Abstract
Background/Aim: The expression of matrix metalloproteinase 9 (MMP9) is elevated in various renal diseases, including renal cell carcinoma. However, the role of MMP9 genotype in this context remains unclear. This study aimed to investigate the association between MMP9 promoter rs3918242 genotypes and the risk of renal cell carcinoma. Materials and Methods: The MMP9 rs3918242 genotypes of 118 patients with renal cell carcinoma and 590 healthy subjects were determined using the polymerase chain reaction-restriction fragment length polymorphism method. Results: The results indicated that individuals carrying the CT or TT genotype of MMP9 rs3918242 did not exhibit an increased risk of renal cell carcinoma compared to wild-type CC carriers (odds ratio=1.20 and 2.68, 95% confidence interval=0.75-1.92 and 0.89-8.03; p=0.5270 and 0.1420, respectively). However, individuals with the CT and TT genotypes had a higher prevalence of renal cell carcinoma than those with the CC genotype when they also had hypertension (p=0.0010), diabetes (p=0.0010), or a family history of cancer (p<0.00001). No correlation was observed between MMP9 rs3918242 genotypic distribution and age (60 years or younger vs. older than 60 years) or sex (both p>0.05). Additionally, no correlation was found between MMP9 rs3918242 genotype and the risk of renal cell carcinoma in individuals with smoking or alcohol consumption habits. Conclusion: Carrying the T allele for MMP9 rs3918242 may predict a higher risk of renal cell carcinoma among individuals diagnosed with hypertension, diabetes, or with a family history of cancer.
Renal cell carcinoma, responsible for approximately 80% of all renal tumors (1), holds the distinction of being worldwide the most predominant malignant solid tumor affecting the kidneys (2, 3). Evidence from epidemiological studies suggests that several factors may contribute to the development of renal cell carcinoma. These factors include diabetes (4, 5), alcoholism (6-8), cigarette consumption (9), hypertension (4, 10) and aging (11-13), as supported by scientific literature. The identification of reliable genomic markers for renal cell carcinoma has been slow and unsatisfactory, despite the existence of several protein markers used to assess the risk of this condition. This delay can be attributed to various factors, including the relatively low prevalence of renal cell carcinoma compared to other types of cancer and the challenges associated with sample collection. Moreover, although several inherited genetic polymorphisms associated with renal cell carcinoma have been uncovered (12-16), the interactions between these genetic variations and lifestyle/environmental factors remain largely unknown.
Matrix metalloproteinases (MMPs) comprise a protein family responsible for the metabolism of extracellular matrix components (17, 18). Dysregulation of MMPs has been implicated in the early stages of tumorigenesis (19). Various types of cancer frequently exhibit altered expression patterns of MMPs and an imbalance in the extracellular microenvironment (20, 21).
MMP9, also known as 92 kDa type IV collagenase, 92 kDa gelatinase, and gelatinase B, plays a crucial role in the development of various human malignancies. It facilitates carcinogenesis by degrading collagen IV in the basement membrane and extracellular matrix, thereby promoting cancer cell proliferation, invasion, migration, and angiogenesis (22-24). Among the numerous genetic variations associated with MMP9, the most extensively studied is the MMP9 rs3918242 polymorphism located in the promoter region. This polymorphism has been investigated for its association with several types of cancer, including lung (25), breast (26), colorectal (27, 28), gastric (29) and prostate cancer (30). Recent research conducted in 2023 revealed the overexpression of MMP9 in circulating tumor cells and clear-cell renal cell carcinoma tissues (31). Furthermore, high MMP9 expression has been associated with a poor prognosis in clear-cell renal cell carcinoma (31). While the involvement of MMP9 rs3918242 genotypic variations remains largely unexplored, it is evident from the available information that the progression of renal cell carcinoma may be influenced by MMP9 overexpression.
In light of the above findings, the current study aimed to evaluate the association between MMP9 rs3918242 and the risk of renal cell carcinoma in a Taiwanese population. Additionally, we also aimed to investigate the potential interactions between MMP9 rs3918242 genotypes and clinical as well as lifestyle factors.
Materials and Methods
Recruit of renal cell carcinoma cases and controls. Data collection for the renal cell carcinoma cases and controls was carried out at China Medical University Hospital, a medical center located in central Taiwan. Renal cell carcinoma diagnoses were made by Dr. Wu’s surgical team, and the histopathological confirmation of tumor grades and types was conducted by skilled pathologists. Among the 139 patients interviewed, 118 (85%) agreed to participate and were included in the study. To establish a control group, five controls per patient with renal cell carcinoma were recruited from the Health Examination Center of China Medical University Hospital. The controls were frequency-matched to the patients based on sex and had an age within ±2 years of each patient. Importantly, the cancer-free controls had no biological relationship with one another and met the inclusion criteria of being Taiwanese citizens with no history of any cancer. Serum carcinoembryonic antigen levels of controls were within the normal range, confirming their cancer-free status. During the pre-screening and matching process, patients with incomplete demographic data regarding smoking, alcohol consumption, hypertension, diabetes, or family history of cancer were excluded. Additionally, potential control candidates displaying symptoms suggestive of renal cell carcinoma, such as hematuria, were also excluded. A total of 590 control subjects were retained for genotyping experiments and subsequent data analysis. Written informed consent was obtained from each participant, and 3-5 ml of venous blood was collected for genotyping experiments under the supervision and guidance of the Institutional Review Board of China Medical University Hospital (DMR98-IRB-209). All clinical investigations and records adhered to the principles outlined in the Declaration of Helsinki. Table I provides a summary of selected demographic and clinical characteristics of all study participants.
Distributions of the frequencies of selected characteristics among the renal cell carcinoma cases and healthy controls.
DNA preparation and storage. DNA extraction was performed on peripheral blood leukocytes obtained from patients with renal cell carcinoma within 24 h of collection using a QIAamp blood mini kit (Qiagen, Valencia, CA, USA) (14, 32). The extracted DNA samples were kept at −80°C for long-term storage. Additionally, the DNA samples were simultaneously diluted, aliquoted, and prepared as a working stock for genotyping purposes, following previously described protocols (33, 34). The working stock DNA samples were stored at −20°C until further analysis.
Methodology for the determination of MMP9 rs3918242 genotype. The MMP9 rs3918242 genotype was determined following the methodology described in our previous publications (35, 36). In brief, the forward and reverse primer sequences used for MMP9 rs3918242 amplification were 5′-TGGTCAACGTAGTGAAACCCCATCT-3′ and 5′-TCCAGCCCCAATTATCACACTTAT-3′, respectively. Restriction fragment length polymorphism analysis was performed using Sph I restriction endonuclease (New England Biolabs, Taipei, Taiwan, ROC) on the polymerase chain reaction products. Following enzyme digestion, the DNA fragments corresponding to the MMP9 rs3918242 CC, CT and TT genotypes were identified as 386 bp, 386+320+66 bp and 320+66 bp, respectively, as DNA adducts after enzyme digestion.
Methodology of statistical analysis. Student’s t-test was utilized to compare the age distributions between the case and control groups. Pearson’s chi-square test with Yates’ correction was applied for all other comparisons. The associations between MMP9 polymorphisms and the risk of renal cell carcinoma were assessed using odds ratios (ORs) with corresponding 95% confidence intervals (CIs) for each individual. Statistical significance was defined as a p-value of less than 0.05.
Results
Analysis of basic characteristics of the renal cell carcinoma and control groups. The frequency distributions of age, sex, and behavioral habits, including smoking and alcohol drinking, were compared between the 118 patients with renal cell carcinoma and the 590 cancer-free control subjects (Table I). The control subjects were matched with the renal cell carcinoma patients in terms of age and sex during participant selection, and no significant differences were observed between the two groups (p>0.05) (Table I). There were no significant differences in the frequencies of diabetes or personal behavioral habits, such as smoking or alcohol consumption, between the two groups (both p>0.05) (Table I). However, it is worth noting that the rate of family history of cancer was higher in the renal cell carcinoma group compared to the control group (9.3% versus 3.1%, p=0.0011), suggesting a potential inherited predisposition to renal cell carcinoma. Furthermore, the percentage of individuals with hypertension was significantly higher in the renal cell carcinoma group (66.9%) compared to the control group (50.2%, p=0.0009). From a histological perspective, 77.1% of patients had the most common subtype of renal cell carcinoma, which is clear-cell renal cell carcinoma. Among the patients, 53.4% had low-grade renal cell carcinoma, while 46.6% had middle- or high-grade renal cell carcinoma (Table I).
Association of MMP9 rs3918242 genotypes with renal cell carcinoma risk. The genotypic results for MMP9 rs3918242 among the 118 patients with renal cell carcinoma and the 590 controls are presented in Table II. Firstly, the genotypic distribution of MMP9 rs3918242 in the control group was found to be in accordance with Hardy–Weinberg equilibrium (p=0.6768). The genotypic frequencies of MMP9 rs3918242 did not significantly differ between the renal cell carcinoma case and control groups (p for trend=0.1631) (Table II). Specifically, the heterozygous CT genotype of MMP9 rs3918242 did not demonstrate an association with renal cell carcinoma risk (OR=1.20, 95% CI=0.75-1.92, p=0.5270). Conversely, the homozygous variant TT genotype was associated with a 2.68-fold increased risk of renal cell carcinoma (OR=2.68, 95% CI=0.89-8.03, p=0.1420), although this did not reach statistical significance. Furthermore, a comparison of individuals carrying the TT genotype with those carrying the CC or CT genotype revealed a 2.57-fold odds ratio for renal cell carcinoma (95% CI=0.86-7.65, p=0.1613, Table II). Similarly, individuals carrying the CT or TT genotypes exhibited a non-significantly elevated risk of renal cell carcinoma (OR=1.31, 95% CI=0.84-2.04, p=0.2861) compared to those with the CC genotype (Table II).
Distribution of matrix metalloproteinase 9 rs3918242 genotypes among the controls and patients with renal cell carcinoma.
Association of MMP9 rs3918242 allelic frequencies with renal cell carcinoma risk. To further validate the preliminary findings presented in Table II, we performed allelic frequency analysis to investigate the association between MMP9 rs3918242 and renal cell carcinoma risk, as shown in Table III. The distribution of variant alleles for MMP9 rs3918242 did not exhibit a significant difference between the renal cell carcinoma case and control groups (p=0.1368). Individuals carrying the variant T allele had a 1.37-fold odds ratio (OR=1.37, 95% CI=0.93-2.02) for renal cell carcinoma compared to those carrying the wild-type C allele, although this finding did not reach statistical significance (Table III). Therefore, the results of the allelic frequency analysis provide support for the conclusion that the MMP9 rs3918242 CT or TT genotypes are not associated with an increased risk of renal cell carcinoma.
Allelic frequencies for matrix metalloproteinase 9 rs3918242 in the control and renal cell carcinoma patient groups.
Interaction analysis of MMP9 rs3918242 genotypes and clinical and lifestyle factors. Firstly, we performed subgroup analysis based on age and observed that the distribution of MMP9 rs3918242 genotypes did not differ significantly between renal cell carcinoma patients older and younger than 60 years of age (p=0.6755). Additionally, when we examined the distribution of genotypes based on sex, no significant difference was observed between males and females (p=0.3643).
Secondly, we investigated potential interactions between MMP9 rs3918242 polymorphism and lifestyle factors such as smoking and alcohol consumption on renal cell carcinoma risk (Table IV). The results indicated that there were no significant differences in the distribution of MMP9 rs3918242 genotypes among smokers, non-smokers, alcohol drinkers, and non-drinkers (Table IV).
Correlation between matrix metalloproteinase 9 rs3918242 genotype and clinicopathological features of 118 patients with renal cell carcinoma.
Thirdly, as hypertension and diabetes are considered risk factors for renal cell carcinoma, we explored whether there was an interaction between MMP9 rs3918242 and these factors in relation to renal cell carcinoma susceptibility. The findings revealed that among individuals carrying the MMP9 rs3918242 variant CT and TT genotypes, there was a higher proportion with hypertension (p=0.0010) and diabetes (p=0.0010) in patients with renal cell carcinoma.
Lastly, there was a notable association between MMP9 rs3918242 genotype and a family history of cancer in relation to renal cell carcinoma susceptibility (p<0.00001).
Discussion
The activity of MMP9 is known to be highly dependent on its expression level (37, 38). Overexpression of MMP9 has been observed in various renal disorders, including membranous nephropathy and renal fibrosis (39, 40). Additionally, MMP9 has been found to be highly expressed in circulating tumor cells and clear-cell renal cell carcinoma tissues (31). In 2023, it was suggested that a high expression of MMP9 contributes to a poor prognosis in clear-cell renal cell carcinoma (31). These findings collectively indicate that MMP9 protein may play a critical role in carcinogenesis, particularly in renal cell carcinoma. It is hypothesized that the dysregulation of MMP9 expression is influenced by its inherited genotype. In other words, individuals with different MMP9 genotypes may have varying susceptibility to renal cell carcinoma. Among the MMP9 polymorphic sites, MMP9 rs3918242 is located in the promoter region, which controls the expression level of MMP9 itself (41). Evidence has shown an association of MMP9 rs3918242 genotype with prostate cancer risk and prognosis (30). It was also observed that higher MMP9 expression levels were associated with shorter 5-year survival time (30). However, as far as we are aware, no investigations have been conducted regarding the role of MMP9 genotypes in renal cell carcinoma.
In our study, we observed that the TT genotype of MMP9 rs3918242 was not significantly associated with an altered risk of renal cell carcinoma (Table II). This finding is consistent with a previous study conducted in the Japanese population by Awakura and colleagues. However, it is worth noting that their study also had a relatively small sample size, consisting of 179 renal cell carcinoma cases and 211 controls (42). In their dataset, the control group had 154 (73%), 49 (23.2%) and 8 (3.8%) individuals with MMP9 rs3918242 CC, CT and TT genotypes, respectively. In the renal cell carcinoma group, these numbers were 126 (70.4%), 50 (27.9%) and 3 (1.7%), respectively. The ORs associated with renal cell carcinoma were 1.25-fold for individuals carrying the CT genotype and 0.46-fold for those carrying the TT genotype. The genotypic distribution for MMP9 rs3918242 in their control group adhered to the Hardy–Weinberg equilibrium (p=0.1137). Notably, in their renal cell carcinoma group, the percentage of individuals with the CT genotype (27.9%) was higher compared to the control group (23.2%), while the TT genotype was less prevalent (1.7% versus 3.8%). This discrepancy may be attributed to the relatively small sample size of the renal cell carcinoma group (42). In our data, although the sample size for the renal cell carcinoma group was also limited, we observed a higher percentage of individuals with the MMP9 rs3918242 CT genotype (23.7%) compared to the control group (21.2%), and a more pronounced prevalence of the TT genotype (4.3% versus 1.7%). The ORs for the MMP9 rs3918242 CT and TT genotypes were 1.20 and 2.68, respectively (Table II). These results suggest that the T allele may contribute to a higher risk of renal cell carcinoma. However, these preliminary findings need to be validated with larger sample sizes, and further investigations are required to elucidate potential differences among various ethnic groups.
In addition to investigating the contribution of MMP9 rs3918242 genotypes to the risk of renal cell carcinoma, our study also explored the association between MMP9 rs3918242 genotypes and lifestyle factors in relation to renal cell carcinoma. We found no significant associations among subgroups stratified by age, sex, smoking status, or alcohol consumption (Table IV). However, our key findings do suggest that genetic variants in the MMP9 gene may increase susceptibility to renal cell carcinoma among individuals with hypertension, diabetes, and a family history of the disease (Table IV). Currently, there is limited solid evidence regarding the effects of MMP9 rs3918242 genotypes combined with hypertension and diabetes on renal cell carcinoma. Nonetheless, it is possible that MMP9 rs3918242 genotypes play a critical role in determining the risk of renal cell carcinoma. Further studies with larger sample sizes, including diverse populations, are urgently needed to better understand the involvement of MMP9 genotypes in renal carcinogenesis.
In conclusion, this study has provided initial evidence suggesting that the T allele of the MMP9 promoter rs3918242 polymorphism may contribute to the risk of renal cell carcinoma. The findings indicate that the MMP9 rs3918242 T allele may have a significant role, particularly among individuals with hypertension, diabetes, or a family history of cancer. Further investigations with larger sample sizes and diverse populations are required to validate and expand upon these findings.
Acknowledgements
The Authors are grateful to Yu-Ting Chin, and Hou-Yu Shih for their technical support. The cooperation of all the participants is highly appreciated. This study was financially supported by Taichung Armed Forces General Hospital to Dr. Liao (grant number: TCAFGH-D-112024), Taichung Veterans General Hospital (TCVGH-1114801B) and China Medical University and Asia University (CMU111-ASIA-02).
Footnotes
Authors’ Contributions
Research design: Liao CH, Bau DT, and Tsai CL; patient and questionnaire summaries: Liao CH and Chang SY; experimental work: Wang YC, Chang WS, and Tsai CW; data clearance and identification: Yang YC, Huang WC, and Chang SY; statistical analysis: Mong MC, Wu WT, and Chen JC; article writing: Liao CH, Tsai CL, Chang SY, Chang WS, and Bau DT; article review and revision: Chang WS and Bau DT.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received June 20, 2023.
- Revision received July 24, 2023.
- Accepted July 25, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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).
