Abstract
Background/Aim: Metalloproteinase-7 (MMP-7) has been previously found to be up-regulated in hepatocellular carcinoma (HCC) specimens and cells, favoring epithelial–mesenchymal transition. However, the contribution of MMP-7 genotypes to HCC has not been revealed to date. The study aimed to evaluate the contribution of MMP-7 promoter A-181G (rs11568818) and C-153T (rs11568819) genotypes on the risk of HCC in Taiwan, where HCC incidence is extremely high compared to worldwide data. Materials and Methods: In this case–control study, MMP-7 genotypes and their association with cigarette smoking and alcohol drinking habits were determined in 298 HCC patients and 889 healthy subjects by a typical polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Results: Ever smokers and alcohol drinkers were represented with higher percentages in the case group compared to the control group. MMP-7 rs11568818 genotypes were not found differentially distributed in case and control groups (p for trend=0.5246). People of the analyzed cohort of the present study were all of CC genotypes at their rs11568819 polymorphic sites, without any CT or TT genotypes. As for gene–lifestyle interactions, people with variant genotypes at MMP-7 rs11568818 had the same odds for HCC development compared to the wild-type AA genotype, no matter whether the subjects belonged to the smoker, non-smoker alcohol drinker, or non-drinker groups. Conclusion: MMP-7 variant genotypes did not present any significance towards being a marker for HCC risk in Taiwanese.
Statistically, hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and is known to be the most common cause of death in cirrhotic cases (1). HCC incidence is extremely higher in Asian-Pacific countries than Europe and the United States, especially in Taiwan (2). There are several factors to cause this regional difference, including infection with hepatitis B (HBV) or C virus (HCV), exposure to aflatoxin, smoking habits, alcohol drinking habits, sex (males) and family cancer history (3, 4). Inherited factors may also contribute to the etiology of HCC, however, a large majority of these genetic factors are unrevealed. Although some biomarkers for HCC have been proposed (5-9), findings still need to be validated. Additionally, the interactions between the genetic and environmental risk factors are largely unsettled.
The matrix metalloproteinases (MMPs, matrixins) are a group of peptidases that play critical roles in inflammation, carcinogenesis, and cancer cell migration via regulation of extracellular matrix (ECM) components (10, 11). Several members of MMPs have been found up-regulated in tissues of various types of cancers and have been highly related to tumor behaviors such as invasion (12, 13). Additionally, MMPs play important roles in metastasis (14, 15). In recent decades, a number of studies have reported that the single nucleotide polymorphisms (SNPs) on the MMP genes may significantly contribute to individual variations in the susceptibility to certain cancer types, including oral (16-18), esophageal (19), colorectal (20), lung (21, 22), bladder cancer (23) and pterygium (24). In humans, MMP-7 is located on chromosome 11 q22.3 (25). Under normal circumstances, MMP-7 is constitutionally expressed in the epithelial cells of skin exocrine glands, salivary glands, pancreas, intestines, reproductive organs, breast tissues, and most of all liver tissues (26). In physiological conditions, MMP-7 expression levels are low in normal tissues, however, MMP-7 has been found overexpressed in malignant tissues such as colorectal cancer (27, 28). From the viewpoint of gene expression, MMP-7 activity was higher in promoter constructs of MMP-7 rs11568818 and rs11568819 (29). In addition, MMP-7 genotypes have been evaluated for their association with several cancer types, including oral, esophageal, gastric, colorectal, gallbladder, lung, breast, bladder, prostate cancer, astrocytoma, renal cell carcinoma, childhood leukemia (30-44), but seldom HCC.
According to the above, we aimed to evaluate the association of MMP-7 rs11568818 and rs11568819 genotypes with HCC risk in a Taiwanese population composed of 298 HCC patients and 889 controls. The plot of chosen MMP-7 polymorphic sites is shown in Figure 1.
The polymorphic sites of matrix metalloproteinase-7 (MMP-7) rs11568818 and rs11568819 on chromosome 11.
Materials and Methods
Recruitment of HCC patients and non-cancer control groups. Patients diagnosed with HCC by expert surgeons were candidates for recruitment at the China Medical University Hospital, Taiwan, between 2004–2010. HCC patients and non-cancerous healthy subjects who agreed to complete a self-administered questionnaire and provide their peripheral blood samples were recruited in the study. Healthy controls were matched for age and sex and selected from the Health Examination Cohort in the same period. The study design including exclusion and inclusion criteria were approved by Institutional Review Board of the China Medical University Hospital (DMR103-IRB-094). Information extracted from questionnaire database of HCC cases and healthy subjects are summarized in Table I.
Summary of selected characteristic data of the 298 patients with hepatocellular carcinoma (HCC) and the matched 889 healthy controls.
MMP-7 genotyping methodology. Peripheral blood leucocytes from each subject in this study were extracted using the DNA applying QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) as per the previous publications (45-48). The primer design, the corresponding restriction endonucleases and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) conditions for MMP-7 genotyping are the same as in our previous publications (23, 49). Each genotyping process was repeatedly conducted by at least two well-trained researchers (Huang TH, Chang WS, Tsai CW, Huang TL, and Chin YT) independently and double-blinded, and the results of the genotyping were 100% concordant to each other.
Statistical analyses. The Student’s t-test was used to compare age indexes between HCC case and the control groups. The Pearson’s Chi-square test was used to evaluate the distribution of the MMP-7 genotypes among the analyzed subgroups. The contribution of the MMP-7 genotypes to HCC risk was also estimated by odds ratios (ORs) and their coupled 95% confidence intervals (CIs). Any p-value less than 0.05 was considered significant.
Results
Demographic data for the HCC populations. The distribution of age, sex, smoking and alcohol drinking behaviors of the investigated population (298 HCC patients together with 889 non-cancer healthy subjects) is shown in Table I. Since we applied a matching strategy to include non-cancer healthy subjects, it is taken for granted that there was no difference in age and sex distributions between cases and controls (both p>0.05) (Table I). There were significantly more ever smokers and ever alcohol drinkers in the case group than the control group (75.2% vs. 65.1% and 69.1% vs. 41.7%, p=0.0017 and p=0.0011, respectively) (Table I). These findings support the idea that individual cigarette smoking and alcohol drinking habits are risk factors for HCC in Taiwan.
The MMP-7 genotyping results of the HCC population. The distribution of the MMP-7 rs11568818 and rs11568819 genotypes for the 298 HCC cases and 889 healthy controls are shown in Table II. The results showed that MMP-7 rs11568818 genotypes were not differentially distributed between case and control groups (p for trend=0.5246) (Table II). In other words, neither the MMP-7 rs11568818 heterozygous variant AG nor the homozygous variant GG were associated with HCC risk, compared to the wild-type AA genotype (OR=1.04 and OR=1.90, 95%CI=0.71-1.52 and 95%CI=0.61-5.83, p=0.9117 and p=0.4217, respectively; Table II). In the dominant model, the data showed that no association between MMP-7 rs11568818 genotype and HCC risk was found (OR=1.09, 95%CI=0.76-1.57, p=0.6982, Table II). In the recessive model, still no positive association was found). People of the analyzed cohort of the present study were all of CC genotypes at their rs11568819 polymorphic sites, without any CT or TT genotypes (Table II).
Genotypic frequency distributions of matrix metalloproteinase-7 rs11568818 and rs11568819 among the 298 hepatocellular carcinoma (HCC) cases and 889 healthy controls.
The MMP-7 allelic frequency distributions of the HCC population. Supporting the findings that neither AG nor GG genotypes of MMP-7 rs11568818 were associated with HCC risk, the variant allele G of MMP-7 rs11568818 was found to be 8.7% in the case group, non-significantly different from that of 7.8% in the control group (OR=1.14, 95% CI=0.81-1.59, p=0.4532, Table III). Simultaneously, the allelic frequency of MMP-7 rs11568819 was 100% of allele C in both the case and the control groups (Table III).
Allelic frequencies for matrix metalloproteinase-7 rs11568818 and rs11568819 polymorphisms among the hepatocellular carcinoma (HCC) cases and healthy controls.
Discussion
In the literature, MMP-1, −2, −9, −13, together with their regulators TIMP-1 and −2 have been reported to play a part in liver fibrosis in 1990’s and 2000’s (50-54), which is one of the mechanisms involved in the initiation of liver cancer. However, no study has evaluated the contribution of MMP-7 to the etiology of HCC. In the current case–control study through investigation of the MMP-7 genotype profiles in a population containing 1,187 subjects (298 HCC cases and 889 controls), the contribution of MMP-7 to HCC risk was evaluated for the first time among Taiwanese. The results showed that neither the genotypes (Table II) nor variant alleles of MMP-7 (Table III) were differentially distributed between HCC case and healthy control groups, indicating that MMP-7 genotypes are not directly associated with HCC risk.
As mentioned above, smoking and alcohol drinking are two risk factors for HCC in Taiwan. We were also interested in evaluating the joint effects of MMP-7 rs11568818, rs11568819 genotypes with cigarette smoking and alcohol drinking behaviors. In the literature, long-term tobacco smoking has been believed to play an important role in the development of HCC (55-58), but little is known on the association of genetic risk factors to HCC development. Therefore, no clinical relevant marker are available to date. Recently, accumulated studies have reported that specific genotypes may interact with cigarette smoking behaviors and contribute to increased HCC risk. Among them, the polymorphisms on CYP1A1 (59) and tumor necrosis factor-alpha (9) genotypes are some examples. Not to our surprise, some of these genotypes lack any positive interaction with cigarette smoking habit affecting HCC risk (5, 8).
In Table I, there are more smokers and alcohol drinkers in the HCC case group than the control group. This supports the concept that smoking and alcohol drinking are risk factors to HCC. In Taiwan, people infected with hepatitis B (HBV) and C virus (HCV) are of rather high percentages, which may contribute to HCC. However, incomplete records of the infection status with HBV or HCV limited us to access the interaction of MMP-7 genotypes with HBV and HCV on HCC risk. In this study, we showed that people with variant genotypes at MMP-7 rs11568818 were under the same risk levels of being attacked by HCC, no matter if the subjects belonged to the smoker or non-smoker groups (data not shown). After adjusting for age, sex and alcohol drinking status, there still was no significant association found (data not shown). This phenomenon was similar for the case of alcohol drinkers and non-drinkers (data not shown).
MMP-7 in mainly in charge of degrading fibronectin, type IV collagen, laminin, nidogen, elastin, and β4-integrin (60-62). These extracellular matrix components have been reported to be produced by gastrointestinal, prostatic, endometrial and breast tumor cells (63). In the literature, MMP-7 is expressed by several types of cancer cells, including esophagus, stomach (64), pancreas (65, 66), lung (67), breast (68), prostate, endometrium (69), head and neck (70), and most noticeable liver (68, 71). Therefore, MMP-7 is supposed to serve as a practical marker and a potential therapeutic intervention target (65, 66, 69, 71, 72). However, the genotyping role of MMP-7 may be quite different in various cancer types, which is interesting and not yet fully clarified. In the current study, our results in a representative population of HCC in Taiwan are consistent with findings of Bialkowska and colleagues, who reported that MMP-7 genotypes were not associated with breast, lung and colorectal cancer risk in a Polish population consisting of 299 breast cancer, 199 lung cancer, 150 colon cancer and 648 non-cancer individuals (44).
The intracellular mechanisms of MMP-7 signaling are not yet clear, since the molecules interacting with MMP-7 are numerous and interactions are complicated. MMP-7 may also be related to differences of the cell behaviors in proliferative patterns, apoptosis, metastasis, angiogenesis etc. In the near future, the involvement of smoking in HCC etiology was accessible with the treatment of cigarette components to liver cells with different MMP-7 genotypes. It is very valuable to investigate whether liver cells with MMP-7 rs11568818 GG genotypes under treatment of BaP 7,8-diol 9,10-epoxide (BPDE), the ultimate carcinogenic metabolite of carcinogen benzo[a]pyrene (BE), are prone to tumorigenesis or not. Overall, the role of MMP-7 genotype and/or phenotype in HCC carcinogenesis requires further study. Of course, the role of other members in MMP family, such as MMP-1 and MMP-2, can team up to determine the risk of cancer. Although they have not been investigated in the same HCC population, they have been examined in other cancer types (73, 74).
The study provided evidence that the MMP-7 genotype may not directly determine a person’s susceptibility to HCC. In the future, further studies are required to reveal the significant alterations of MMP expression levels in HCC carcinogenesis.
Acknowledgements
The Authors are grateful to Tai-Lin Huang and Yu-Ting Chin for their excellent technical assistance. All the participants, including those who belonged to the control group of the study, are appreciated. This study was supported by Taichung Armed Forces General Hospital (Grant number: TCAFGH-D-109019), Chang Bing Show Chwan Memorial Hospital (Grant number: BRD-109025 and BRD-110025) and China Medical University and Hospital (Grant number: DMR-111-144). The funders had no role in study design, patient collection, experiment conduction, statistical analysis, data annotation, or decision to publish or preparation of the manuscript.
Footnotes
↵* These Authors contributed equally to this study.
Authors’ Contributions
Research design: Yueh TC, Tsao HY, and Chien WC; patient and questionnaire summary: Chen CP, Chen CC and Hung YC; experimental work: Tsai CW, Wu MH, Wang ZH and Chang WS; statistical analysis: Pei JS, Mong MC and Yang YC; article writing: Chang WS and Bau DT; manuscript checking and discussing: Yueh TC, Tsao HY, Chien WC, Tsai CW, Pei JS, Wu MH, Chen CP, Chen CC, Wang ZH, Mong MC, Yang YC, Hung YC, Bau DT and Chang WS.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received August 19, 2022.
- Revision received August 27, 2022.
- Accepted September 22, 2022.
- Copyright © 2022 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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