Expression and genetic polymorphism of Foxp3 gene and its association to breast cancer susceptibility

Introduction and Aim: Breast cancer is responsible for more deaths in women than all other types of cancer combined, and it is also responsible for thirty percent of all new cancer diagnoses. FOXP3 is the key transcription factor necessary for the development of regulatory T cells (Treg), and it functions as a selection marker for regulatory T cells. The purpose of this study was to establish a correlation between genetic polymorphism and the degree to which breast cancer genes are expressed in the patients who participated in the study. Materials and Methods: This study included 40 breast cancer patients who had been histologically and clinically verified to have the disease, as well as 40 healthy subjects. Blood drawn from each participant was subjected to RNA extraction. The extracted RNA was converted to its cDNA and SNP rs3761547 genotyped. RT-PCR was used to estimate the Foxp3gene expression levels in all samples. Results: The discrepancy between the TT genotype in the control group and the patients (40% vs 12.5%, respectively) was accompanied by a low odd ratio that was statistically significant (Odds=0.21, under p-value = 0.01). Additionally, the TC genotype was more prevalent in the control group (37.5% vs 10%, respectively) than in the patient group, with a significant Odds ratio (Odds=0.19, p-value=0.008). On the other hand, the CC genotype was significantly more common in patients (77.5% vs 22.5%, respectively) than in controls (Odds=11.86). Foxp3 gene expression was significantly greater in patients (7.098) compared to the control (1.375) under level (P-value=0.001). Conclusion: There is a possibility that breast cancer is associated with variants in the SNP rs3761547, which would result in an elevated level of Foxp3 gene expression.


INTRODUCTION
reast cancer is by far and away the most frequent form of the disease, accounting for thirty percent of all cancer diagnoses (1).The rise in the number of women being diagnosed with breast cancer around the world is most noticeable in less developed nations (2).Although recent advances in cancer treatment, particularly immunotherapy, have resulted in a significant drop in the mortality rate associated with this disease in recent years (3), there is still a significant amount of variation in patients' responses to treatment due to factors such as the heterogeneity of tumors and the development of resistance to drugs (4).Forkhead box P3 (FoxP3) mRNA expression is highly characteristic of T lymphocytes (FOXP3).FOXP3 is a marker for Tregs and plays an important role in maintaining their immunosuppressive function (5).Negative regulation of the immune system by T cells, namely through the downregulation of the proliferation and activity of immunological effector cells, is crucial to the establishment of autoimmune tolerance (6).The transcription factor FOXP3 is critical for the development of Tregs and is used as a select marker for these cells (7).Reducing FOXP3 expression has been demonstrated to modify T cell immunosuppressive activity and increase the body's antineoplastic immune response capacity (6), according to previous studies.Tumors that overproduce FOXP3 are really bad news for cancer patients (7)(8)(9).The human FOXP3 gene, which has been located at p11.23-13.3 on the short arm of the X chromosome, consists of 12 exons and 11 introns (10).There are several polymorphisms in the promoter region of the FOXP3 gene that have been shown to affect gene expression.These polymorphisms either rearrange the order in which transcription factorbinding sites are located or alter the rate at which transcription takes place.For example, the functional ramifications of the variant rs3761548 were discovered to have an effect on the inhibitory function of Tregs (11).The purpose of this research was to examine the association of SNP rs3761548 with breast cancer risk and its relationship to FOXP3 gene expression in breast cancer patients.

MATERIALS AND METHODS
40 participants who were histologically confirmed to have breast cancer and had their diagnosis confirmed at B Baghdad Medical Hospital between July 2021 and March 2022 were included in this study.Another 40 healthy individuals were used as control subjects in this study.Before subjects were recruited for the study, the hospital's ethics committee approved the protocol, ensuring that it followed the guidelines for human research outlined in the revised version of the Helsinki Declaration, published in June 2021.Blood (2 ml) was withdrawn from each participant and transferred into an EDTA tube.Subsequently, 100μl of blood was transferred into a sterile 1.5 ml tube containing 200μl of DNA/RNA shield (Cat# R1100-250, Zymo, research, USA).

DNA extraction and amplification
The DNA was extracted with the use of a commercial kit (Miniprep genomic DNA, manufactured by Zymo research in the United States), and the extraction process was carried out in accordance with the instructions that were included with the kit.Applied Biosystem's pre-designed rs1050171 SNP genotyping assay (C276605710) was utilized to successfully complete the genotyping process.The amplification by PCR is done for diagnosis of different genes (12)(13)(14)(15)(16).For each sample the following components were added to a new sterilize 0.2 ml tube; 10 µl of Maxima Probe qPCR Master Mix (2X), 0.2 µl of working assay, 5 µl of eluted sample, then the volume completed to 20 µl by nuclease free water.After the completion of preparation, the tubes were sealed and placed into the thermal cycler which had programmed by the following temperatures steps; 10 min of denaturation at 95C, followed by 40 cycles of; 95C for 10 sec, 60C for 20 sec, fluorescent capturing occur during the annealing step of each cycle.

Foxp3 gene expression
Total RNA was isolated from the blood samples using a commercial kit (RNeasy Micro Kit, Qiagen).Following elution, RNA was transformed to cDNA with the use of a commercial kit (High-Capacity RNAto-cDNA™ Kit, Cat: 4387406).To perform the cDNA synthesis reaction, for each sample 10.0 µL of RT Buffer Mix, 0.1 µL RT Enzyme Mix, RNA sample up to 5 μL and finally Nuclease-free H2O up to 20 μL were added.Then the tubes were placed in a thermal cycler machine and programmed to keep the tubes at 37C for 30 minutes, after 30 min completed the cDNA were ready to use.A specific region within the Foxp3 gene were targeted by primer sequences as follow; sense, 5'-CTCATGATAGTGCC TGTGTCCTCAA-3' and the anti-sense; 5'-AGGGCCAGCATAGGTGCAAG-3' (17).The housekeeping gene GAPDH was amplified using the primer sequences; sense; 5'-AATGGGCAGCCGT TAGGAAA-3 and anti-sense; 5'-GCGCCCAATA CGACCAAATC-3' (18).The QRT-PCR reaction mixture was done by including (10 μL) of KAPA SYBR green FAST qPCR Universal, (0.4 μL) of each primer, and 4 μL of prepared cDNA, then the volume completed to 20 by Nuclease-free water.The tube is placed in the real time-thermal cycler which has been programmed as 5 min at 95°C then 40 cycles of 30 sec for each step 95°C, 60°C and 72°C.

Statistical analysis
SPSS version 24.0 was used for all statistical testing.Important associations between clinic pathology data and marker categories were analyzed using the chisquare test (or Fisher's exact test, for smaller samples).For continuous variables, the parametric Student's t test was used.The SNP genotypes frequency and Odds ratio was calculated.

RESULTS
Table 1 provides a comparison of the participants' mean ages and body mass indices (BMI) between the sick group and the control group.Both groups were included in the study.As can be observed from the table, the participants in the patient group had a much lower mean age than those in the control group; however, this difference did not reach statistical significance.Like the previous finding, the difference in BMI between the two groups was not statistically significant.The participants in the patient and control group were divided further based on their blood group and family history of breast cancer which is summarized in Table 2. Most breast cancer patients (14/40) belonged to the O+ blood group and had no family history of the cancer (Table 2).The distribution pattern of the study subjects based on blood group shows a non-significant difference (p value=0.554) between the two groups.However, the distribution of participants based on family history showed a significant difference for participants in patient and control groups (p-value= 0.05).
The breast cancer patients in this study were further categorized based on their tumor stage, lymph node spread and metastasis (Table 3).The results revealed that tumor stage T-II (22.5%) accounted for the highest number of patients, followed by stages T-III and T-IV (20.0%), stage T0 (17.5%) and stage T1 (15%).Stage Tx, which had the lowest frequency was discovered in only 5% of the patients.Based on the spread to the lymph nodes, the distribution of patients was seen to be highest in N4 (32.5%), followed by N2 (27.5%),N3 (25%), and the lowest percentage seen in N1 and N0 (Table 3).In addition, metastasis stage 1(M1) was recorded in 60% of the breast cancer patients.The SNP rs3761547 has been detected by using real time-PCR and to detect gene expression levels of Foxp3 gene.The QRT-PCR results for the SNP (rs3761547) and Foxp3 gene amplification are shown in Fig1.Each single curve represents single-sample amplification.
To test the amplification specificity, a melting curve analysis was performed, and the result shown in Fig. 2. The presence of a single peak indicates high specific results for the genes in QRT-PCR.
The frequencies of the rs3761547 SNP's various genotypes are shown in Table 4, which can be found here.According to the data, the TT genotype is found in the control group at a considerably higher frequency than it is in the ill group (Odds = 0.21, P-value = 0.01).
The frequency of the TC genotype was considerably higher in the control group than it was in the ill group, as shown by an Odds value of 0.19 and a p-value of 0.008 respectively (37.5 percent VS 10 percent, respectively).On the other hand, patients had a considerably higher frequency of the CC genotype than did the controls (77.5 %vs.22.5 %, Odds = 11.86,P0.001).

Fig. 3. Comparison of Foxp3gene expression level in patient and control group
Our study revealed a significant increase in Foxp3 gene expression levels in patients (7.058) in comparison to the control (1.375) group (Fig. 3).
The Foxp3 gene expression level was also studied within each genotype of the SNP (rs3761547) and the results are presented in Fig. 4.Among genotypes, a significant elevated level of the Foxp3 gene expression was observed among patients with the CC genotype (7.82), followed by patients with TC genotype (7.157).
Patients with the TT genotype expressed the lowest Foxp3 expression (Fig. 4).Similarly, in the control group the Foxp3 gene was marginally elevated in individuals with the genotype CC (1.95), followed by TC (1.58) and TT (0.84) (Fig. 4).

Fig. 1 :Fig. 2 :
Fig. 1: A; FAM channel detecting T allele, B; HEX targeting to detect the c allele, C; amplification of GAPDH (reference gene), D; amplification of the target FOXp3gene

Table 1 :
The mean age and BMI of participants in the patient and control group

Table 2 :
Distribution of participants based on blood groups and family history

Table 3 :
Distribution of breast cancer patients based on tumor, lymph node and metastasis

Table 4 :
Frequency of the three genotypes associated withSNP rs3761547in breast cancer patients and controls