Tumor Biol. (2014) 35:11289–11293 DOI 10.1007/s13277-014-2428-5
RESEARCH ARTICLE
Genetic variations of CAV1 gene contribute to HCC risk: a case–control study Xixue Zhao & Guozheng Pan & Qingzhong Yuan & Dongpo Mu & Jun Zhang & Tao Cui & Jian Zhang & Linghai Zhang
Received: 2 July 2014 / Accepted: 31 July 2014 / Published online: 13 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Hepatocellular carcinoma (HCC) is the sixth common cancer and the third common cause of cancer mortality worldwide. However, the exact molecular mechanism of HCC remains uncertain. Caveolin-1 (CAV1) is the main protein in the caveolin family and plays an important role in tumorigenesis signaling. However, the contribution of CAV1 genetic variants to HCC is still unknown. The purpose of this study was to evaluate the association between the tagSNPs of the CAV1 gene and HCC risk. In this case–control study, we enrolled 1,000 HCC patients and 1,000 cancer-free controls, which were frequency-matched by age, gender, and HBV infection status. We found that CAV1 rs729949 was statistically associated with increased risk of HCC (odds ratio (OR)= 1.28; 95 % confidence interval (CI), 1.11–1.48; P=8.53× 10−4), even after Bonferroni correction (P=5.97×10−3); the expression levels of CAV1 in cancer tissues were significantly lower than those in adjacent normal tissues (P=0.012). We also detected a significant association for CAV1 rs3807989 under the log-additive model (OR=0.85; 95 % CI, 0.74–0.98; P=0.026). Significant associations were also detected for CAV1 rs6466583 (GG vs AA: OR=2.53; 95 % CI, 1.24– 5.17; P=0.011) and CAV1 rs3807986 (AG vs AA: OR=3.16; 95 % CI, 1.68–5.91; P=3.36×10−4) among genotype comparisons. These findings indicated that genetic variants n CAV1 might contribute to HCC susceptibility.
Keywords Polymorphism . Hepatocellular carcinoma . Caveolin-1 . Genetic
X. Zhao : G. Pan : Q. Yuan (*) : D. Mu : J. Zhang : T. Cui : J. Zhang : L. Zhang Shengli Oilfield Central Hospital, Jinan Road No.31, Dongying, Shandong 257034, China e-mail: [email protected]
Introduction Hepatocellular carcinoma (HCC), a highly malignant disease with an extremely poor prognosis, is one of the most common cancer types in China, with an incidence rate of 53/100,000 per year and a death rate of 37–55/100,000 annually [1–3]. HCC is the sixth common cancer and the third common cause of cancer mortality worldwide [4]. A number of candidate genes have been investigated by genetic association studies to evaluate their roles in HCC susceptibility [5]. However, up to now, the exact molecular mechanism of HCC remains uncertain. The major caveolin proteins, including caveolin-1 (CAV1), serve as scaffolding proteins in charge of recruiting related signaling molecules to the caveolae and regulating their activity, thus modulating several biological functions, including cell growth, apoptosis, and angiogenesis. Also, the scaffolding protein encoded by CAV1 is the main component of the caveolae plasma membranes found in most cell types [6]. CAV1 acts as a key regulator in cancer onset and progression through its role as a tumor suppressor [7]. CAV1 can also promote cell proliferation, survival, and metastasis as well as chemo- and radioresistance [7–11]. More recently, considerable studies have specified that genetic variations, especially single nucleotide polymorphisms (SNPs), could contribute to HCC risk. It has been reported that SNPs, which could alter gene expression, were associated with cancer susceptibility, prognosis, and survival. Many epidemiological studies have investigated the association between the genotype of CAV1 and the risk for several types of cancer, including hepatoma, nasopharyngeal carcinoma, non-small cell lung carcinoma, prostate, breast, oral, colorectal, and bladder cancer [6, 7]. However, there seems to be few literatures investigating the associations of CAV1 genotypes and HCC risk, except for only one study with limited sample size [12]. In this study, the specific aim was
11290
to determine the associations of polymorphisms in the CAV1 gene with HCC risk in a Chinese Han population.
Materials and methods Study population Totally, 1,000 patients pathologically diagnosed with HCC were recruited at Shengli Oilfield Central Hospital, Dongying, Shandong Province, China, between 2008 and 2012. All patients voluntarily participated, completed a selfadministered questionnaire, and provided their peripheral blood samples. Each subject was interviewed face-to-face by trained personnel using a formatted questionnaire to obtain demographic data and overall health characteristics. The equal number of noncancer healthy individuals was selected by matching for age, gender, and HBV infection status after initial random sampling from the Health Examination Cohort of the hospital as controls. The present study was approved by the institutional review board and informed written consent was obtained from each subject. SNP selection and genotyping Seven tagSNPs of CAV1 gene were selected using Hapmap (rs729949, rs6466583, rs3779512, rs3807989, rs3807988, rs1049337, and rs3807986). The SNPs were genotyped using TaqMan OpenArray SNP Genotyping Platform (Applied Biosystems). Samples from matched case–control pairs were handled identically and genotyped in the same batch in a blinded fashion. All included SNPs had call rates and concordance rates of at least 95 % among duplicates within each platform as well as across genotyping platforms. Laboratory personnel were blinded to the case–control and QC status of all samples. Quantitative real-time reverse transcription–polymerase chain reaction assay
Tumor Biol. (2014) 35:11289–11293
P0.05). None of the distribution of genotypes among control subjects deviates from the Hardy–Weinberg equilibrium (P>0.05). Table 2 showed the genotypic frequencies for tagSNPs of CAV1 gene and associations between CAV1 polymorphisms and HCC risk among Chinese Han people. Among the seven SNPs investigated, the genotypic pattern of CAV1 rs729949 was differentially distributed among the HCC patients and noncancer controls, and the P value for trend was significant (P=8.53×10−4). The difference remains
Table 1 Distribution of demographic characteristics for HCC cases and controls Category
Cases (N=1,000)
Controls (N=1,000)
P value
Age (year)
50.6±7.6
50.3±7.0
0.359
752 (75.2 %) 69 (6.9 %) 102 (10.2 %) 145 (14.5 %) 584 (58.4 %) 23.7±3.2
749 (74.9 %) 26 (2.6 %) 112 (11.2 %) 157 (15.7 %) 579 (57.9 %) 23.6±3.4
0.877 0.617 0.469 0.454 0.821 0.498
Statistical analyses
Gender (male) Family history of cancer Ever smoker Ever drinker HBV infection (HBsAg+) Body mass index (kg/m2)
All statistical analyses were conducted with SAS version 9.2 (SAS Institute Inc.). All statistical tests were 2-tailed, and
Continuous variables: mean values±standard deviation, P value from t tests. Categorical variables: numbers and percentages, P values from χ2 test
A total of 100 HCC tumor tissues and the adjacent normal tissues were obtained from surgically removed specimens. Then RNAs were isolated from ~100 mg tissues using mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA). The expression level of CAV1 was examined by TaqMan MicroRNA Assays (Applied Biosystems). The comparative CT method comparing to the transcription level of bactin was used to evaluate the expression levels.
Tumor Biol. (2014) 35:11289–11293
11291
Table 2 Association between CAV1 SNPs and HCC risk
rs729949 GG GA AA A vs G P value trend rs6466583 AA AG GG G vs A P value trend rs3779512 GG GT TT T vs G P value trend rs3807989 GG GA AA A vs G P value trend rs3807988 AA AC CC C vs A P value trend rs1049337 TT TC CC C vs T P value trend rs3807986 AA AG GG G vs A
Cases (N=1,000)
Controls (N=1,000)
Age- and sex-adjusted OR
621 250 129
642 297 61
1.00 (reference) 0.87 (0.71–1.06) 2.18 (1.59–3.01) 1.28 (1.11–1.48) 8.53×10−4
810 165 25
820 170 10
1.00 (reference) 0.98 (0.78–1.24) 2.53 (1.24–5.17) 1.15 (0.93-1.41) 0.190
795 194 11
800 192 8
1.00 (reference) 1.02 (0.81–1.27) 1.38 (0.56–3.44) 1.04 (0.85–1.28) 0.681
598 332 70
550 367 83
1.00 (reference) 0.83 (0.69–1.00) 0.78 (0.55–1.09) 0.85 (0.74–0.98) 0.026
621 311 68
633 300 67
1.00 (reference) 1.06 (0.87–1.28) 1.03 (0.73–1.48) 1.04 (0.89–1.21) 0.620
412 421 167
400 425 175
1.00 (reference) 0.96 (0.79–1.17) 0.93 (0.72–1.19) 0.96 (0.84–1.09) 0.515
632 331 37
647 341 12
1.00 (reference) 0.99 (0.82–1.20) 3.16 (1.68–5.91) 1.14 (0.97–1.33)
P value trend
0.109
P value in italics means statistically significant
significant after Bonferroni correction (P=8.53×10−4 ×7= 5.97×10−3). Compared with individuals with the GG genotype, the age and sex adjusted OR for developing HCC was
2.18 (95 % CI, 1.59–3.01) among those with the AA genotype. Under the log-additive model, each additional copy of minor allele A was associated with a 1.28-fold increased risk of HCC (OR=1.28; 95 % CI, 1.11–1.48). We also detected a significant association for CAV1 rs3807989 under the logadditive model, with a 0.85-fold decreased risk of HCC (OR= 0.85; 95 % CI, 0.74–0.98; P=0.026). Besides, for CAV1 rs6466583, compared with individuals with the AA genotype, those with the GG genotype have a higher HCC risk (OR=2.53; 95 % CI, 1.24–5.17; P=0.011); for CAV1 rs3807986, compared with individuals with the AA genotype, those with the AG genotype have a higher HCC risk (OR=3.16; 95 % CI, 1.68–5.91; P=3.36×10−4). As for CAV1 rs3779512, rs3807988, and rs1049337, there was no significant difference between patient and control groups in the genotypic or allelic frequency of these SNPs. After adjusting for additional potentially confounding factors such as smoking status, alcohol consumption, body mass index, HBV infection status, and family history of cancer, the results did not change materially. To validate our findings of CAV1 rs729949 and explore its influence on CAV1 expression, we used quantitative real-time PCR to examine the messenger RNA (mRNA) levels of CAV1 in 100 HCC cases. As shown in Fig. 1, the mRNA levels of CAV1 in HCC tissues were significantly lower than that of their adjacent normal tissues (P=0.012). Genotypes AA seemed to have lower CAV1 mRNA levels in both tumor tissues and their adjacent normal tissues than genotype GG.
Discussion In this large population-based case–control study, we investigated the associations of genetic variants in CAV1 gene with the risk of HCC in a Chinese population. We found that CAV1 rs729949 was statistically associated with increased risk of HCC, even after Bonferroni correction; we also detected a significant association for CAV1 rs3807989 under the logadditive model. Besides, significant associations were also detected for CAV1 rs6466583 and CAV1 rs3807986 among genotypes comparisons. To validate this finding, we also observed that the G to A substitution in CAV1 substantially altered CAV1 expression. These provide evidence to implicate CAV1 polymorphisms as novel susceptibility factors for HCC risk. CAV1, an integral membrane 178-amino acid protein of 21–22 kD, is located on chromosome 7 in region q31.1 at the D7S522 locus [13]. In 1992, Rothberg et al. [14] named the protein caveolin and identified it as a unique array of filaments or strands that form striated coatings decorating the cytoplasmic surfaces of caveolae. Previous studies showed that Cav1 expression was downregulated in a wide range of human tumors and cell lines, which hinted at its tumor suppressor
11292 Fig. 1 Association between the CAV1 rs729949 and CAV1 expression
Tumor Biol. (2014) 35:11289–11293
0.9 0.8 0.7 0.6 0.5
Tumor ssue
0.4
Normal ssue
0.3 0.2 0.1 0 GG abilities [15–23]. Many oncogenes, such as SRC, RAS, and BCR-ABL, transcriptionally downregulate Cav1 expression [7]. Hayashi et al. first reported a somatic punctiform Cav1 mutation at codon 132 (the P132L change that converts proline-132 into leucine) in 16 % of the cases of primary human breast cancers that were examined [24]. Since then, many epidemiological studies have investigated the association between the genotype of CAV1 and the risk for several types of cancer, including hepatoma, nasopharyngeal carcinoma, non-small cell lung carcinoma, prostate, breast, oral, colorectal, and bladder cancer. Few literatures investigated the association of CAV1 genotype and HCC risk, with only one study with limited sample size [12]. In the current study, we selected seven tagSNPs of the CAV1 gene to represent its genetic variation. We found that CAV1 rs729949 was statistically associated with increased risk of HCC (OR=1.28; 95 % CI, 1.11–1.48; P= 8.53× 10−4), even after Bonferroni correction (P= 5.97× 10−3); we also detected a significant association for CAV1 rs3807989 under the log-additive model (OR=0.85; 95 % CI, 0.74–0.98; P=0.026). Significant associations were also detected for CAV1 rs6466583 (GG vs AA: OR=2.53; 95 % CI, 1.24–5.17; P=0.011) and CAV1 rs3807986 (AG vs AA: OR= 3.16, 95 % CI: 1.68-5.91; P=3.36×10−4) among genotypes comparisons. All of the evidences above implicate CAV1 polymorphisms as novel susceptibility factors for HCC risk. This study had several limitations. First, selection bias might have occurred through the selection of hospital-based control subjects when the sampling is not random within the subpopulations of cancer and cancer-free subjects, though we have try our best to control it through the whole process of the study; since this study was restricted to a Chinese Han population, it is uncertain whether our findings can be replicated by other ethnic groups. The controls in our study were matched to
AA the cases by age, sex, and HBV infection status, which might minimize the subject selection bias. Second, in spite of the relatively large sample size, the power to elucidate gene– environment interactions was limited because of the small magnitudes of the overall associations. In conclusion, polymorphisms in the CAV1 gene highly associated with HCC risk among Chinese population. The results suggest that the CAV1 polymorphisms may be new biomarkers for PC susceptibility. Validations with larger population-based studies in different ethnic groups, further research into the function of CAV1 SNPs, and its potential biological mechanism association may be warranted. Acknowledgments We thank all the staffs who were involved in the subject recruitment, telephone interviews, sample preparation, and laboratory assays for their hardworks. Conflict of interest None.
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Tumor Biol. (2014) 35:11289–11293 6. Lin CH, Lin CC, Tsai CW, Chang WS, Yang CW, Bau DT. Association of caveolin-1 genotypes with gastric cancer in Taiwan. Anticancer Res. 2014;34:2263–7. 7. Senetta R, Stella G, Pozzi E, Sturli N, Massi D, Cassoni P. Caveolin-1 as a promoter of tumour spreading: when, how, where and why. J Cell Mol Med. 2013;17:325–36. 8. Wiechen K, Diatchenko L, Agoulnik A, Scharff KM, Schober H, Arlt K, et al. Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene. Am J Pathol. 2001;159:1635–43. 9. Sugie S, Tsukino H, Yamauchi T, Mukai S, Fujii M, Shibata N, et al. Functional polymorphism in the cav1 T29107A gene and its association with prostate cancer risk among Japanese men. Anticancer Res. 2013;33:1023–7. 10. Trimmer C, Bonuccelli G, Katiyar S, Sotgia F, Pestell RG, Lisanti MP, et al. Cav1 suppresses tumor growth and metastasis in a murine model of cutaneous SCC through modulation of MAPK/AP-1 activation. Am J Pathol. 2013;182:992–1004. 11. Vizkeleti L, Ecsedi S, Rakosy Z, Begany A, Emri G, Toth R, et al. Prognostic relevance of the expressions of CAV1 and TES genes on 7q31 in melanoma. Front Biosci (Elite Ed). 2012;4:1802–12. 12. Hsu CM, Yang MD, Tsai CW, Ho CY, Chang WS, Chang SC, et al. The contribution of caveolin-1 genotype and phenotype to hepatocellular carcinoma. Anticancer Res. 2013;33:671–7. 13. Engelman JA, Zhang XL, Lisanti MP. Genes encoding human caveolin-1 and −2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers. FEBS Lett. 1998;436:403–10. 14. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. Caveolin, a protein component of caveolae membrane coats. Cell. 1992;68:673–82.
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RESEARCH ARTICLE
Genetic variations of CAV1 gene contribute to HCC risk: a case–control study Xixue Zhao & Guozheng Pan & Qingzhong Yuan & Dongpo Mu & Jun Zhang & Tao Cui & Jian Zhang & Linghai Zhang
Received: 2 July 2014 / Accepted: 31 July 2014 / Published online: 13 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Hepatocellular carcinoma (HCC) is the sixth common cancer and the third common cause of cancer mortality worldwide. However, the exact molecular mechanism of HCC remains uncertain. Caveolin-1 (CAV1) is the main protein in the caveolin family and plays an important role in tumorigenesis signaling. However, the contribution of CAV1 genetic variants to HCC is still unknown. The purpose of this study was to evaluate the association between the tagSNPs of the CAV1 gene and HCC risk. In this case–control study, we enrolled 1,000 HCC patients and 1,000 cancer-free controls, which were frequency-matched by age, gender, and HBV infection status. We found that CAV1 rs729949 was statistically associated with increased risk of HCC (odds ratio (OR)= 1.28; 95 % confidence interval (CI), 1.11–1.48; P=8.53× 10−4), even after Bonferroni correction (P=5.97×10−3); the expression levels of CAV1 in cancer tissues were significantly lower than those in adjacent normal tissues (P=0.012). We also detected a significant association for CAV1 rs3807989 under the log-additive model (OR=0.85; 95 % CI, 0.74–0.98; P=0.026). Significant associations were also detected for CAV1 rs6466583 (GG vs AA: OR=2.53; 95 % CI, 1.24– 5.17; P=0.011) and CAV1 rs3807986 (AG vs AA: OR=3.16; 95 % CI, 1.68–5.91; P=3.36×10−4) among genotype comparisons. These findings indicated that genetic variants n CAV1 might contribute to HCC susceptibility.
Keywords Polymorphism . Hepatocellular carcinoma . Caveolin-1 . Genetic
X. Zhao : G. Pan : Q. Yuan (*) : D. Mu : J. Zhang : T. Cui : J. Zhang : L. Zhang Shengli Oilfield Central Hospital, Jinan Road No.31, Dongying, Shandong 257034, China e-mail: [email protected]
Introduction Hepatocellular carcinoma (HCC), a highly malignant disease with an extremely poor prognosis, is one of the most common cancer types in China, with an incidence rate of 53/100,000 per year and a death rate of 37–55/100,000 annually [1–3]. HCC is the sixth common cancer and the third common cause of cancer mortality worldwide [4]. A number of candidate genes have been investigated by genetic association studies to evaluate their roles in HCC susceptibility [5]. However, up to now, the exact molecular mechanism of HCC remains uncertain. The major caveolin proteins, including caveolin-1 (CAV1), serve as scaffolding proteins in charge of recruiting related signaling molecules to the caveolae and regulating their activity, thus modulating several biological functions, including cell growth, apoptosis, and angiogenesis. Also, the scaffolding protein encoded by CAV1 is the main component of the caveolae plasma membranes found in most cell types [6]. CAV1 acts as a key regulator in cancer onset and progression through its role as a tumor suppressor [7]. CAV1 can also promote cell proliferation, survival, and metastasis as well as chemo- and radioresistance [7–11]. More recently, considerable studies have specified that genetic variations, especially single nucleotide polymorphisms (SNPs), could contribute to HCC risk. It has been reported that SNPs, which could alter gene expression, were associated with cancer susceptibility, prognosis, and survival. Many epidemiological studies have investigated the association between the genotype of CAV1 and the risk for several types of cancer, including hepatoma, nasopharyngeal carcinoma, non-small cell lung carcinoma, prostate, breast, oral, colorectal, and bladder cancer [6, 7]. However, there seems to be few literatures investigating the associations of CAV1 genotypes and HCC risk, except for only one study with limited sample size [12]. In this study, the specific aim was
11290
to determine the associations of polymorphisms in the CAV1 gene with HCC risk in a Chinese Han population.
Materials and methods Study population Totally, 1,000 patients pathologically diagnosed with HCC were recruited at Shengli Oilfield Central Hospital, Dongying, Shandong Province, China, between 2008 and 2012. All patients voluntarily participated, completed a selfadministered questionnaire, and provided their peripheral blood samples. Each subject was interviewed face-to-face by trained personnel using a formatted questionnaire to obtain demographic data and overall health characteristics. The equal number of noncancer healthy individuals was selected by matching for age, gender, and HBV infection status after initial random sampling from the Health Examination Cohort of the hospital as controls. The present study was approved by the institutional review board and informed written consent was obtained from each subject. SNP selection and genotyping Seven tagSNPs of CAV1 gene were selected using Hapmap (rs729949, rs6466583, rs3779512, rs3807989, rs3807988, rs1049337, and rs3807986). The SNPs were genotyped using TaqMan OpenArray SNP Genotyping Platform (Applied Biosystems). Samples from matched case–control pairs were handled identically and genotyped in the same batch in a blinded fashion. All included SNPs had call rates and concordance rates of at least 95 % among duplicates within each platform as well as across genotyping platforms. Laboratory personnel were blinded to the case–control and QC status of all samples. Quantitative real-time reverse transcription–polymerase chain reaction assay
Tumor Biol. (2014) 35:11289–11293
P0.05). None of the distribution of genotypes among control subjects deviates from the Hardy–Weinberg equilibrium (P>0.05). Table 2 showed the genotypic frequencies for tagSNPs of CAV1 gene and associations between CAV1 polymorphisms and HCC risk among Chinese Han people. Among the seven SNPs investigated, the genotypic pattern of CAV1 rs729949 was differentially distributed among the HCC patients and noncancer controls, and the P value for trend was significant (P=8.53×10−4). The difference remains
Table 1 Distribution of demographic characteristics for HCC cases and controls Category
Cases (N=1,000)
Controls (N=1,000)
P value
Age (year)
50.6±7.6
50.3±7.0
0.359
752 (75.2 %) 69 (6.9 %) 102 (10.2 %) 145 (14.5 %) 584 (58.4 %) 23.7±3.2
749 (74.9 %) 26 (2.6 %) 112 (11.2 %) 157 (15.7 %) 579 (57.9 %) 23.6±3.4
0.877 0.617 0.469 0.454 0.821 0.498
Statistical analyses
Gender (male) Family history of cancer Ever smoker Ever drinker HBV infection (HBsAg+) Body mass index (kg/m2)
All statistical analyses were conducted with SAS version 9.2 (SAS Institute Inc.). All statistical tests were 2-tailed, and
Continuous variables: mean values±standard deviation, P value from t tests. Categorical variables: numbers and percentages, P values from χ2 test
A total of 100 HCC tumor tissues and the adjacent normal tissues were obtained from surgically removed specimens. Then RNAs were isolated from ~100 mg tissues using mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA). The expression level of CAV1 was examined by TaqMan MicroRNA Assays (Applied Biosystems). The comparative CT method comparing to the transcription level of bactin was used to evaluate the expression levels.
Tumor Biol. (2014) 35:11289–11293
11291
Table 2 Association between CAV1 SNPs and HCC risk
rs729949 GG GA AA A vs G P value trend rs6466583 AA AG GG G vs A P value trend rs3779512 GG GT TT T vs G P value trend rs3807989 GG GA AA A vs G P value trend rs3807988 AA AC CC C vs A P value trend rs1049337 TT TC CC C vs T P value trend rs3807986 AA AG GG G vs A
Cases (N=1,000)
Controls (N=1,000)
Age- and sex-adjusted OR
621 250 129
642 297 61
1.00 (reference) 0.87 (0.71–1.06) 2.18 (1.59–3.01) 1.28 (1.11–1.48) 8.53×10−4
810 165 25
820 170 10
1.00 (reference) 0.98 (0.78–1.24) 2.53 (1.24–5.17) 1.15 (0.93-1.41) 0.190
795 194 11
800 192 8
1.00 (reference) 1.02 (0.81–1.27) 1.38 (0.56–3.44) 1.04 (0.85–1.28) 0.681
598 332 70
550 367 83
1.00 (reference) 0.83 (0.69–1.00) 0.78 (0.55–1.09) 0.85 (0.74–0.98) 0.026
621 311 68
633 300 67
1.00 (reference) 1.06 (0.87–1.28) 1.03 (0.73–1.48) 1.04 (0.89–1.21) 0.620
412 421 167
400 425 175
1.00 (reference) 0.96 (0.79–1.17) 0.93 (0.72–1.19) 0.96 (0.84–1.09) 0.515
632 331 37
647 341 12
1.00 (reference) 0.99 (0.82–1.20) 3.16 (1.68–5.91) 1.14 (0.97–1.33)
P value trend
0.109
P value in italics means statistically significant
significant after Bonferroni correction (P=8.53×10−4 ×7= 5.97×10−3). Compared with individuals with the GG genotype, the age and sex adjusted OR for developing HCC was
2.18 (95 % CI, 1.59–3.01) among those with the AA genotype. Under the log-additive model, each additional copy of minor allele A was associated with a 1.28-fold increased risk of HCC (OR=1.28; 95 % CI, 1.11–1.48). We also detected a significant association for CAV1 rs3807989 under the logadditive model, with a 0.85-fold decreased risk of HCC (OR= 0.85; 95 % CI, 0.74–0.98; P=0.026). Besides, for CAV1 rs6466583, compared with individuals with the AA genotype, those with the GG genotype have a higher HCC risk (OR=2.53; 95 % CI, 1.24–5.17; P=0.011); for CAV1 rs3807986, compared with individuals with the AA genotype, those with the AG genotype have a higher HCC risk (OR=3.16; 95 % CI, 1.68–5.91; P=3.36×10−4). As for CAV1 rs3779512, rs3807988, and rs1049337, there was no significant difference between patient and control groups in the genotypic or allelic frequency of these SNPs. After adjusting for additional potentially confounding factors such as smoking status, alcohol consumption, body mass index, HBV infection status, and family history of cancer, the results did not change materially. To validate our findings of CAV1 rs729949 and explore its influence on CAV1 expression, we used quantitative real-time PCR to examine the messenger RNA (mRNA) levels of CAV1 in 100 HCC cases. As shown in Fig. 1, the mRNA levels of CAV1 in HCC tissues were significantly lower than that of their adjacent normal tissues (P=0.012). Genotypes AA seemed to have lower CAV1 mRNA levels in both tumor tissues and their adjacent normal tissues than genotype GG.
Discussion In this large population-based case–control study, we investigated the associations of genetic variants in CAV1 gene with the risk of HCC in a Chinese population. We found that CAV1 rs729949 was statistically associated with increased risk of HCC, even after Bonferroni correction; we also detected a significant association for CAV1 rs3807989 under the logadditive model. Besides, significant associations were also detected for CAV1 rs6466583 and CAV1 rs3807986 among genotypes comparisons. To validate this finding, we also observed that the G to A substitution in CAV1 substantially altered CAV1 expression. These provide evidence to implicate CAV1 polymorphisms as novel susceptibility factors for HCC risk. CAV1, an integral membrane 178-amino acid protein of 21–22 kD, is located on chromosome 7 in region q31.1 at the D7S522 locus [13]. In 1992, Rothberg et al. [14] named the protein caveolin and identified it as a unique array of filaments or strands that form striated coatings decorating the cytoplasmic surfaces of caveolae. Previous studies showed that Cav1 expression was downregulated in a wide range of human tumors and cell lines, which hinted at its tumor suppressor
11292 Fig. 1 Association between the CAV1 rs729949 and CAV1 expression
Tumor Biol. (2014) 35:11289–11293
0.9 0.8 0.7 0.6 0.5
Tumor ssue
0.4
Normal ssue
0.3 0.2 0.1 0 GG abilities [15–23]. Many oncogenes, such as SRC, RAS, and BCR-ABL, transcriptionally downregulate Cav1 expression [7]. Hayashi et al. first reported a somatic punctiform Cav1 mutation at codon 132 (the P132L change that converts proline-132 into leucine) in 16 % of the cases of primary human breast cancers that were examined [24]. Since then, many epidemiological studies have investigated the association between the genotype of CAV1 and the risk for several types of cancer, including hepatoma, nasopharyngeal carcinoma, non-small cell lung carcinoma, prostate, breast, oral, colorectal, and bladder cancer. Few literatures investigated the association of CAV1 genotype and HCC risk, with only one study with limited sample size [12]. In the current study, we selected seven tagSNPs of the CAV1 gene to represent its genetic variation. We found that CAV1 rs729949 was statistically associated with increased risk of HCC (OR=1.28; 95 % CI, 1.11–1.48; P= 8.53× 10−4), even after Bonferroni correction (P= 5.97× 10−3); we also detected a significant association for CAV1 rs3807989 under the log-additive model (OR=0.85; 95 % CI, 0.74–0.98; P=0.026). Significant associations were also detected for CAV1 rs6466583 (GG vs AA: OR=2.53; 95 % CI, 1.24–5.17; P=0.011) and CAV1 rs3807986 (AG vs AA: OR= 3.16, 95 % CI: 1.68-5.91; P=3.36×10−4) among genotypes comparisons. All of the evidences above implicate CAV1 polymorphisms as novel susceptibility factors for HCC risk. This study had several limitations. First, selection bias might have occurred through the selection of hospital-based control subjects when the sampling is not random within the subpopulations of cancer and cancer-free subjects, though we have try our best to control it through the whole process of the study; since this study was restricted to a Chinese Han population, it is uncertain whether our findings can be replicated by other ethnic groups. The controls in our study were matched to
AA the cases by age, sex, and HBV infection status, which might minimize the subject selection bias. Second, in spite of the relatively large sample size, the power to elucidate gene– environment interactions was limited because of the small magnitudes of the overall associations. In conclusion, polymorphisms in the CAV1 gene highly associated with HCC risk among Chinese population. The results suggest that the CAV1 polymorphisms may be new biomarkers for PC susceptibility. Validations with larger population-based studies in different ethnic groups, further research into the function of CAV1 SNPs, and its potential biological mechanism association may be warranted. Acknowledgments We thank all the staffs who were involved in the subject recruitment, telephone interviews, sample preparation, and laboratory assays for their hardworks. Conflict of interest None.
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