Genetic association study of intron variants in the forkhead box protein P3 gene in Chinese patients diagnosed with cervical cancer

Abstract The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high‐risk human papilloma virus (HR‐HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR‐HPV infection and CC malignant lesions (TC vs. TT: OR = 0.71, 95% CI = 0.51–0.99); the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions (TC+CC vs. TT: OR = 0.69, 95% CI = 0.50–0.95; C vs. T: OR = 0.78, 95% CI = 0.63–0.97). The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR‐HPV infection and CC malignant lesions (GA vs. GG: OR = 0.70, 95% CI = 0.51–0.96; GA+AA vs. GG: OR = 0.69, 95% CI = 0.51–0.94; A vs. G: OR = 0.75, 95% CI = 0.58–0.96). Patients with CC and HR‐HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T‐C‐A decreased the risk of HR‐HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR‐HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC.

than those in developed countries, particularly in lower-resource countries. The highest number of CC cases was reported in China (106,000 cases), whereas the highest number of CC-related deaths was reported in India (60,000 cases); furthermore, China and India together accounted for 35% of cases and deaths globally worldwide. 2 High-risk human papillomavirus (HR-HPV) infection is an important cause of CC, and 99.7% of patients with CC carry this virus. 3 In the initial stage of infection, patients may have no obvious clinical manifestations. However, persistent HPV infection is the main cause of high-grade squamous intraepithelial lesions of the cervical epithelium and CC. Furthermore, most HPV subtypes can be cleared by the immune system, and only approximately 10% of HPV-infected individuals will subsequently develop into precancerous lesions and CC. 4 Nevertheless, HPV infection alone is not sufficient to induce CC onset; smoking, oral contraceptives and multiple sexual partners are also important factors affecting the development of CC. 5 The susceptibility to and pathogenesis of CC are associated with persistent HPV infection and host-reaction interactions. Thus, host genetic variants may affect the development of CC. 6 Indeed, growing evidence has indicated that variants of methylenetetrahydrofolate reductase, Toll-like receptor (TLR) 4 and TLR9 are closely associated with the risk of CC. 7,8 Therefore, further studies of the relationships between genetic variants and the pathogenesis of CC are warranted.
The forkhead box protein 3 (FOXP3) gene is located on the X chromosome at Xp11. 23 and encodes the FOXP3 protein, a member of the forkhead lineage-transcription factor family. FOXP3 is characteristically expressed in regulatory T cells (Tregs) and is involved in the activation, proliferation, differentiation and regulation of Tregs. 9 As an important component of the tumour microenvironment, Tregs are responsible for the downregulation of autoimmune reactions and promotion of immunological tolerance. Recent studies on the roles of Tregs in tumours have demonstrated that the regulatory mechanisms mediated by Tregs contribute to immune evasion against tumour immunotherapy. 10 Therefore, FOXP3 is an essential factor that induces and maintains the unique immunosuppressive properties of Tregs.
Single nucleotide variants (SNVs) are the most common type of genetic mutation, and variants in the promoter region of a gene can lead to abnormal recognition by RNA polymerase Ⅱ, leading to abnormal gene expression. Furthermore, variants in the intron region of a gene may affect mRNA splicing. 11 Importantly, FOXP3 is regarded as tumour initiation in CC cells, 12 and variants in the FOXP3 gene have been shown to be associated with HPV infection and precancerous lesions. 13 However, the relationships between Accordingly, in this study, we evaluated the effects of FOXP3 variants on the occurrence of CC among Chinese individuals. In addition, we explored the role of immune-infiltrating cells in the prognosis of patients with CC.

| Study design and population
In total, 350 patients with CC diagnosed at the Affiliated Cancer Hospital of Guangxi Medical University and the Affiliated Hospital of YouJiang Medical University from 2018 to 2019 were randomly enrolled as the case group, and 350 age-matched healthy women were randomly enrolled as the control group. Before sample collection, all participants were informed of the purpose of the study and provided written informed consent for participation in the study.
The inclusion criteria were as follows: all patients were diagnosed with CC, and the diagnosis was confirmed pathologically. The exclusion criteria were as follows: patients who had a history of radiotherapy and chemotherapy for treatment or a history of immune diseases or other cancers. The control participants had no history of immune disease, cervical lesions or HPV infection. The study was approved by the Ethics Committee of the Affiliated Hospital of YouJiang Medical University for Nationalities.

| Sample collection
Cervical epithelial cytology samples were collected using cytobrushes and stored in 2 ml TE buffer at −4℃ until HPV detection and genotyping once a week. Peripheral blood was collected with EDTA2+ anticoagulant and stored at −80℃ until FOXP3 gene variants genotyping. In addition, serum was separated from peripheral blood at 2500 g for 10 min and stored at −80℃ until analysis of serum FOXP3 protein levels.

| HPV DNA detection and genotyping
Genomic DNA for HPV DNA detection and genotyping was extracted from cytobrushes using DNAzol (YaNeng Bioscience) according to the manufacturer's instructions, and HPV subtypes were

| Genomic DNA extraction and FOXP3 genetic genotyping
Genomic DNA for FOXP3 variants genotyping was extracted from peripheral blood using a TIANGEN kit according to the manufacturer's guidelines. ImLDR multiple single nucleotide polymorphism genotyping technology was performed to detect rs2280883, rs2294021 and rs3761549 in the FOXP3 gene.
For rs2280883 and rs2294021, thymine (T) was replaced with cytosine (C), whereas in rs3761549, guanine (G) was replaced with adenine (A). The amplified primer sequences for

| Serum FOXP3 detection
Serum FOXP3 protein levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer's instructions. The absorbance value was measured using an enzyme-labelled metre at 450 nm (RT-6000, China), and the concentration of serum FOXP3 protein was analysed using a standard curve.

| Immune cells infiltration and prognosis in patients with CC
To reliably evaluate immune cells infiltration in CC, we used

| Statistical analysis
Pearson's chi-square (χ 2 ) test was used to analyse differences in so-

| Basic sociodemographic and clinical characteristics
In this study, 700 women (350 patients with CC and 350 healthy individuals) were recruited. The flow chart of all subject selection through each stage of the analysis was presented in Figure 1. As shown in Table 1, the mean ages of patients with CC and control there was no significant difference (p = 0.690). However, women with an age at first intercourse of <18 years (p = 0.002) and who had a history of abortion (p = 0.029) had a higher risk of CC. Persistent HR-HPV infection was the main cause of CC. Thus, the CC group was divided into HR-HPV-positive and -negative groups according to HPV infection status. We found that women with an age at first intercourse of less than 18 years (p = 0.016) and who had a history of abortion (p = 0.036) were more susceptible to persistent HR-HPV infection ( Table 2). Serum FOXP3 levels were significantly higher in the HR-HPV-positive and CC groups than in the HR-HPV-negative and control groups, respectively (p = 0.004 and p < 0.001, respectively; Figure 2).

| FOXP3 gene variants and susceptibility to HR-HPV/CC
The variants of rs2280883, rs2294021 and rs3761549 in the  Table 4).

| Haplotype analysis of the FOXP3 gene
Haplotype analysis of rs2280883, rs2294021 and rs3761549 genetic variants in FOXP3 was performed in HR-HPV and CC. There were three possible haplotypes in the HR-HPV group and six     Table 5). However, no significant association was found between haplotype and susceptibility to CC, although there was a strong tendency for the haplotype T-C-A to be associated with decreased risk of CC (p = 0.055; Table 6).

| Associations of rs2294021 and rs3761549 with clinical characteristics
The rs2294021 and rs3761549 variants were associated with the we did observe a trend of correlation between rs2294021 genotype and lymph node metastasis of CC (Tables 7 and 8).

| Evaluation of immune cells infiltration in CC
Next, differences in immune responses between the high-FOXP3 expression and low-FOXP3 expression groups were evaluated ( Figure S1). The results also showed that groups with high FOXP3 expression exhibited higher levels of immune cells infiltration. Finally, we analysed the expression of immune  As shown in Figure S2A, the expression levels of multiple immune checkpoint-related genes were significantly different between the high-and low-FOXP3 expression groups, as were the infiltration levels of resting memory CD4 T cells, memory CD4 + T cells, follicular helper T cells, Tregs, natural killer cells, M0 macrophages, monocytes, M1 macrophages, dendritic cells, mast cells and neutrophils ( Figure S2B).

| DISCUSS ION
FOXP3 was initially identified as an immune mediator involved in the differentiation and maturation of Tregs and has since been shown to play important roles in immune diseases. Furthermore, FOXP3 is involved in antitumour immune responses and the epithelialmesenchymal transition in cancer 14   with HR-HPV infection and CC risk. Recently, a case-control study revealed that the rs2280883 variants, specifically the CC genotype, was significantly associated with the risk of colorectal cancer and was correlated with the clinical characteristics of lymph node metastasis and TNM stage in a Chinese population. 19 Another study showed that patients with hepatocellular carcinoma exhibited higher  immunity. 28 One study reported that dendritic cells were positively related to prognosis in patients with glioma. 29 In addition, mature dendritic cells have been shown to be related to favourable immune infiltrates and improved prognosis in patients with ovarian carcinoma. 30 Macrophages are involved in the regulation of inflammation and the progression of tumourigenesis and are abundant in various cancers, including breast cancer, colorectal cancer and CC. 31,32 Moreover, relatively high levels of M0 macrophages are related to a high risk of relapse in digestive system cancers. 33 Mast cells act in a protumourigenic manner in most cancers 34,35 and are associated with cancer prognosis. Bioinformatics analyses have demonstrated that mast cells are positively related to overall survival in patients with glioma 29 and clear cell renal cell carcinoma. 36 Monocytes, as precursors of macrophages and dendritic cells, mainly originate from the bone marrow and account for 10% of leukocytes in human blood. 37 Studies have shown that high infiltration of macrophages in the tumour microenvironment is generally associated with a poor prognosis in cancer. [38][39][40] Neutrophils are released from the bone marrow and account for 50-70% of leukocytes, making them the most abundant immune cells in human blood. Neutrophils have been manipulated to adapt to tumour behaviour, and high infiltration of neutrophils indicates poor prognosis in the majority of cancers. 41 CD4 + and CD8 + T cells are considered positive prognostic factors in tumours, and during cancer progression, CD4 + and CD8 + T cells are suppressed by Tregs; a higher ratio of CD8 + T cells to Tregs is generally related to a favourable prognosis in cancer, whereas the reverse ratio is related to a poor prognosis. 42 In the current study, we demonstrated that mast cells, neutrophils and resting memory CD4 T cells were associated with a poor prognosis in patients CC.

TA B L E 5 Haplotype distributions and the susceptibility of HR-HPV
By contrast, memory CD4 + T cells and CD8 + T cells were protective factors in patients with CC.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.