Polymorphisms of microRNA-Binding Sites in Integrin Genes Are Associated with Oral Squamous Cell Carcinoma Susceptibility and Progression

Abstract

Integrins, which act as an important role in the connection between cells and extra-cellular environments, are important cell surface receptors. Integrins have been demonstrated to play critical roles in many aspects of the progression of oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in microRNA-binding sites of integrin genes and the susceptibility and progression of OSCC in Chinese Han Population. We recruited 167 OSCC patients and 200 cancer-free controls from three independent medical centers. Genotyping was completed successfully for the five selected integrin SNPs: rs1062484 (integrin α3), rs11902171 (integrin αv), rs17468 (integrin β1), rs3809865 (integrin β3), and rs2675 (integrin β5). The results demonstrated that the A allele of rs3809865 T/A (a T-to-A nucleotide change), a functional polymorphism in the 3′UTR of integrin β3 gene, was associated with OSCC risk (p < 0.05). In addition, the association analysis between this SNP and integrin β3 mRNA expression level in the patients’ peripheral blood mononuclear cells indicated that OSCC patients carrying the A allele would have a lower integrin β3 expression level (p = 0.047). Meanwhile, survival analysis showed that the C allele of rs2675 A/C (nucleotide change from A to C), another 3′UTR polymorphism in integrin β5 gene, was related with progression of OSCC. Overall, our results suggest that rs3809865 and rs2675 may contribute to OSCC risk and progression in Chinese Han Population. These two SNPs may be used as potential diagnostic and prognostic biomarkers for OSCC in future.

Introduction

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the world, accounting for about 40% of head and neck cancers. OSCC is characterized by low survival rate and high morbidity (Funk et al. 2002; Okamoto et al. 2002). The 5-year survival rate of OSCC is slightly above 50%, which is in part due to the lack of effective biomarkers for both early diagnosis and optimal treatment (Weinberg and Estefan 2002). A large number of genetic variants have been associated with OSCC (Ratajczak-Wrona et al. 2013), but the contribution of these gene interactions in the development of OSCC remains unclear. Therefore, there is an urgent need for new and effective biomarkers to improve the prognosis of OSCC.

Integrins are important transmembrane proteins that act as cell surface receptors. Cells use integrins to attach and respond to the extra-cellular environment. There are 24 combinations of integrins formed by non-covalently association between 18α and 8β glycoprotein subunits (Hood and Cheresh 2002; Hynes 2002). Recent studies have demonstrated that integrins play critical roles in many aspects of cancer progression, including proliferation, cell survival, tumor invasion and metastasis. Lamb et al. (2011) reported that integrins could promote cell survival in prostate cancer. Integrins are also proven to have an effect on the tumor-induced angiogenesis by promoting the proliferation of vascular endothelial cells (Kumar 2003). Moreover, in breast cancer, integrins could promote invasion, migration and metastasis of cancer cell by mechanically linking extracellular matrix (ECM) ligands to the cell (Cordes and Park 2007). Integrins also contribute to the development of OSCC. For example, integrin αvβ6 facilitated OSCC invasion through interaction with cyclooxygenase-2 (Nystrom et al. 2006) and integrin α9β1 enhanced the migration of an OSCC cell line through changing the expression of matrix-degrading proteases (Roy et al. 2011). In addition, integrin β1 acts as a prelecan receptor in oral prelecan lesions prior to invasion, thereby affecting cell differentiation and proliferation in carcinoma in situ (CIS) (Ahsan et al. 2011).

A number of cancer-associated integrin genes can be regulated by microRNAs (miRNAs). miRNAs are 21-23 nucleotide strands of endogenous single-stranded RNAs that post-transcriptionally regulate the expression of many genes (Subramanian et al. 2008). The seed-region, which contains 2-7 nucleotides in the 5′ sequence, is the functional region of miRNAs. miRNAs bind to the 3′-untranslated region (3′UTR) of mRNA through the seed-region and activate the degradation of mRNA, thus repressing translation (Cho 2010a, b). As the binding between mRNA and miRNA is based on complementary base pairing, a single nucleotide polymorphism (SNP) in the 3′UTR or seed region would affect the binding efficiency, which may lead to change of the expression level of mRNA. When the miR-related SNPs exist in intergrins genes, such SNPs may contribute to the risk and severity of cancers by influencing the intergrin expression and function.

Previous studies have provided evidence on the association between sequence variations in miRNA-binding sites and cancer susceptibility. Kontorovich et al. (2010) reported that the SNP in miRNA-binding site of the ATF1 gene, encoding activating transcription factor 1, could increase the risk of developing breast cancer. The SNP, rs10771184, was suggested to have significant association with the risk of ovarian cancer by affecting the binding efficiency between KRAS and has-miR-544 (Liang et al. 2010). Similar associations were also observed in other cancers, such as non-small cell lung cancer (Chin et al. 2008), papillary thyroid carcinoma (Jazdzewski et al. 2008), gastric adenocarcinoma (Wang et al. 2013) and breast cancer (Saetrom et al. 2009).

It has been reported that SNPs in 3′UTRs of integrin genes are associated with the susceptibility of breast cancer (Brendle et al. 2008). We thus hypothesized that such SNPs might be related with OSCC. A hospital-based case-control study was conducted to investigate the association of miRNA-related SNPs in integrin genes with OSCC susceptibility in a Han Chinese cohort. Research subjects were recruited from three different regions in China in order to account for the genetic heterogeneity in the Han population.

Materials and Methods

Study population

A total of 167 case subjects were recruited from three medical facilities in China, including Guangdong Provincial Stomatological Hospital; School of Stomatology, Capital Medical University and West China Hospital of Stomatology from 2009 to 2012. All these subjects were sporadic cases with the pathological diagnosis of OSCC during the period from 2009 to 2012 by two qualified stomatologists. A follow-up study was conducted for all the OSCC patients until July 2013. In addition, 200 cancer-free control subjects, matched with case subjects in gender and age distribution, were enrolled from the three medical facilities above during the same period. The distribution of all subjects was presented in Table 1. Patients with immune disease or any histopathologic diagnosis other than OSCC were excluded. Clinical and epidemiology information was collected obtained for all case subjects, such as tumor stage, tumor site, classify of TNM, alcohol use and smoking status. 1 mL peripheral blood of all subjects was collected and then frozen at −80°C. The research protocol in this study was approved by the Ethics Committee of State Key Laboratory of Oral Diseases. Written informed consent was obtained from all the enrolled subjects.

Table 1.

Basic Characteristics in the OSCC Patients and Control Subjects.

SNPs selection

SNPs investigated in this study were selected by the following method as previously described (Brendle et al. 2008). First, 12 SNPs in 3′UTR were selected from all SNPs in ten integrin (ITG) genes, including integrin α3 (ITGA3), integrin α5 (ITGA5), integrin α6 (ITGA6), integrin αv (ITGAv), integrin β1 (ITGB1), integrin β3 (ITGB3), integrin β4 (ITGB4), integrin β5 (ITGB5), integrin β6 (ITGB6), and integrin β8 (ITGB8) according to the standard of Minor Allele Frequency (MAF) higher than 10%. Then the test for linkage disequilibrium was performed to inspect if there are more than one SNP in single gene and 5 of 12 SNPs were excluded according to the result. In addition, two SNPs, rs17664 (ITGA6) and rs743554 (ITGB4), were excluded, as the PCR condition was not appropriate for genotyping. At last, rs1062484 (ITGA3), rs11902171 (ITGAv), rs17468 (ITGB1), rs3809865 (ITGB3), and rs2675 (ITGB5) were investigated in the study. Putative miRNA-binding sites corresponding to the 5 SNPs above were predicted by using microInspecter (http://mirna.imbb.forth.gr/microinspector/), PicTar (http://pictar.bio.nyu.edu/), and TargetScan (http://www.targetscan.org/). Information about these 5 SNPs was shown in Table 2.

Table 2.

The Characteristics of the Five Predicted SNPs.

DNA isolation and SNP genotyping

Genomic DNA was extracted from peripheral blood sample usingQIAmp DNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. After extraction, DNA purity and concentration was determined by Nano-drop (GE Healthcare) and Nucleic acid electrophoresis. Genotyping of the selected SNPs was conducted with the Sequenom Mass ARRAY & iPLEX assay. Primers for PCR and iPLEX reaction were designed by Genotyping Tools & Mass ARRAY Assay Design software. Genotyping result was analyzed using TYPER 4.0 software.

Expression level of integrin β3 mRNA

Quantitative RT-PCR was used to detect integrin β3 mRNA expression level. Total RNA was extracted from frozen peripheral blood from 40 OSCC patients using RiboPure™-Blood Kit (Applied Biosystems). Reverse transcription was done using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. qRT-PCR was performed by Quantitative real-time PCR using SYBR® Select Master Mix (Applied Biosystems) and ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The primers for integrin β3 were: 5′-CAGGTGACCCGCTTCAAT-3′ (forward) and 5′-CCTTCCGTCCAATGCTATATGA-3′ (reverse). PCR reaction was initiated by the polymerase activation step for 5 min at 95°C, followed by 40 cycles of 15 s at 95°C and annealing/extension step at 60°C for 1 minute. Each sample was analyzed in duplicate. Endogenous β-actin was used as an internal control and its mRNA expression level was used to normalize the expression level of integrin β3 with the formula ratio of Ct _{integrin β3}/Ct _β-actin*100% (Liu et al. 2011).

Statistical analysis

Student’s t test was carried out to analyze the differences in of subjects’ characteristics and integrin β3 mRNA expression of integrin β3 for each genotype. Pearson χ² test or Fisher’s exact test was used to determine the difference of genotype and allele distribution of each SNP between the two groups. Hardy-Weinberg equilibrium was tested in control subjects for each SNP. Odds ratio (OR) and 95% confidence interval (CI) were estimated with the risk option of crosstabs. The Kaplan-Meier analysis was used to examine the association between genotypes and progression-free survival time of OSCC patients. All statistical analyses above were performed using the SPSS 19.0 statistical software package. All p-values reported were 2-sided.

Results

Demographic characteristics of the subjects

In this study, 167 OSCC patients and 200 cancer-free control subjects were recruited from three different regions in China (Table 1). The mean age (± s.d.) of patients and controls was 58.45 (± 12.02) and 57.28 (± 10.01) years, respectively. No statistically significant difference was detected in the distribution of gender and age between patients and control subjects, either in single-center population or the whole research population (p > 0.05, Table 1).

Association of SNPs with OSCC susceptibility

The genotype and allele frequencies of five selected SNPs and the association of these SNPs with OSCC susceptibility are shown in Table 3. All five SNPs conformed to the Hardy-Weinberg equilibrium in control group (p > 0.05). We noticed that rs3809865 in integrin β3 was significantly associated with OSCC occurrence. Patients carrying AA genotype had a decreased risk of OSCC (OR = 0.274, 95% CI = 0.094, 0.802). The allele frequencies of rs3809865 in cases and controls were also significantly different (p = 0.0004). The frequency of the A allele was lower in OSCC patients than in controls, with an odds ratio of 0.508 (95% CI = 0.349, 0.742). These results indicated that such allele might act as a protective factor in OSCC. No significant difference was observed in genotype or allele frequencies of the other four SNPs between patients and controls (p > 0.05).

In addition, subgroup analysis was performed to assess the association between integrin SNPs and OSCC susceptibility in different regions. Table 4 listed the genotype and allele frequencies of selected SNPs of cases and controls from three medical facilities. The A allele of rs3809865 was significantly associated with OSCC risk in all regions (p < 0.05). Statistical analysis of all participants was corrected using multicentre testing by Bonferroni method (Table 4).

Table 3.

Association of Predicted SNP Genotypes/Alleles in Integrin Genes and Risk of OSCC among All the Participators.

OR, odds ratio; CI, confidence interval.

P-values less than 0.05 are presented in bold.

Table 4.

Association of SNP rs3809865 Genotypes/Alleles in ITGB3 Genes and Risk of OSCC in Three Centers.

OR, odds ratio; CI, confidence interval.

The bold numbers mean the p-value is less than 0.05.

*Adjusted for multicentres testing by Bonferroni method.

Correlation of integrin β3 expression with genotype in OSCC

PBMCs from 40 OSCC patients were collected in this study. The association of SNP rs3809865 with integrin β3 expression level in PBMCs was investigated. The normalized Ct value was used to represent the mRNA expression level. Sample with higher expression level had lower normalized Ct value. As shown in Fig. 1, the mean normalized Ct values for patients carrying TT, TA or AA genotype were 129.75, 143.86 and 148.63, respectively. The expression level of integrin β3 in patients carrying TT genotype was significantly higher than those carrying AA genotype (p = 0.044). In addition, a significant difference of integrin β3 expression was observed between patients with TA or AA genotype and those with TT genotype, indicating that OSCC patients carrying the A allele would have a lower integrin β3 expression level (p = 0.047). No significant difference of integrin β3 expression was detected between patients with TT genotype and those with TA genotype (p > 0.05).

Fig. 1.

The association between integrin β3 genotypes and mRNA expression level in OSCC.

Integrin β3 related mRNA expression level in 40 OSCC patients with known integrin β3 genotypes as assessed by RT-qPCR. β-actin gene was used as the internal reference to normalize the Ct values. Normalized Ct value represents the relative expression level. The lower normalized Ct value means higher integrin β3 expression. Normalized Ct value of TT genotype was significant lower than that of AA genotype, indicating that integrin β3 expression is higher in PBMCs of OSCC patients with TT genotype than that of AA genotype (p = 0.044). Also, a significant difference in the normalized Ct value was observed between patients with TA/AA genotype and TT genotype, indicating that OSCC patients harbored A allele would have a lower integrin β3 expression level (p = 0.047).

Association between SNPs and OSCC progression

Sixty-three of the OSCC patients finished the follow-up and 37 of them had cancer progression. The mean time of follow-up and progression-free survival was 26.09 ± 2.18 months and 12.33 ± 1.77 months, respectively. The possible association between OSCC progression and the five selected SNPs was shown in Table 5. Compared with patients carrying genotype AA, patients carrying AC or CC genotype at rs2675 on integrin β5 gene had a higher risk of OSCC progression (HR = 1.957, 95% CI = 1.008, 3.797, P = 0.047) and shorter progression-free survival time (Fig. 2). No significant association was observed between OSCC progression and the other 4 SNPs.

Table 5.

Progression-free survival analysis of SNPs on Integrin in OSCC patients.

*No follow-up information about patients with CT genotype of ITGA3 rs1062484.

HR, hazard ratio; CI, confidence interval.

P-values less than 0.05 are presented in bold.

This is a univariable analysis.

Fig. 2.

The association between integrin β5 genotype and OSCC progression.

Kaplan-Meier curves of progression-free survival time of OSCC patients carrying AA and AC+CC genotypes of rs2675 on integrin β5. Y-axis represents the cumulative survival proportion. X-axis represents the progression-free survival time, which is calculated from the surgery day until the date of reoccurrence or death. Green line represents cases carrying variant alleles (CA+CC genotype) of rs2675 and blue line represents cases with wild-type genotype (AA genotype) of rs2675. Based on the follow-up study, OSCC patients with CA/CC genotype of rs2675 show a higher risk in progression than those with AA genotype.

Discussion

In this study, we investigated the association between genetic variants in 3′UTR region of integrins and OSCC risk in Chinese Han Population. Five SNPs from five integrin subunits were selected and genotyped by Sequenom Mass ARRAY & iPLEX assay. rs3809865, a functional SNP in 3′UTR of integrin β3, was found to be associated with OSCC risk. Also, we found rs2675, another 3′UTR SNP in integrin β5 gene, to be related with OSCC progression.

Integrin β3 subunit, which is encoded by integrin β3 gene, usually forms a vitronectin/RGD receptor with integrin αv subunit. Up-regulation of integrin αvβ3 expression has been detected in many kinds of cancers and is suggested to contribute to the invasion and migration of tumor cells. Gasparini et al. (1998) reported that integrin αvβ3 could be a prognostic indicator in breast cancer since it was highly expressed in breast tumor but not in normal tissue. It has been reported that overexpression of integrin αvβ3 could enhance the migration of prostatic carcinoma cells through a focal adhesion kinase pathway (Zheng et al. 1999). The association between integrin αvβ3 expression and tumor metastasis has also been proven in ovarian adenocarcinomas (Partheen et al. 2009) and small cell lung cancer (Li et al. 2012). As the important subunit of integrin αvβ3, integrin β3 also has great effort on the several kinds of cancers. However, the relationship between expression level and function of integrin β3 in OSCC is still unclear. In this study, we enrolled research subjects of Han ethnicity from three different regions in China. We found that SNP rs3809865 on integrin β3 gene was related with the risk of OSCC, both in single-center population and the whole research population. These results indicated that rs3809865 might be a universal OSCC-related SNP in Chinese Han Population. Furthermore, the expression of integrin β3 was detected in the peripheral blood of OSCC patients, suggesting that mRNA expression of integrin β3 might be associated with SNP rs3809865.

Previous studies on cancers and other diseases indicated that polymorphisms at miRNA-binding site would affect the expression level of mRNA. For example, the base transition of T to C at rs8126, a polymorphism located in the miRNA-binding site of TNFAIP2, was suggested to contribute in the up-regulation of TNFAIP2 in Head and Neck Squamous Cell Carcinoma (HNSCC) (Liu et al. 2011). Another study in systemic lupus erythematosus showed that rs1057233 in the 3′UTR of SPI1 was associated with the elevation of SPI1 mRNA level (Hikami et al. 2011). Similar results were also found in ovarian cancer (Wynendaele et al. 2010) and breast cancer (Zhang et al. 2011). The existence of SNPs in miRNA targeting sites may affect the binding affinity of miRNA, thus leads to alter expression of mRNA (Medina and Slack 2008). In this study, we observed that integrin β3 expression level in PBMCs from patients carrying the TA or AA genotype was significantly different from those carrying the TT genotype. Considering that SNP rs3809865 was located in the miRNA-binding site of integrin β3 and several miRNAs such as mir-26b and mir-330 were putatively predicted to target this SNP, we assumed that the miRNA-mRNA binding affinity would be different between rs3809865T and rs3809865A variants. Thus, it could cause the alteration of integrin β3 expression.

Previously, Zhang et al. (2013) reported that miR-124 could influence the expression of integrin β3 in asthma by targeting rs3809865 of integrin β3. However, contrary to our results, the T-to-A change at rs3809865 led to higher expression of integrin β3 in their study. As expression of one mRNA species could be regulated by several microRNAs and single microRNA could bind to different mRNAs (He and Hannon 2004), the miRNA targeting rs3809865 of integrin β3 might be different in OSCC and asthma, and then caused the opposite results. In our study, patients carrying the A allele at rs3809865 had a lower expression level of integrin β3. We speculated that different genotypes at rs3809865 might affect the function of integrin β3 by regulating integrin β3 expression, and then contribute to the development of OSCC. However, functional experiment is still required to confirm the mechanism of rs3809865 on the function of integrin β3 and OSCC susceptibility in future.

In addition, the survival analysis indicated that there was a possible association between rs2675 in integrin β5 and OSCC progression. We found that patients carrying variant genotype of rs2675 had an increased risk of OSCC progression in Chinese Han Population, compared with those carrying wild-type genotype. Recently, several studies have demonstrated that integrin β5 expression level was related with the patients’ clinical outcome and survival, thus integrin β5 might be used as a potential prognostic predictor in gastric cancer (Xu et al. 2010) and tongue squamous cell carcinoma (Kurokawa et al. 2008). As rs2675 was located in the 3′UTR of integrin β5, we assumed that this SNP might affect integrin β5 expression by binding with specific microRNA in the post-transcriptional regulation, and then contribute to OSCC progression. However, the exact mechanism still needs to be clarified.

Taken together, our data have provided evidence for the association between SNP rs3809865 in integrin β3 and OSCC risk in Chinese Han Population. We observed that rs3809865 could affect the mRNA expression level of integrin β3, indicating that rs3809865 might contribute to the development and progression of OSCC by regulating integrin β3 expression. These results suggested that rs3809865 in integrin β3 could be a potential biomarker for OSCC. Moreover, SNP rs2675 in integrin β5 may be related with progression of OSCC, indicating that rs2675 could be a potential prognostic marker for OSCC. Further functional studies are required to validate our speculation and unravel the role of integrins in the etiopathogenesis of OSCC.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81321002, 81061120531, 30930100, 81200791, 81102060, 81001208), Doctoral Program of the Ministry of Education of China (No. 20110181110055, 20100181120057, 20120181120011) and the International Science and Technology Cooperation Program of China (No. 2012DFA31370).

Conflict of Interest

The authors declare no conflict of interest.

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