Association of phosphatase and tension homologue deleted on chromosome ten polymorphism rs1903858, but not serum levels with the risk of non–small‐cell lung cancer: A case‐control study

Background To investigate the association between phosphatase and tension homologue deleted on chromosome ten (PTEN) gene polymorphisms and non–small‐cell lung cancer (NSCLC) and further identify whether these polymorphisms influence serum PTEN levels. Methods A total of 152 NSCLC patients and 124 healthy controls were included in the study. PTEN gene rs11202586 (T > C) and rs1903858 (A > G) polymorphisms were detected using the multiple single‐base extension technique (SNaPshot). The serum PTEN levels were determined using an enzyme‐linked immunosorbent assay (ELISA) kit. Results The rs1903858 AG, GG genotypes, and G allele were associated with a higher risk of NSCLC (odds ratio (OR) =2.079, 95% confidence interval (CI) = 1.087‐3.974, P = .027; OR = 1.897, 95%CI = 1.053‐3.419, P = .033; OR = 1.505, 95%CI = 1.065‐2.126, P = .020). Stratified analysis reveal that the rs1903858 GG genotype and G allele were associated with an increased risk of squamous cell carcinoma (SCC) (OR = 3.226, 95%CI = 1.075‐9.678, P = .037; OR = 1.873, 95%CI = 1.092‐3.212, P = .023). Among smokers, the rs1903858 G allele carriers have an increased risk of NSCLC (OR = 1.916, 95%CI = 1.023‐3.589, P = .042), but a decreased risk of NSCLC was found with the AT haplotype. With respect to the serum PTEN levels, no significant difference was noted between NSCLC patients and healthy controls in this study. Conclusions The study indicated that the rs1903858 gene polymorphism is associated with increased risk of NSCLC, particularly in SCC and smoker, and the haplotype AT was a protective factor for NSCLC. The serum PTEN levels were not associated with NSCLC.


| INTRODUC TI ON
Lung cancer is the most common malignant; according to statistics in 2018, there were about 2.1 million new cases and 1.8 million deaths worldwide. 1 Lung cancer has become the major cause of death in China, with the rising incidence and mortality. 2 Lung cancer is triggered by a variety of factors. The major risk factors are environmental, cigarette smoking, and genetic.
Polymorphisms in deoxyribonucleic acid (DNA) sequences caused by single-base variations at the genome level are called single nucleotide polymorphisms (SNPs). The mutation of the SNP site may affect the function of the gene and lead to a change in its biological function and the occurrence of disease. 3 As a tumor-suppressor gene, phosphatase and tension homologue deleted on chromosome ten (PTEN) located on chromosome 10q23.3 and with a total length of 200 kb. Its encoded protein engages in the activities of both a protein phosphatase and a lipid phosphatase. PTEN is the tumor-suppressor gene with bispecific phosphatase activity. 4 Research suggests that PTEN has a variety of biological functions, for instance inhibiting angiogenesis, regulating cell proliferation, differentiation, and apoptosis. Thus, several studies have revealed a correlation between PTEN gene polymorphisms and various types of cancers, such as hepatocellular carcinoma, chronic myeloid leukemia (CML), and breast cancer. [5][6][7] To date, three studies have found that PTEN gene polymorphisms were associated with the overall survival and sensitivity to chemoradiotherapy of NSCLC. [8][9][10] Nevertheless, there are few studies concerning the linkage between PTEN SNPs and susceptibility to NSCLC.
Thus far, numerous studies have revealed that the change in PTEN activity is related to the occurrence, development, and metastasis of several cancers, such as gastric carcinoma, NSCLC, and breast carcinoma. [11][12][13] Alimonti et al reported that the decrease in PTEN levels was associated with an increase in cancer susceptibility. 14

| Study population
A total of 152 NSCLC patients and 124 healthy controls were included in the study. The NSCLC patients were consecutively recruited between August 2018 and January 2019 from the First Affiliated Hospital of Guangxi Medical University (Guangxi, China).
For the disease group, each patient was diagnosed with histologically confirmed NSCLC, including SCC and adenocarcinoma (AD), and excluded other concomitant tumors. 16 The healthy control group matched the gender and age of the disease group, they did not have cancer or other serious illness, and healthy controls were recruited from healthy volunteers at the same hospital during the same time. Information was collected by questionnaire and electronic medical records. After interpretation of the study, all participants provided written informed consent. The study was approved by the ethics committee of the First Affiliated Hospital of Guangxi Medical University.

| SNP selection
Single nucleotide polymorphisms genotype information was retrieved from the dbSNP data. The selected SNPs frequency were >0.05 and were previously described to be association with cancer. 17

| DNA extraction and polymorphism genotyping
About 2 mL of venous blood was collected from each participant and stored at −20°C before DNA extraction. DNA was isolated using the AxyPrep blood genomic DNA small dose kit (Axygen Biotech Co., Ltd.). DNA concentration and purity were determined by spectrophotometry and then stored at −80℃ before analysis.    Biotech Company, Shanghai, China) following the manufacturer's instructions. The detection levels for PTEN range from 7.5 to 240 ng/ mL, and the inter-and intra-assay variation coefficients of the used kit in our study were 9% and 11%, respectively. To analyze serum PTEN levels, absorbance was read at 450 nm using an ELISA reader (680; Bio-Rad).

| Statistical analysis
All data were analyzed using statistical software SPSS version 23.0 (IBM Corp). A two-sided P < .05 was accepted as statistically significant. Normally distributed variables were expressed as means ± standard deviations (SD), and the median and interquartile range (IQR) were used for the skewed variables. A Kruskal-Wallis test was conducted to compare the differences among groups. The goodness-of-fit chi-square test was used to estimate the Hardy-Weinberg equilibrium (HWE). Genotype and allele frequencies of the two SNPs were compared between patients with NSCLC and controls by the chi-square test (χ 2 ). Binary logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) after adjusting for gender, age, and smoking status, to assess the relative risk conferred by a particular allele and genotype.

| Demographic and clinical characteristics of all the participants
A total of 152 NSCLC patients and 124 healthy controls were included in the study. The basic demographic and clinical characteristics of all the participants in the study are shown in Table 2. No significant difference was found between the two groups regarding gender and mean age. However, when considering the smoking status, the number of smokers in patients with NSCLC was higher than in healthy controls (P < .05).

| PTEN rs11202586 and rs1903858 polymorphisms and the risk of NSCLC
The genotype distributions of the SNPs rs11202586 and rs1903858 in the PTEN gene were found to be consistent with the HWE in the controls (P > .05).
The genotype and allele frequencies of the PTEN rs11202586 and rs1903858 between NSCLC patients and healthy controls are presented in Table 3

| Stratified analysis
The subjects were stratified according to pathological subtypes and smoking status to assess the effect of confounders on the association  Table 4.

| Haplotype analysis
Haplotypes of the PTEN rs11202586 and rs1903858 polymorphisms were derived to detect haplotypes specifically correlated with NSCLC by using SHEsis software. The PTEN rs11202586 and rs1903858 haplotype frequencies (>3%) are listed in Table 6.
Three haplotypes were observed, and the frequency of haplotype AT was associated with reduced NSCLC risk (OR = 0.661, 95%CI = 0.471-0.928, P = .017). Haplotype AT may be protective for NSCLC.

| Serum PTEN levels
As serum PTEN levels of the two groups were non-normal distributed, they were presented as median ± IQR.

| D ISCUSS I ON
In recent years, although significant advances have been made in the diagnosis and treatment of NSCLC, due to the lack of typical which is beyond the optimal treatment period, by the time the diagnosis is confirmed. 18 Phosphatase and tension homologue deleted on chromosome ten has been shown to control the cell cycle and prevent excessive prolif- Therefore, our study assesses this association. Interleukin-6 (IL-6), and others. 32,33 The study has several potential limitations. First, the current study researched only two SNPs in the PTEN gene. Secondly, all subjects were from the same hospital and two ethnicities (Han and Zhuang) in Guangxi. Finally, the sample size in each subgroup was small when stratified analysis was performed. These limitations may influence the results of this study. Therefore, we need multi-site and multi-center samples to verify the experimental results.
In conclusion, our results indicate that the PTEN rs1903858 polymorphisms contribute to an increased risk of NSCLC, and the AT haplotype reduces the risk of NSCLC, which is a protective factor against NSCLC. However, there was no statistical difference of serum PTEN levels between patients with NSCLC and healthy controls, and no association between rs1903858 and rs11202586 polymorphisms and PTEN serum levels.

CO N FLI C T S O F I NTE R E S T
None.

AUTH O R S ' CO NTR I B UTI O N
The design and writing of this paper were completed by Zhen Liang. Yuzhu Tang and Hao Li performed the experiments. Youjun Xie analyzed the data. The manuscript was reviewed by Lingling Zhan.