the PLCE1 rs2274223 polymorphism with risk in subgroups based on clinical characteristics. The associations between the PLCE1 variants and CRC risk were estimated by calculating the odds ratios (OR) and 95% confidence intervals (CI), using both univariate and multivariate logistic regression analysis with adjustment for age, sex, smoking status and alcohol consumption status. Paired-sample Student’s ttest was used for the comparison of mRNA levels between pair-matched tissue samples from patients with different PLCE1 genotypes. Two-sided tests were used for statistical analysis and a P-value < 0.05 was considered statistically significant. The statistical power was calculated using Power
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and Sample Size Calculation software, version 3.0 (Department of Biostatistics, Vanderbilt University, Nashville, TN, USA).32
Results A total of 417 Han Chinese patients with newly diagnosed and histopathologically confirmed CRC and 416 cancer-free healthy control subjects were enrolled in the study. Their clinical and demographic
characteristics are summarized in Table 1. There was no significant difference between the two groups in terms of age or sex distribution (two-sided 2-test; Wilcoxon’s test), suggesting that the case–control matching based on age and sex was adequate. In addition, there was no significant difference between the two groups in terms of smoking and alcohol drinking status (two-sided 2-test). Of the 417 patients with CRC, 266 (63.8%) had tumours in the rectum, 67 (16.1%) in the left colon and
Table 1. Clinical and demographic characteristics of Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416) enrolled in this study to investigate associations between phospholipase C epsilon 1 gene polymorphisms and risk of colorectal cancer. Characteristic Age, years 65 >65 Sex Male Female Tobacco smoking statusa Yes No Alcohol consumptionb Yes No Pathological tumour stagec I–II III–IV Tumour differentiation status Poor Moderate Well Tumour site Rectum Left colon Right colon
Patients with colorectal cancer (n ¼ 417)
Healthy control subjects (n ¼ 416)
62.9 12.6 218 (52.3) 199 (47.7)
62.6 13.2 213 (51.2) 203 (48.8)
225 (54.0) 192 (46.0)
232 (55.8) 184 (44.2)
135 (32.4) 282 (67.6)
136 (32.7) 280 (67.3)
91 (21.8) 326 (78.2)
96 (23.1) 320 (77.0)
246 (59.0) 171 (41.0) 149 (35.7) 238 (57.1) 30 (7.2) 266 (63.8) 67 (16.1) 84 (20.1)
Data presented as mean SD for age; other data presented as number (%) of patients or controls. a ‘No’ for smoking includes non-smoker and former smoker. b ‘No’ for alcohol consumption includes former alcohol drinkers and those who had never drunk alcohol. c Graded according to the WHO Classification of Tumours of the Digestive System.30
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Table 2. Logistic regression analysis of the association between phospholipase C epsilon 1 (PLCE1) genotypes and risk of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416).
Variants/genotypes
Patients with colorectal cancer, n (%)
Healthy control Subjects, n (%)
Crude OR (95% CI)
Statistical significancea
Adjusted OR (95% CI)b
Statistical significanceb
PLCE1 rs2274223 AA AG GG AG þ GG
228 (54.7) 161 (38.6) 28 (6.7) 189 (45.3)
269 (64.7) 128 (30.8) 19 (4.6) 147 (35.3)
1.00 1.46 (1.08, 1.98) 1.74 (0.91, 3.38) 1.46 (1.09, 1.94)
P ¼ 0.012 NS P ¼ 0.009
1.00 1.48 (1.11, 1.99) 1.74 (0.95, 3.20) 1.52 (1.15, 2.00)
P ¼ 0.008 NS P ¼ 0.003
a 2
-test for genotype distribution between patients and control subjects. Adjusted for age, sex, tobacco, smoking and alcohol drinking status in logistic regression models. OR, odds ratio; CI, confidence interval; A, adenine; G, guanine; NS, no statistically significant difference (P 0.05). b
84 (20.4%) in the right colon. Pathological examinations showed that 30 (7.2%) tumours were well differentiated, 238 (57.1%) were moderately differentiated and 149 (35.7%) were poorly differentiated. Distribution of pathological tumour stage was as follows: stage I–II, 246 (59.0%); and stage III–IV, 171 (41.0%). Age, sex, smoking status and alcohol drinking status were further adjusted for use in later logistic regression analysis. The genotype and allele distributions of the PLCE1 gene rs2274223 polymorphism in patients with CRC and control subjects are shown in Table 2. The observed genotype frequencies for the PLCE1 gene rs2274223 polymorphism agreed with that expected according to the Hardy–Weinberg equilibrium in the control subjects. The genotype distribution between the patients with CRC and the control subjects was significantly different for rs2274223 (P ¼ 0.0118; 2-test of the difference between the three genotype groups). After adjustment for age, sex, smoking and alcohol drinking status, a significantly increased risk of CRC was associated with the variant genotypes AG þ GG of rs2274223 with an adjusted OR of 1.517 (95% CI 1.148, 2.004) compared with the AA genotypes.
Stratification analysis was performed to evaluate the potential association of genetic variants of the PLCE1 rs2274223 polymorphism with risk in subgroups based on clinical characteristics. As shown in Table 3, the stratification analysis indicated that the risk associated with the rs2274223 variant AG þ GG genotypes was more evident in younger patients (65 years; adjusted OR 2.287, 95% CI 1.553, 3.368), men (adjusted OR 1.680, 95% CI 1.145, 2.467), and those that did not drink alcohol (adjusted OR 1.538, 95% CI 1.113, 2.125) compared with those patients without any variant genotypes (AA). The association between the PLCE1 rs2274223 polymorphism and the clinical characteristics of CRC was also examined (Table 4). Results showed that the AG þ GG genotype was associated with advanced pathological stage (adjusted OR 1.765, 95% CI 1.211, 2.575), moderately differentiated tumours (adjusted OR 1.709, 95% CI 1.221, 2.396), and tumours located in the rectum and left colon (adjusted OR 1.549, 95% CI 1.118, 2.148 and adjusted OR 1.774, 95% CI 1.019, 3.087, respectively). To investigate the potential association between PLCE1 SNP rs2274223 variants and PLCE1 mRNA levels, samples of
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Table 3. Stratification analysis of the association between phospholipase C epsilon 1 (PLCE1) genotype and risk of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416). PLCE1 genotype (patients/controls) Characteristic Age, years 65 >65 Sex Male Female Tobacco exposurea Yes No Alcohol useb Yes No
AA
AG þ GG
Adjusted ORa (95% CI)
Statistical significanceb
95/136 133/133
123/77 66/70
2.29 (1.55, 3.37) 0.96 (0.63, 1.46)
P < 0.001 NS
120/152 108/117
105/80 84/67
1.68 (1.15, 2.47) 1.44 (0.94, 2.19)
P ¼ 0.008 NS
69/88 159/181
66/48 123/99
1.80 (1.10, 2.96) 1.45 (1.03, 2.05)
P ¼ 0.020 P ¼ 0.036
45/59 183/210
46/37 143/110
1.68 (0.92, 3.05) 1.54 (1.11, 2.13)
NS P ¼ 0.009
a
Obtained in logistic regression models with adjustment for age, sex, smoking and alcohol drinking status. P-value for heterogeneity test using 2-test-based Q-test. OR, odds ratio; CI, confidence interval; A, adenine; G, guanine; NS, no statistically significant difference (P 0.05). b
Table 4. Stratification analysis of the association between phospholipase C epsilon 1 (PLCE1) genotypes and clinical characteristics of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417). PLCE1 genotypes
Adjusted ORa (95% CI)
Statistical significanceb
Characteristic
AA n (%)
AG þ GG n (%)
Control subjects, n ¼ 416 Patients, n ¼ 417 Pathological tumour stagec I–II, n ¼ 246 III–IV, n ¼ 171 Tumour differentiation status Well, n ¼ 30 Moderate, n ¼ 238 Poor, n ¼ 149 Tumour site Right colon, n ¼ 84 Rectum, n ¼ 266 Left colon, n ¼ 67
269 (64.7) 228 (54.7)
147 (35.3) 189 (45.3)
1.00 (reference)
141 (57.3) 87 (50.9)
105 (42.7) 84 (49.1)
1.36 (0.97, 1.91) 1.77 (1.21, 2.58)
P < 0.001 NS P ¼ 0.002
22 (73.3) 123 (51.7) 83 (55.7)
8 (26.7) 115 (48.3) 66 (44.3)
0.67 (0.25, 1.60) 1.71 (1.22, 2.40) 1.45 (0.98, 2.17)
NS P ¼ 0.001 NS
50 (59.5) 144 (54.1) 34 (50.8)
34 (40.5) 122 (45.9) 33 (49.2)
1.24 (0.74, 2.06) 1.55 (1.12, 2.15) 1.77 (1.02, 3.09)
NS P ¼ 0.006 P ¼ 0.040
a
Obtained in logistic regression models with adjustment for age, sex, smoking and alcohol drinking status. P-value for heterogeneity test using the 2-test-based Q-test. c Tumours were graded according to the WHO Classification of Tumours of the Digestive System.30 A, adenine; G, guanine; OR, odds ratio; CI, confidence interval; NS, no statistically significant difference (P 0.05). b
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Discussion
Figure 1. Phospholipase C epsilon 1 (PLCE1) gene expression levels, measured using reverse transcription–polymerase chain reaction, are presented as the PLCE1 mRNA levels in colorectal cancer (CRC) tissue matched tissue adjacent to the tumour. Data points represent fold differences between each pair-matched sample set. Horizontal bars represent the calculated medians of the overall group and each subgroup. Levels of PLCE1 mRNA were downregulated in CRC tissues compared with pair-matched normal tissues. ‘Total’ includes the data from patients (n ¼ 36) with different PLCE1 genotypes and the three subgroups show the samples stratified by the three different PLCE1 rs2274223 genotypes (n ¼ 12 per group). The levels of PLCE1 mRNA were determined relative to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A, adenine; G, guanine. *P < 0.05, two-sided Student’s t-test.
tumour and pair-matched noncancerous tissues adjacent to the tumour from patients with different PLCE1 genotypes were used to determine PLCE1 mRNA levels using RT–PCR (Figure 1). A total of 36 CRC tumour samples and their corresponding noncancerous tissues were selected at random to quantify PLCE1 mRNA levels. PLCE1 mRNA levels were significantly lower in the CRC tumours than in the adjacent noncancerous tissues (n ¼ 36; twosided Student’s t-test; P < 0.001). Lower PLCE1 mRNA levels were observed in both AG and GG genotype carriers than in those patients that were AA genotype
This hospital-based case–control study investigated the association between a potentially functional SNP rs2274223 in the PLCE1 gene with the risk of CRC in a Han Chinese population. The current results showed that the SNP rs2274223 polymorphic genotype AG þ GG of the PLCE1 gene (combined genotypes) might contribute to the risk of CRC, and this risk was more evident in younger patients (65 years), men and those who did not drink alcohol. To the best of our knowledge, this is the first report on the relationship between the SNP rs2274223 polymorphic genotype AG þ GG of the PLCE1 gene and the risk of CRC. Our study also demonstrated a role of the PLCE1 gene and its potential functional variant rs2274223 in the aetiology of CRC at the mRNA level in pair-matched tissue samples from patients with different PLCE1 genotypes. The PLCE1 gene is a member of the phospholipase C family.14–16 Structural analysis revealed that the PLCE1 protein contains one cell division cycle 25 domain at the N-terminus and two Ras-associating domains at the C-terminus, which are all associated with Harvey-Ras and Ras-related protein 1 A activity.14,16 Therefore, PLCE1 acts as an effector of the Ras family small guanine triphosphatases and plays a critical role in regulating cell growth, differentiation and development.14,16,17 Other studies have found that some mutations in the PLCE1 gene lead to early onset nephritic syndrome and isolated diffuse mesangial sclerosis in humans,33,34 but this gene might also be linked to carcinogenesis.16 Research has also revealed that the PLCE1 gene was downregulated in tumour tissue from patients with CRC and that it might play a crucial
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role in CRC formation.28,29 To date, there have been no reports of studies that have investigated the association between genetic variations of the PLCE1 gene and susceptibility to CRC. In the present study, the PLCE1 rs2274223A >G SNP was demonstrated to be associated with the risk of CRC. This SNP causes an amino acid change from histidine to arginine in the calcium-dependent lipidbinding C2 domain of the PLCE1 protein.18 The rs2274223 SNP in the PLCE1 gene was first identified as a risk factor for oesophageal squamous cell carcinoma and gastric cancer in Chinese populations in two independent genome-wide association studies.23,24 These results were reproduced by others.18,19,25,35 Although Chinese patients formed the majority of the study populations in these studies,18,19, 35 the association between the PLCE1 rs2274223 SNP and the risk for cancer has also been demonstrated in Caucasian populations.25,26 These results and the findings of the current study suggest that the rs2274223 SNP of the PLCE1 gene might be a crucial risk factor for tumours of the digestive system (including CRC), and that it could be used as a biomarker for the diagnosis of these cancers. Individuals carrying PLCE1 rs2274223 AG þ GG genotypes had higher survival rates than those carrying the AA genotype, suggesting that the rs2274223 G allele might be associated with prognosis in patients with gastric cancer.27 However, in this current study, survival information for the patients with CRC was not included. Further studies focusing on the association between the rs2274223 SNP of the PLCE1 gene with prognosis of patients with CRC are needed. In the stratification analysis undertaken in this current study, the rs2274223 SNP of the PLCE1 gene was more evident in younger patients (65 years), in men and in those who did not drink alcohol. These current findings were consistent with the concept that individuals with high-risk
genetic susceptibility are more likely to develop cancer when they are exposed to a low level of alcohol exposure.19 For alcohol drinkers, the effect of genetic variations may be overwhelmed by the strong impact of environmental carcinogens. In contrast, for those who have been exposed to a low level of alcohol, genetic variations may play a dominant role in the initiation of carcinogenesis. Cancer is a complex and multifactorial disease: gene–gene and gene– environment interactions may occur, and a single genetic variant is usually insufficient to predict the overall risk. Further research is required to elucidate the role of other functional SNPs in the PLCE1 gene and to investigate the impact of other related genes involved in similar biological pathways that might be involved in the aetiology of CRC. To test the hypothesis that the rs2274223 SNP of the PLCE1 gene might play a functional role in the aetiology of CRC through altering the PLCE1 gene expression level, the mRNA levels from paired samples of CRC tumours and adjacent noncancerous tissue were measured using real-time RT–PCR. Overall, the current results showed that the PLCE1 mRNA level was much lower in the tumours than in adjacent noncancerous tissue samples, which was consistent with the results from two other studies of CRC.28,29 Such findings were also observed in Chinese patients with oesophageal squamous cell carcinoma or gastric adenocarcinoma.18,19 These results suggest that the PLCE1 gene might play a suppressive role in the carcinogenesis of a variety of cancers including CRC. Although this current study demonstrated that the G allele was related to lower PLCE1 mRNA levels, studies with larger population sizes are needed to validate this finding. Higher PLCE1 protein levels have been demonstrated in oesophageal squamous cell carcinoma tumour tissue compared with matched normal tissue.24 Although protein levels generally parallel the mRNA level,
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post-transcriptional regulation could also alter PLCE1 protein levels.18 Studies that measure PLCE1 protein levels are required, to validate these current PLCE1 mRNA findings. This current study had a number of limitations. First, it was a hospital-based study, so inherent biases in this type of study might lead to unreliable results. However, the G allele frequency in the control subjects was similar to that in the haplotype map database36 and the genotype distributions of the polymorphism in the control subjects conformed to the Hardy–Weinberg equilibrium. Therefore, the currents results are not thought to be subject to selection bias. Secondly, the sample size was relatively modest in the present study, but the study had 83.7% power to detect a minimal OR of 1.517 with a minor allele frequency of 35.3% using the current sample size. Thirdly, the PLCE1 rs2274223 SNP was the only polymorphism investigated in this study; SNPs at other loci that may be associated with susceptibility to CRC were neglected. Finally, the survival rates of individuals with different PLCE1 rs2274223 genotypes were not available for analysis. Future research should address whether the rs2274223 SNP contributes to the survival of patients with CRC. In conclusion, these current data show that the genotype distribution of the PLCE1 rs2274223 polymorphism was significantly different between patients with CRC and healthy control subjects. Larger populationbased and in-depth molecular studies are needed to validate these current findings and to elucidate the functional roles of the rs2274223 polymorphism in the aetiology of CRC.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This work was supported by the Shaoxing Major Scientific and Technological Projects (no. 2011A11013).
Acknowledgements We thank Michael Brownstein (ZhejiangCalifornia International Nanosystems Institute) for his critical reading of our manuscript and Xutao Hong (Zhejiang-California International Nanosystems Institute) for his technical support.
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Journal of International Medical Research 42(2) of colorectal cancer in the Czech Republic. Mutat Res 2008; 638: 146–153. Kiyohara C. Genetic polymorphism of enzymes involved in xenobiotic metabolism and the risk of colorectal cancer. J Epidemiol 2000; 10: 349–360. Michaud DS, Fuchs CS, Liu S, et al. Dietary glycemic load, carbohydrate, sugar, and colorectal cancer risk in men and women. Cancer Epidemiol Biomarkers Prev 2005; 14: 138–147. Eaden JA and Roberts-Thomson IC. Gastrointestinal: giant gastric ulcers. J Gastroenterol Hepatol 2001; 16: 573. Bunney TD and Katan M. Phospholipase C epsilon: linking second messengers and small GTPases. Trends Cell Biol 2006; 16: 640–648. Rhee SG. Regulation of phosphoinositidespecific phospholipase C. Annu Rev Biochem 2001; 70: 281–312. Katan M. New insights into the families of PLC enzymes: looking back and going forward. Biochem J 2005; 391: e7–e9. Song C, Hu CD, Masago M, et al. Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras. J Biol Chem 2001; 276: 2752–2757. Hu H, Yang J, Sun Y, et al. Putatively functional PLCE1 variants and susceptibility to esophageal squamous cell carcinoma (ESCC): a case-control study in eastern Chinese populations. Ann Surg Oncol 2012; 19: 2403–2410. Wang M, Zhang R, He J, et al. Potentially functional variants of PLCE1 identified by GWASs contribute to gastric adenocarcinoma susceptibility in an eastern Chinese population. PLoS One 2012; 7: e31932. Bai Y, Edamatsu H, Maeda S, et al. Crucial role of phospholipase C" in chemical carcinogen-induced skin tumor development. Cancer Res 2004; 64: 8808–8810. Ou L, Guo Y, Luo C, et al. RNA interference suppressing PLCE1 gene expression decreases invasive power of human bladder cancer T24 cell line. Cancer Genet Cytogenet 2010; 200: 110–119. Bourguignon LY, Gilad E, Brightman A, et al. Hyaluronan–CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/
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Ras co-activation, phospholipase C epsilonCa2þ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells. J Biol Chem 2006; 281: 14026–14040. Abnet CC, Freedman ND, Hu N, et al. A shared susceptibility locus in PLCE1 at 10q23 for gastric adenocarcinoma and esophageal squamous cell carcinoma. Nat Genet 2010; 42: 764–767. Wang LD, Zhou FY, Li XM, et al. Genomewide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54. Nat Genet 2010; 42: 759–763. Palmer AJ, Lochhead P, Hold GL, et al. Genetic variation in C20orf54, PLCE1 and MUC1 and the risk of upper gastrointestinal cancers in Caucasian populations. Eur J Cancer Prev 2012; 21: 541–544. Ma H, Wang LE, Liu Z, et al. Association between novel PLCE1 variants identified in published esophageal cancer genome-wide association studies and risk of squamous cell carcinoma of the head and neck. BMC Cancer 2011; 11: 258. Luo D, Gao Y, Wang S, et al. Genetic variation in PLCE1 is associated with gastric cancer survival in a Chinese population. J Gastroenterol 2011; 46: 1260–1266. Wang X, Zhou C, Qiu G, et al. Phospholipase C epsilon plays a suppressive role in incidence of colorectal cancer. Med Oncol 2012; 29: 1051–1058. Wang X, Zbou C, Qiu G, et al. Screening of new tumor suppressor genes in sporadic colorectal cancer patients. Hepatogastroenterology 2008; 55: 2039–2044. Bosman FT. World Health Organization, International Agency for Research on Cancer: WHO Classification of Tumours of the Digestive System, 4th edn. Lyon: ARC Press, 2010. Mou X, Wan S, Li Y, et al. Phenotypic pattern-based assay for dynamically monitoring host cellular responses to Salmonella infections. PLoS One 2011; 6: e26544. Power and Sample Size Calculation version 3.0, 2009. Department of Biostatistics,
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Vanderbilt University, Nashville, TN, USA. http://biostat.mc.vanderbilt.edu/twiki/bin/ view/Main/PowerSampleSize (accessed 9 June 2013). 33. Hinkes B, Wiggins RC, Gbadegesin R, et al. Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet 2006; 38: 1397–1405. 34. Gbadegesin R, Hinkes BG, Hoskins BE, et al. Mutations in PLCE1 are a major cause of isolated diffuse mesangial sclerosis
(IDMS). Nephrol Dial Transplant 2008; 23: 1291–1297. 35. Gu H, Ding G, Zhang W, et al. Replication study of PLCE1 and C20orf54 polymorphism and risk of esophageal cancer in a Chinese population. Mol Biol Rep 2012; 39: 9105–9111. 36. The National Center for Biotechnology Information. dbSNP. Short Genetic Variations, http://www.ncbi.nlm.nih.gov/ projects/SNP/snp_ref.cgi?rs¼2274223 (accessed 17 June 2013).
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Association between phospholipase C epsilon gene (PLCE1) polymorphism and colorectal cancer risk in a Chinese population
Journal of International Medical Research 2014, Vol. 42(2) 270–281 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513492484 imr.sagepub.com
Qi Wang1, Ping Chen1, Dafang Chen1, Fanlong Liu2 and Weihuo Pan3
Abstract Objective: To investigate the association between a single nucleotide polymorphism rs2274223 (adenine [A] to guanine [G]) in the phospholipase C epsilon 1 (PLCE1) gene and susceptibility to colorectal cancer (CRC). Methods: The PLCE1 rs2274223 polymorphism was genotyped in patients with CRC and age- and sex-matched cancer-free control subjects from a Chinese population in a case–control study. PLCE1 mRNA levels in pair-matched tumour and adjacent noncancerous tissue were evaluated by real-time quantitative reverse transcription–polymerase chain reaction. Results: A total of 417 patients with CRC and 416 control subjects were enrolled in the study. The AG and GG genotypes of PLCE1 rs2274223 were associated with a significantly increased risk of CRC (for AG þ GG versus AA: adjusted odds ratio 1.52, 95% confidence interval 1.15, 2.00). PLCE1 mRNA levels were significantly lower in tumours than in adjacent noncancerous tissue. Lower levels of PLCE1 mRNA were observed in both AG and GG genotype carriers compared with the AA genotype carriers. Conclusions: These results indicate that the PLCE1 rs2274223 A > G change might reduce gene expression and that the variant G genotype might contribute to the increased risk of CRC.
Keywords Phospholipase C epsilon 1, single nucleotide polymorphism, colorectal cancer, real-time quantitative reverse transcription–polymerase chain reaction 3
Department of Colorectal Surgery, Shaoxing People’s Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, Zhejiang Province, China
1 Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou, Zhejiang Province, China 2 Department of Anus, Rectum and Colon Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
Corresponding author: Dr Weihuo Pan, Department of Colorectal Surgery, Shaoxing People’s Hospital, 568 Zhongxing North Road, Shaoxing 312000, Zhejiang Province, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without furtherDownloaded permission provided the original work is attributed ason specified on2015 the SAGE and Open Access page from imr.sagepub.com at GEORGIAN COURT UNIV March 11, (http://www.uk.sagepub.com/aboutus/openaccess.htm).
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Date received: 1 April 2013; accepted: 13 April 2013
Introduction Colorectal cancer (CRC) is one of the most aggressive cancers and causes approximately 1 million deaths worldwide each year.1 It is a frequent cause of cancer-related death in developed countries.1 With improvements in the standard of living and changes in lifestyle, food and the environment, the incidence of CRC is constantly increasing in China, where it is now the third most frequent malignancy.2 Although advances in surgical techniques, improved chemotherapy and early detection have decreased the mortality rate of CRC,1 there is still an urgent need for early diagnosis and effective cancer prevention because of the poor prognosis and lack of novel treatments for this disease. A number of theories have been put forward to explain the aetiology of CRC, such as the classical adenoma–carcinoma sequence model.3 Both environmental and genetic factors appear to contribute to the tumorigenesis of CRC.3 Environmental risk factors (such as an unbalanced diet, nutritional deficiencies, environmental exposure to carcinogens, and infectious agents such as viruses and bacteria) may also contribute to the carcinogenesis of CRC.4–8 Genetic polymorphisms in a variety of genes have been shown to be involved in the susceptibility to CRC, particularly for those genes involved in cell-cycle control,9 DNA repair,10 metabolism,11 insulin resistance, obesity and glucose level regulation,12 and inflammation.13 Phospholipase C epsilon 1 (PLCE1), encoded by the PLCE1 gene on chromosome 10q23, belongs to the phospholipase family of enzymes and catalyses the hydrolysis of polyphosphoinositides to generate the secondary messengers inositol 1,4,5triphosphate and diacylglycerol, thereby
contributing to intracellular signalling.14–16 PLCE1 protein initiates a cascade of intracellular responses that result in cell growth, differentiation and gene expression.14,16,17 PLCE1 has been reported to play a critical role in carcinogenesis and progression of a variety of human cancers including those of the oesophagus,18 stomach,19 skin,20 bladder,21 and head and neck.22 Two large-scale genome-wide association studies simultaneously showed that a new and notable lowpenetrance susceptibility single nucleotide polymorphism (SNP) rs2274223, located in exon 26 of the PLCE1 gene, was strongly associated with increased risk of oesophageal squamous cell carcinoma and gastric cancer in Chinese populations.23,24 Subsequent research confirmed that the SNP rs2274223 was a critical risk factor for the incidence of oesophageal squamous cell carcinoma and gastric cancer.19,25–27 There is also evidence that the PLCE1 SNP rs2274223 adenine (A) >guanine (G) change might reduce gene expression, and that variant G genotypes might contribute to the increased risk of oesophageal squamous cell carcinoma.18 PLCE1 mRNA levels were also shown to be significantly lower in gastric tumours compared with adjacent normal tissue, which might be associated with the SNP rs2274223.19 Research has demonstrated that PLCE1 gene expression was downregulated in sporadic CRC and that this gene exhibited a suppressive role on the incidence of CRC.28,29 However, no association between the potentially functional variants of the PLCE1 gene and susceptibility to CRC has been reported. The aim of the present study was to evaluate the possible correlation between the SNP rs2274223 in the PLCE1 gene and
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susceptibility to CRC in a Chinese population. The PLCE1 mRNA levels in pairmatched tissue samples were analysed and the results stratified according to different rs2274223 genotypes.
Patients and methods Study population This hospital-based case–control study enrolled consecutive Han Chinese patients with newly diagnosed and histopathologically confirmed CRC, receiving care at the Department of Anus, Rectum and Colon Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China between May 2007 and May 2012. In addition, 416 Han Chinese subjects from Zhejiang Province, who were healthy according to a physical examination, were recruited from several clinics at the First Affiliated Hospital, Zhejiang University School of Medicine between May 2007 and May 2012 with the selection criteria including no history of cancer. These cancer-free control subjects were matched to the patients based on age (5 years) and sex. At recruitment, each study participant was interviewed to gather demographic data (such as age and sex) and environmental exposure history, including smoking status (‘no’ for smoking included nonsmokers and former smokers) and alcohol consumption (‘no’ for alcohol consumption included former alcohol drinker and those who had never drunk alcohol). Tumours were graded according to the WHO Classification of Tumours of the Digestive System.30 This case–control study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (no. 20120118-01). Written informed consent was provided by all of the study participants.
Genotyping Blood samples (about 3 ml) were collected from the antecubital veins of CRC patients or healthy donors after an overnight fast during the first examination. Genomic DNA was extracted immediately from 200 ml of ethylenediaminetetra-acetic acid (20 g/l) anticoagulated venous blood using the QIAampÕ DNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) and stored at 80 C until analysis. The rs2274223 SNP was genotyped using the TaqManÕ SNP Genotyping Assay for the rs2274223 SNP, which contained the appropriate primers and probes (sequence information was not provided by the manufacturer; Applied Biosystems, Foster City, CA, USA). The polymerase chain reaction (PCR) was performed with 50 ng of genomic DNA, 0.2 mM of each primer, 0.1 mM of each probe, 200 mM of each deoxyribonucleotide, 3 mM MgCl2 and 1 U PlatinumÕ Taq DNA polymerase (Applied Biosystems). The PCR amplification was carried out with an initial denaturation at 95 C for 2 min, followed by 40 cycles of denaturation at 95 C for 15 s and annealing at 65 C for 30 s, using an Applied BiosystemsÕ 7900HT Fast Real-Time PCR System according to the manufacturer’s instructions. The ABI PRISMÕ 7900 Sequence Detection System software version 2.3 (Applied Biosystems) was used for data analysis. Approximately 5% of the samples were randomly selected for repeated genotyping for confirmation and the results were 100% concordant.
Quantitative real-time RT–PCR Noncancerous colorectal tissue was collected from an area of mucosa 15 cm away from the tumour when the tumour samples were collected. Tissue samples were divided into two parts: one was for histopathological examination and the other was transferred into a cryotube and immediately
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frozen in liquid nitrogen before further analysis. Total RNA was isolated from 50 mg of tumour tissue and pair-matched noncancerous tissue adjacent to the tumour using the RNeasyÕ Mini kit (QIAGEN) according to the manufacturer’s instructions. The quality and quantity of the RNA were assessed using a NanoDropTM 1000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA). A total of 36 CRC tumour samples and their corresponding noncancerous tissues were selected to quantify PLCE1 mRNA levels. These samples were selected at random from the total samples (12 with AA, 12 with AG, 12 with GG): we only identified the genotypes of blood samples whose genotypes we considered as the same as the genotypes in tumour or healthy tissues. Quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR) was used to measure the amount of PLCE1 mRNA in pair-matched tissue samples from patients with different PLCE1 genotypes. For each sample, 1 mg of total RNA was subjected to reverse transcription using the PrimeScriptTM RT Master Mix System (TaKaRa, Dalian, China). A SYBRÕ Green real-time PCR method was used to assess the PLCE1 mRNA levels in colon tissue. Primer sequences for RT–PCR for the PLCE1 gene were 50 -CCTGGG CATAAGCACTACCAAG-30 (forward) and 50 -GTCTTGAGGATCAGAACCACT CC-30 (reverse).19 Primer sequences for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were 50 -GCACCGTCAA GGCTGAGAAC-30 (forward) and 50 -ATG GTGGTGAAGACGCCAGT-30 (reverse). All the primers were obtained from Invitrogen (Shanghai, China). Real-time PCR was performed in a Bio-Rad CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using 1 ml of the resulting cDNA from the RT step, 10 ml of SYBRÕ Green PCR Master Mix (TaKaRa) and 4 pmol each of the forward
and reverse primers. The cycling programme involved preliminary denaturation at 95 C for 30 s, followed by 40 cycles of denaturation at 95 C for 15 s and annealing at 60 C for 30 s; amplification fluorescence was monitored at 60 C at the end of each cycle. Three replicates were performed for each sample and the quantitative real-time PCR products were analysed using Bio-Rad CFX ManagerTM software, version 3.0 (Bio-Rad) using the 2CT method.31
Statistical analyses All statistical analyses were performed using the SPSSÕ software package, version 16.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ . The Hardy–Weinberg equilibrium was tested by a goodness-of-fit 2-test to compare the observed genotype frequencies compared with the expected frequencies among the control subjects. Differences in the distributions of demographic characteristics, selected variables, and the genotypes of the PLCE1 rs2274223 SNP between patients with CRC and control subjects were evaluated using a 2-test-based Q-test and the Wilcoxon signed-rank test. Stratification analysis was performed to evaluate the potential association of genetic variants of the PLCE1 rs2274223 polymorphism with risk in subgroups based on clinical characteristics. The associations between the PLCE1 variants and CRC risk were estimated by calculating the odds ratios (OR) and 95% confidence intervals (CI), using both univariate and multivariate logistic regression analysis with adjustment for age, sex, smoking status and alcohol consumption status. Paired-sample Student’s ttest was used for the comparison of mRNA levels between pair-matched tissue samples from patients with different PLCE1 genotypes. Two-sided tests were used for statistical analysis and a P-value < 0.05 was considered statistically significant. The statistical power was calculated using Power
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and Sample Size Calculation software, version 3.0 (Department of Biostatistics, Vanderbilt University, Nashville, TN, USA).32
Results A total of 417 Han Chinese patients with newly diagnosed and histopathologically confirmed CRC and 416 cancer-free healthy control subjects were enrolled in the study. Their clinical and demographic
characteristics are summarized in Table 1. There was no significant difference between the two groups in terms of age or sex distribution (two-sided 2-test; Wilcoxon’s test), suggesting that the case–control matching based on age and sex was adequate. In addition, there was no significant difference between the two groups in terms of smoking and alcohol drinking status (two-sided 2-test). Of the 417 patients with CRC, 266 (63.8%) had tumours in the rectum, 67 (16.1%) in the left colon and
Table 1. Clinical and demographic characteristics of Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416) enrolled in this study to investigate associations between phospholipase C epsilon 1 gene polymorphisms and risk of colorectal cancer. Characteristic Age, years 65 >65 Sex Male Female Tobacco smoking statusa Yes No Alcohol consumptionb Yes No Pathological tumour stagec I–II III–IV Tumour differentiation status Poor Moderate Well Tumour site Rectum Left colon Right colon
Patients with colorectal cancer (n ¼ 417)
Healthy control subjects (n ¼ 416)
62.9 12.6 218 (52.3) 199 (47.7)
62.6 13.2 213 (51.2) 203 (48.8)
225 (54.0) 192 (46.0)
232 (55.8) 184 (44.2)
135 (32.4) 282 (67.6)
136 (32.7) 280 (67.3)
91 (21.8) 326 (78.2)
96 (23.1) 320 (77.0)
246 (59.0) 171 (41.0) 149 (35.7) 238 (57.1) 30 (7.2) 266 (63.8) 67 (16.1) 84 (20.1)
Data presented as mean SD for age; other data presented as number (%) of patients or controls. a ‘No’ for smoking includes non-smoker and former smoker. b ‘No’ for alcohol consumption includes former alcohol drinkers and those who had never drunk alcohol. c Graded according to the WHO Classification of Tumours of the Digestive System.30
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Table 2. Logistic regression analysis of the association between phospholipase C epsilon 1 (PLCE1) genotypes and risk of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416).
Variants/genotypes
Patients with colorectal cancer, n (%)
Healthy control Subjects, n (%)
Crude OR (95% CI)
Statistical significancea
Adjusted OR (95% CI)b
Statistical significanceb
PLCE1 rs2274223 AA AG GG AG þ GG
228 (54.7) 161 (38.6) 28 (6.7) 189 (45.3)
269 (64.7) 128 (30.8) 19 (4.6) 147 (35.3)
1.00 1.46 (1.08, 1.98) 1.74 (0.91, 3.38) 1.46 (1.09, 1.94)
P ¼ 0.012 NS P ¼ 0.009
1.00 1.48 (1.11, 1.99) 1.74 (0.95, 3.20) 1.52 (1.15, 2.00)
P ¼ 0.008 NS P ¼ 0.003
a 2
-test for genotype distribution between patients and control subjects. Adjusted for age, sex, tobacco, smoking and alcohol drinking status in logistic regression models. OR, odds ratio; CI, confidence interval; A, adenine; G, guanine; NS, no statistically significant difference (P 0.05). b
84 (20.4%) in the right colon. Pathological examinations showed that 30 (7.2%) tumours were well differentiated, 238 (57.1%) were moderately differentiated and 149 (35.7%) were poorly differentiated. Distribution of pathological tumour stage was as follows: stage I–II, 246 (59.0%); and stage III–IV, 171 (41.0%). Age, sex, smoking status and alcohol drinking status were further adjusted for use in later logistic regression analysis. The genotype and allele distributions of the PLCE1 gene rs2274223 polymorphism in patients with CRC and control subjects are shown in Table 2. The observed genotype frequencies for the PLCE1 gene rs2274223 polymorphism agreed with that expected according to the Hardy–Weinberg equilibrium in the control subjects. The genotype distribution between the patients with CRC and the control subjects was significantly different for rs2274223 (P ¼ 0.0118; 2-test of the difference between the three genotype groups). After adjustment for age, sex, smoking and alcohol drinking status, a significantly increased risk of CRC was associated with the variant genotypes AG þ GG of rs2274223 with an adjusted OR of 1.517 (95% CI 1.148, 2.004) compared with the AA genotypes.
Stratification analysis was performed to evaluate the potential association of genetic variants of the PLCE1 rs2274223 polymorphism with risk in subgroups based on clinical characteristics. As shown in Table 3, the stratification analysis indicated that the risk associated with the rs2274223 variant AG þ GG genotypes was more evident in younger patients (65 years; adjusted OR 2.287, 95% CI 1.553, 3.368), men (adjusted OR 1.680, 95% CI 1.145, 2.467), and those that did not drink alcohol (adjusted OR 1.538, 95% CI 1.113, 2.125) compared with those patients without any variant genotypes (AA). The association between the PLCE1 rs2274223 polymorphism and the clinical characteristics of CRC was also examined (Table 4). Results showed that the AG þ GG genotype was associated with advanced pathological stage (adjusted OR 1.765, 95% CI 1.211, 2.575), moderately differentiated tumours (adjusted OR 1.709, 95% CI 1.221, 2.396), and tumours located in the rectum and left colon (adjusted OR 1.549, 95% CI 1.118, 2.148 and adjusted OR 1.774, 95% CI 1.019, 3.087, respectively). To investigate the potential association between PLCE1 SNP rs2274223 variants and PLCE1 mRNA levels, samples of
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Table 3. Stratification analysis of the association between phospholipase C epsilon 1 (PLCE1) genotype and risk of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417) and healthy control subjects (n ¼ 416). PLCE1 genotype (patients/controls) Characteristic Age, years 65 >65 Sex Male Female Tobacco exposurea Yes No Alcohol useb Yes No
AA
AG þ GG
Adjusted ORa (95% CI)
Statistical significanceb
95/136 133/133
123/77 66/70
2.29 (1.55, 3.37) 0.96 (0.63, 1.46)
P < 0.001 NS
120/152 108/117
105/80 84/67
1.68 (1.15, 2.47) 1.44 (0.94, 2.19)
P ¼ 0.008 NS
69/88 159/181
66/48 123/99
1.80 (1.10, 2.96) 1.45 (1.03, 2.05)
P ¼ 0.020 P ¼ 0.036
45/59 183/210
46/37 143/110
1.68 (0.92, 3.05) 1.54 (1.11, 2.13)
NS P ¼ 0.009
a
Obtained in logistic regression models with adjustment for age, sex, smoking and alcohol drinking status. P-value for heterogeneity test using 2-test-based Q-test. OR, odds ratio; CI, confidence interval; A, adenine; G, guanine; NS, no statistically significant difference (P 0.05). b
Table 4. Stratification analysis of the association between phospholipase C epsilon 1 (PLCE1) genotypes and clinical characteristics of colorectal cancer in Han Chinese patients with colorectal cancer (n ¼ 417). PLCE1 genotypes
Adjusted ORa (95% CI)
Statistical significanceb
Characteristic
AA n (%)
AG þ GG n (%)
Control subjects, n ¼ 416 Patients, n ¼ 417 Pathological tumour stagec I–II, n ¼ 246 III–IV, n ¼ 171 Tumour differentiation status Well, n ¼ 30 Moderate, n ¼ 238 Poor, n ¼ 149 Tumour site Right colon, n ¼ 84 Rectum, n ¼ 266 Left colon, n ¼ 67
269 (64.7) 228 (54.7)
147 (35.3) 189 (45.3)
1.00 (reference)
141 (57.3) 87 (50.9)
105 (42.7) 84 (49.1)
1.36 (0.97, 1.91) 1.77 (1.21, 2.58)
P < 0.001 NS P ¼ 0.002
22 (73.3) 123 (51.7) 83 (55.7)
8 (26.7) 115 (48.3) 66 (44.3)
0.67 (0.25, 1.60) 1.71 (1.22, 2.40) 1.45 (0.98, 2.17)
NS P ¼ 0.001 NS
50 (59.5) 144 (54.1) 34 (50.8)
34 (40.5) 122 (45.9) 33 (49.2)
1.24 (0.74, 2.06) 1.55 (1.12, 2.15) 1.77 (1.02, 3.09)
NS P ¼ 0.006 P ¼ 0.040
a
Obtained in logistic regression models with adjustment for age, sex, smoking and alcohol drinking status. P-value for heterogeneity test using the 2-test-based Q-test. c Tumours were graded according to the WHO Classification of Tumours of the Digestive System.30 A, adenine; G, guanine; OR, odds ratio; CI, confidence interval; NS, no statistically significant difference (P 0.05). b
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Discussion
Figure 1. Phospholipase C epsilon 1 (PLCE1) gene expression levels, measured using reverse transcription–polymerase chain reaction, are presented as the PLCE1 mRNA levels in colorectal cancer (CRC) tissue matched tissue adjacent to the tumour. Data points represent fold differences between each pair-matched sample set. Horizontal bars represent the calculated medians of the overall group and each subgroup. Levels of PLCE1 mRNA were downregulated in CRC tissues compared with pair-matched normal tissues. ‘Total’ includes the data from patients (n ¼ 36) with different PLCE1 genotypes and the three subgroups show the samples stratified by the three different PLCE1 rs2274223 genotypes (n ¼ 12 per group). The levels of PLCE1 mRNA were determined relative to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A, adenine; G, guanine. *P < 0.05, two-sided Student’s t-test.
tumour and pair-matched noncancerous tissues adjacent to the tumour from patients with different PLCE1 genotypes were used to determine PLCE1 mRNA levels using RT–PCR (Figure 1). A total of 36 CRC tumour samples and their corresponding noncancerous tissues were selected at random to quantify PLCE1 mRNA levels. PLCE1 mRNA levels were significantly lower in the CRC tumours than in the adjacent noncancerous tissues (n ¼ 36; twosided Student’s t-test; P < 0.001). Lower PLCE1 mRNA levels were observed in both AG and GG genotype carriers than in those patients that were AA genotype
This hospital-based case–control study investigated the association between a potentially functional SNP rs2274223 in the PLCE1 gene with the risk of CRC in a Han Chinese population. The current results showed that the SNP rs2274223 polymorphic genotype AG þ GG of the PLCE1 gene (combined genotypes) might contribute to the risk of CRC, and this risk was more evident in younger patients (65 years), men and those who did not drink alcohol. To the best of our knowledge, this is the first report on the relationship between the SNP rs2274223 polymorphic genotype AG þ GG of the PLCE1 gene and the risk of CRC. Our study also demonstrated a role of the PLCE1 gene and its potential functional variant rs2274223 in the aetiology of CRC at the mRNA level in pair-matched tissue samples from patients with different PLCE1 genotypes. The PLCE1 gene is a member of the phospholipase C family.14–16 Structural analysis revealed that the PLCE1 protein contains one cell division cycle 25 domain at the N-terminus and two Ras-associating domains at the C-terminus, which are all associated with Harvey-Ras and Ras-related protein 1 A activity.14,16 Therefore, PLCE1 acts as an effector of the Ras family small guanine triphosphatases and plays a critical role in regulating cell growth, differentiation and development.14,16,17 Other studies have found that some mutations in the PLCE1 gene lead to early onset nephritic syndrome and isolated diffuse mesangial sclerosis in humans,33,34 but this gene might also be linked to carcinogenesis.16 Research has also revealed that the PLCE1 gene was downregulated in tumour tissue from patients with CRC and that it might play a crucial
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role in CRC formation.28,29 To date, there have been no reports of studies that have investigated the association between genetic variations of the PLCE1 gene and susceptibility to CRC. In the present study, the PLCE1 rs2274223A >G SNP was demonstrated to be associated with the risk of CRC. This SNP causes an amino acid change from histidine to arginine in the calcium-dependent lipidbinding C2 domain of the PLCE1 protein.18 The rs2274223 SNP in the PLCE1 gene was first identified as a risk factor for oesophageal squamous cell carcinoma and gastric cancer in Chinese populations in two independent genome-wide association studies.23,24 These results were reproduced by others.18,19,25,35 Although Chinese patients formed the majority of the study populations in these studies,18,19, 35 the association between the PLCE1 rs2274223 SNP and the risk for cancer has also been demonstrated in Caucasian populations.25,26 These results and the findings of the current study suggest that the rs2274223 SNP of the PLCE1 gene might be a crucial risk factor for tumours of the digestive system (including CRC), and that it could be used as a biomarker for the diagnosis of these cancers. Individuals carrying PLCE1 rs2274223 AG þ GG genotypes had higher survival rates than those carrying the AA genotype, suggesting that the rs2274223 G allele might be associated with prognosis in patients with gastric cancer.27 However, in this current study, survival information for the patients with CRC was not included. Further studies focusing on the association between the rs2274223 SNP of the PLCE1 gene with prognosis of patients with CRC are needed. In the stratification analysis undertaken in this current study, the rs2274223 SNP of the PLCE1 gene was more evident in younger patients (65 years), in men and in those who did not drink alcohol. These current findings were consistent with the concept that individuals with high-risk
genetic susceptibility are more likely to develop cancer when they are exposed to a low level of alcohol exposure.19 For alcohol drinkers, the effect of genetic variations may be overwhelmed by the strong impact of environmental carcinogens. In contrast, for those who have been exposed to a low level of alcohol, genetic variations may play a dominant role in the initiation of carcinogenesis. Cancer is a complex and multifactorial disease: gene–gene and gene– environment interactions may occur, and a single genetic variant is usually insufficient to predict the overall risk. Further research is required to elucidate the role of other functional SNPs in the PLCE1 gene and to investigate the impact of other related genes involved in similar biological pathways that might be involved in the aetiology of CRC. To test the hypothesis that the rs2274223 SNP of the PLCE1 gene might play a functional role in the aetiology of CRC through altering the PLCE1 gene expression level, the mRNA levels from paired samples of CRC tumours and adjacent noncancerous tissue were measured using real-time RT–PCR. Overall, the current results showed that the PLCE1 mRNA level was much lower in the tumours than in adjacent noncancerous tissue samples, which was consistent with the results from two other studies of CRC.28,29 Such findings were also observed in Chinese patients with oesophageal squamous cell carcinoma or gastric adenocarcinoma.18,19 These results suggest that the PLCE1 gene might play a suppressive role in the carcinogenesis of a variety of cancers including CRC. Although this current study demonstrated that the G allele was related to lower PLCE1 mRNA levels, studies with larger population sizes are needed to validate this finding. Higher PLCE1 protein levels have been demonstrated in oesophageal squamous cell carcinoma tumour tissue compared with matched normal tissue.24 Although protein levels generally parallel the mRNA level,
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post-transcriptional regulation could also alter PLCE1 protein levels.18 Studies that measure PLCE1 protein levels are required, to validate these current PLCE1 mRNA findings. This current study had a number of limitations. First, it was a hospital-based study, so inherent biases in this type of study might lead to unreliable results. However, the G allele frequency in the control subjects was similar to that in the haplotype map database36 and the genotype distributions of the polymorphism in the control subjects conformed to the Hardy–Weinberg equilibrium. Therefore, the currents results are not thought to be subject to selection bias. Secondly, the sample size was relatively modest in the present study, but the study had 83.7% power to detect a minimal OR of 1.517 with a minor allele frequency of 35.3% using the current sample size. Thirdly, the PLCE1 rs2274223 SNP was the only polymorphism investigated in this study; SNPs at other loci that may be associated with susceptibility to CRC were neglected. Finally, the survival rates of individuals with different PLCE1 rs2274223 genotypes were not available for analysis. Future research should address whether the rs2274223 SNP contributes to the survival of patients with CRC. In conclusion, these current data show that the genotype distribution of the PLCE1 rs2274223 polymorphism was significantly different between patients with CRC and healthy control subjects. Larger populationbased and in-depth molecular studies are needed to validate these current findings and to elucidate the functional roles of the rs2274223 polymorphism in the aetiology of CRC.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This work was supported by the Shaoxing Major Scientific and Technological Projects (no. 2011A11013).
Acknowledgements We thank Michael Brownstein (ZhejiangCalifornia International Nanosystems Institute) for his critical reading of our manuscript and Xutao Hong (Zhejiang-California International Nanosystems Institute) for his technical support.
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