Abstract
Osteosarcoma (OS) is one of the most common bone malignancies in children and adolescents. To date, inaugural mechanism of OS was considered as a complex process and was still not clear. The p53 gene, most important tumor suppressors, was associated with risk of many tumors, including OS. In current study, we evaluated the relationship between genetic variation of the p53 binding site and the OS susceptibility through a two-stage case-control study in Chinese population. We found that rs1295925 (OR = 0.85; 95 CI = 0.76-0.94; P = 0.003) and rs3787547 (OR = 1.27; 95 CI = 1.11-1.45; P = 4.0 × 10−4) was significantly with OS susceptibility. Compared with those with rs1295925-TT genotype, and the risk of OS was significantly lower in individuals with CT genotype (OR = 0.77; 95 CI = 0.65-0.92) and CC genotype (OR = 0.75; 95 CI = 0.60-0.93). Compared with those with rs3787547-GG genotype, and the risk of OS was significantly higher in individuals with AG genotype (OR = 1.32; 95 CI = 1.10-1.58) and AA genotype (OR = 1.46; 95 CI = 1.11-1.92). To sum up, our results prove that SNP rs1295925 and rs3787547 play an important role in the etiology of OS, suggesting them as the potential genetic modifier for OS development.
Introduction
Osteosarcoma (OS), one of the most common bone malignancies in children and adolescents with an annual incidence of approximately 5/1,000,000, always has detectable metastases at diagnosis frequently leading to a poor prognosis generally [Citation1, Citation2].
As one of the most important tumor suppressors, p53 is found to be associated with risk of many diseases, including OS [Citation3-5]. As a transcription factor, p53 mainly functions through the target gene specific DNA sequence, and transcription regulation of the p53 target gene plays important role in tumor inhibition [Citation6]. Therefore, the genetic variation which located on the binding site of the p53 target gene is likely to influence the transcription of the corresponding target gene by altering the binding power of p53. Then, they may play an important role in the susceptibility of OS [Citation7].
The emergence of ChIP-seq technology, which combines chromatin immune precipitation (ChIP) with a new generation sequencing, makes it possible for researchers to find the transcription factor binding sites at the genome level [Citation8]. Considering the essential role of p53 in the development and progression of OS and the tissue specificity of p53 pathway in different cancer tissues, we downloaded the ChIP-seq data of OS cell line, while the p53 target gene binding domain was analyzed using various bioinformatics methods through the public database. We aimed to reveal the relationship between the single nucleotide polymorphisms (SNPs) located on the binding site of the p53 target genes and the risk of OS through a large sample size association study in Chinese population.
Objects and methods
Study Subjects
A total of 1,300 cases of OS cases and 1,300 healthy controls were included in this two-stage case-control study. All subjects were Han, and the subjects did not have any blood relationship with each other. The cases were confirmed by histopathological diagnosis of new cases by two experienced pathologists before the collection of blood samples, and the case did not carry out any radiotherapy and chemical treatment. The main rule of exclusion criteria: a history of previous tumors, the tumor is transferred from other parts of the primary tumor. The control group and the case group were frequency matched by age (± 5 years). During the epidemiological survey, all subjects were informed of the purpose of the study and signed the informed consent form. After obtaining the informed consent form, 5 ml of peripheral venous blood and corresponding demographic data (including age, gender, family history of cancer, tumor location, and Enneking stages) were collected.
Screening of specific genetic variants and genotyping
In this study, we first downloaded the data of the p53 ChIP-seq dataset, then the binding motif was found in the p53 binding peak and compared with the NCBI dbSNP database to get the SNPs with minor allele frequency (MAF)> 0.05. Finally, a total of 3 loci (rs3787547, rs290392 and rs1295925) were included in the analyses. Genomic DNA of the study subjects was extracted from the Genomic DNA Extraction Kit of Genoa Biotechnology (Beijing) Co., Ltd. Genotyping of the loci was performed using TaqMan (7900HT Fast Real-Time PCR System, USA ABI) genotyping system. Genotyping was performed blindly, and 5% random samples were selected for quality control. Finally, the response rates of all loci were above 99%.
Statistical analysis
Pearson χ2 test and t test were used to compare the classification variables and continuity variables in case group and control group respectively. The H-W genetic balance test was performed on the genotypes of each genetic variation in the control group by fitting the goodness χ2 test. The distribution of genotype frequencies in the case group and the control group was analyzed by χ2 test. The unconditional logistic regression model was used to calculate the OR value and the 95% CI. SAS 9.2 software to complete a variety of statistical analysis, all tests were bilateral, P <0.05 for the difference was statistically significant.
Results
General demographic characteristics
As shown in , the age and gender distribution of the case group and the control group was not statistically significant in two stages (all P value > 0.05). however, the family history of cancer in the case group and the control group had significant difference in the distribution (P <0.001). In the case group, 406 cases were located at long tubular bones, and 94 cases were located at axial skeleton in stage I, accounting for 81.2% and 18.8% of all cases; 52 cases of Enneking stage I, 375 cases of Enneking stage II, and 73 of Enneking stage III, account for 10.4%, 75%, 14.6% of all cases, respectively. The component ratio was similar in stage II.
Table 1 Demographic characteristics of OS cases and healthy controls.
Association between SNPs at the p53 target gene binding region with OS susceptibility in stage I
As shown in , the genotype of the three loci (rs3787547, rs290392 and rs1295925) in the control group was in accordance with the HW genetic balance (P>0.05). In the three loci, the allele distribution of rs1295925 on the VMP1 gene (OR = 0.81; 95 CI = 0.68-0.97; P = 0.024), and rs3787547 on the BCAS1 gene (OR = 1.31; 95 CI = 1.06-1.61; P = 0.012) were statistically significant, after adjusted for age, gender, and family history of cancer. Compared with those with rs1295925-TT genotype, and the risk of OS was significantly lower in individuals with CT genotype (OR = 0.74; 95 CI = 0.56-0.99) and CC genotype (OR = 0.70; 95 CI = 0.49-0.99). Compared with those with rs3787547-GG genotype, and the risk of OS was significantly higher in individuals with AG genotype (OR = 1.37; 95 CI = 1.02-1.82) and AA genotype (OR = 1.53; 95 CI = 0.99-2.35). While the frequency distribution for rs290392 was not statistically significant, indicating no evidence of association with OS risk.
Table 2. Associations between candidate SNPs and OS susceptibility in stage I
Replication of the SNPs at the p53 target gene binding region with OS susceptibility in stage II
Further, we replicated the above findings in an independent stage II samples (). Both rs1295925 and rs3787547 were validated for statistically significant association (P<0.05). When pooled together, the minor alleles of rs1295925 (OR = 0.85; 95 CI = 0.76-0.94; P = 0.003) and rs3787547 (OR = 1.27; 95 CI = 1.11-1.45; P = 4.0 × 10−4) was significantly with OS susceptibility. Compared with those with rs1295925-TT genotype, and the risk of OS was significantly lower in individuals with CT genotype (OR = 0.77; 95 CI = 0.65-0.92) and CC genotype (OR = 0.75; 95 CI = 0.60-0.93). Compared with those with rs3787547-GG genotype, and the risk of OS was significantly higher in individuals with AG genotype (OR = 1.32; 95 CI = 1.10-1.58) and AA genotype (OR = 1.46; 95 CI = 1.11-1.92).
Table 3. Associations between candidate SNPs and OS susceptibility in stage II
Discussion
In this study, bioinformatics analysis was used to screen the polymorphic sites which were located in the p53-binding region and could affect the binding of p53. Then, the relationship between the genetic variation and the OS susceptibility was analyzed by a two-stage, case-control association study. The rs1295925 locus on the VMP1 gene, and rs3787547 locus on the BCAS1 gene were identified to be associated with the OS susceptibility in the Chinese population, while rs290392 on the BCAS1 gene did not show an significant association with the risk of OS. Independent replication in an independent stage II samples found both rs1295925 and rs3787547 were validated for statistically significant association with OS susceptibility. These results indicated that
rs1295925 and rs3787547 may be involved in tumorigenesis of OS. To the best of our knowledge, it is the first study to investigate the possible role of genetic variants in p53 binding sites as a risk factor for OS and find their relationship with susceptibility to OS development in the Han Chinese population.
The VMP1 gene is a stress-inducible gene [Citation9], and its encoded vesicle envelope protein is an important regulatory factor for autophagy of cells [Citation10, Citation11]. Normal VMP1 protein expression is necessary to maintain normal tissue homeostasis and integrity [Citation12]. At present, a large number of studies have shown that VMP1 involves the development of many tumors, including hepatocellular carcinoma [Citation13, Citation14], pancreatic cancer[15-17], colorectal cancer [Citation18-20], ovarian cancer [Citation21], and breast cancer [Citation22]. SNP rs1295925 was located in the p53 transcriptional binding region and its mutation might affect the binding of p53 and eventually lead to different tumor susceptibility. This study showed that rs1295925-C allele decreased the susceptibility of OS. The results indicated that p53 may be involved in a large number of pathways, one of which is likely to affect the autophagy process of cells, and promote or inhibit cell autophagy. The BCAS1 (Breast carcinoma amplified sequence 1, also named NABC1) gene, which was located at 20q13, was amplified in a variety of tumor types and associated with more aggressive tumor phenotypes [Citation23-25]. Correa et al [Citation24] found that BCAS1 was overexpressed in breast tumors and both NABC1 and NABC1_5B were down-regulated in colorectal tumors. In current study, rs3787547-A allele was identified to increase the susceptibility of OS.
Our study have several strengths. First, large sample size provided enough statistical power to detect common variants; second, significant signals were captured by both single SNP in two independent datasets. Meanwhile, there are limitations in this study. This study is a typical hospital-based case-control study which often produced selection biases and information biases. Additional follow-up studies are required to confirm these results in other ethnic populations.
In summary, we first analyzed associations of the genetic variation of p53 binding region with OS susceptibility within Chinese population by using large sample case-control study. Strong associations between SNP rs1295925, rs3787547 and OS risk was revealed through several sets of analyses in two stages. Further functional studies are desired to uncover the exact effect on OS development, which could facilitate potential applications in the clinic.
Disclosure statement
The authors declare that they have no conflict of interest.
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