Cancer is a category of diseases that cause the body's cells to change and proliferate uncontrollably. Ultimately, most types of malignant cells form a mass known as a tumor, which is named for the area of the body where it first appears. The majority of breast cancers start in the ducts that connect the milk glands to the nipples or in the tissue of the breast that contains lobules, which are glands that produce milk. Sebaceous, lymphatic, and connective tissues make up the remainder of the breast. The breast tumor has become a main intimidation to woman health in Iraq, where it is the most crucial reason for mortality after cardiovascular sickness among females, with a tumor-related death rate of 23%. It has been thegreatest-categorized tumors among the Iraqi population since 1986. The latest Iraqi Cancer Registry in 2015 exposed that among an assessed population size of 32,500,000, a total of 21,101 new patients of sarcomawere recorded in 2012; 9,268 were in males and 11,833 were females [1]. The CXCR4, chemokine receptor, is an important element in themigration, invasiveness, metastasis and proliferation of tumor cells of breast [2], and is the most commonly produced on tumor cellsand has been noticed in 23 different types of malignancy. CXCR4 signaling has been associated with invasiveness and the developing of metastasis, withcells manifesting the receptor targeting to tissues producing SDF-1(stromacell-deriving factor-1) [3]. In 1996 SDF-1, later named CXCL12, was recognized as a CXCR4 ligand [4-6]. Previous studies have illustrated that CXCR4 and CXCL12 geneticpolymorphisms, as well as their levels of expressionhave been correlatedwith the development and prognosis of tumor. The current study intends toexamine the impact of CXCR4 rs2228014 and CXCL12 rs1801157 SNPs in breast cancer and control samples and their serum levels amongpostmenopausal woman and body mass index (BMI)

1. Patients and Controls

Two groups of subjects were enrolled in the present study during the period August 2017 - October 2018. The first included 60 cases of Breast cancer with an age range of 20-75 years. These cases were ascertained through Oncology Center at Al Medicine City in Baghdad province and Middle Euphrates Cancer in Najaf Province, in which the diagnosis made. For the purpose of comparison 61 cases age and gender matched were enrolled as a control group of healthy individuals.

1. DNA Extraction

The isolation of DNA (genomic) from the whole frozen blood that saved in EDTA tubes having anticoagulant, was implemented through using the DNA Isolation Kit (Genekam Universal). The extraction process of DNA as achieved according to four major steps:

• WBCs and their nuclei were lyze by using lysing buffer.

• The removing of cellular proteins was obtained by a salt out precipitation step using proteinase K and absolute ethanol.

• The concentration of genomic DNA was carried out and desalting process was achieved by W1 buffer and wash buffer (ethanol added).

• The elution buffer or TE buffer was used to rehydrate the genomic DNA

2.3 Agarose Gel Electrophoresis

The electrophoresis of Agarose gel was implemented to confirm the existence and integrity of the isolated DNA [7].

2.3.1 Reagents for the Electrophoresis process of Gel

• The powder of agarose

• The TBE Buffer 1X

• The loading buffer which is Bromophenol Blue in 1 % glycerol(loading buffer)

• The intercalating agent which is the Ethidium Bromide (EB)

 2.3.2 Agarose Gel Preparation

1. The adding of the agarose powder (0.5 gm) to 50 ml TBE buffer (1X)

was obtained.

2. The mixture was warmed to the boiling temperature using the microwave till the dissolving of all particles of gel.

3. The mixing of the agarose by stirring was achieved and this process was done also to avoid bubbles formation.

4. The cooling down of solution was fulfilled at 50 – 60 ºC.

5. Final step was to add the EB (0.5 μl) of (10mg/ml) to the agarose mix.

2.3. 4 The Loading of DNA and the process of Electrophoresis

The DNA (9 μl) was blended with (3 μl) of bromophenol blue dye (loading dye) and loaded cautiously into the single wells of the gel. Then electrical current was switched on at 70 volts for 30minutes, subsequently the DNA progressed from the cathode (-) to the anode (+) poles. The dye, Ethidium bromide, marked the bands in the gel and they were envisaged by UV transiluminator at 350 nm and the photos were taken

2.4 Estimation of DNA Concentration (Nanodrop)

The DNA samples that presented an acceptable completeness were diluted to 1:10 or 1:20; afterwards the reading of the optical density was obtained by the spectrophotometer at wave length of 260 nm. The calculations of the concentration of the DNA in μg /ml of samples were implemented according to the next equation: The OD.260 nm x the factor of dilution x 50 μg /ml = ( ) μg /ml

To do the measuring of the DNA purity, reading was taken at wave length 280 nm as: The purity of DNA = A 260 / A 280 = ~ 2.

2.5 Primers Selection

The primers employed to increase the CXCL12 rs1801157 coding gene and the CXCR4 rs2228014 gene are shown in table 2-1

Table (2-1): Primers sequences [8]

2.6 Polymerase Chain Reaction (PCR)

2.6.1 PCR Components

The compositions of the reaction of PCR and mixture amounts are intable (2-7).

Table (2-2): The components of PCR

2.6.2 PCR Programs

The optimizing of PCR reaction was achieved after numerous trials, therefore the following programs were adopted. The reaction of PCR was obtained as shown in tables (2-3) and (2-4).

Table (2-3): PCR Programs to amplify CXCL12gene

Table (2-4): PCR Programmers to amplify CXCR4 gene

2.7 Purification of PCR Product for RFLP analysis

The purifications for the productions of the PCR were achieved through RFLP analysis throughout the utilizing of an EZ-10 spinning columns of the kit for DNA cleanup mini-preps. This kit is used to DNA cleanup from the reactions caused by the enzymes and to remove the nucleotides and primers (>40-mer).

2.8 PCR Products Sequencing

The remaining (40 μl ) of the purified PCR product for 200 samples of both CXCL12 and CXCR4 genes and primers sent to NICEMCompany ( South Korea) for sequencing. NICME is a highly focused healthcare and biotechnology company as in:  http://eng.nice.co.kr/main.nice 

2.9 Data Analysis

Data files were analyzed by Applied Biosysteems’ the software program named as Sequence Scanner MEGA X on the website: www.megasoftware.net to do alignment to every exon. Sequenced data was lined up and compared with genomic data of the editor of alignment and sequence analyzing using NCBI website for confirmation as well as on BioEdit. The Genotypes for both genes (CXCL12 rs1801157 and CXCR4 rs2228014) allele frequency was calculated as: (1 (h12H))/2N, where h acts for the heterozygous genotype, while H is representing the homozygous genotype, and N the size of the sample for every population. SNP allele’s incidences were examined by using Hardy–Weinberg Equilibrium through the linking of the observed with the anticipated genotypic incidences using a w2 test. The data of the genotype were inspected by the w2 test with the significantly level set at P<0.05. For the purpose of the multiple nonindependent statistical tests accounting, Fisher’s exact test was used through WinPepi Compare 3.85 for all clusters and genotypic collections simultaneously with the significant level set at P<0.05

3.1 Molecular Identification of CXCR4 and CXCL12 Breast cancer women

The molecular part of this study focused on the analysis of extracted DNA using specific primers PCR, purification and digestion of CXCL12 and CXCR4 by MspI and BccI restriction enzyme through RFLP technique to determine the fragments. Then the genotype sequence was obtained to confirm the findings.

3.1.1 DNA Extraction

The DNA genome which was obtained from blood of Breast cancer and control women through extraction process showed good band in electrophoresis gel (Fig. 3-1). 

Figure 3-1: DNA bands obtained from blood samples of breast tumor and control groups upon 1% agarose at 70 voltages for 30 minutes, visualized under U.V light after staining with ethidium bromide.

3.1.2 Polymerase Chain Reaction (PCR)

The PCR technique was determined using specific primers to amplify some regions of the CXCL12 and CXCR4 genes that include specific SNPs that could have a responsibility in pathogenesis in breast cancer. The PCR findings exposed that identical bands related to the CXCL12 and CXCR4 genes were present in all samples (patient and control). The amplified regions of CXCL12 and CXCR4 genes showed a molecular weight of 209 bp and 309 bp respectively (Figures 3-2 and 3-3).

Fig. 3-2: PCR products upon 2% of the gel of agarose at 70 V. for one hour/ Amplification of CXCL12 gene for patient and control group.

Fig. 3-3: PCR products on 2% of the gel of agarose at 70 V. for one hour/ Amplification of CXCR4 gene for patient and control group

3.2. CXCL12 rs1801157 PCR–RFLP

Genotyping of CXCL12 gene for single nucleotide polymorphism (SNP) of rs1801157 analyzed by PCR-RFLP. The products of PCR were break downed by MspI (HpaII) restriction enzyme. The MspI restriction enzyme was used to identify the sequence CCGG. The results should have Homozygous G/G producing dual bands: at 92 bp and 117 bp, the G/A heterozygous G/A should produce three bands of 92 bp, 117 bp and 209 bp, while the homozygous genotype should only yield 209 bp band, as this allelic type is not digested. The result of PCR-RFLP illustrate that only the homozygous A/A state was presented. All samples produce 209 bp band after using MspI enzyme and this indicated that the enzyme wasn't effectively affecting.

Fig. 3-4: PCR–RFLP products upon 2% the gel of agarose at 70 V. for one hour/Amplification of CXCL12 gene for patient and control group.

3.3 CXCR4 rs2228014 PCR–RFLP

The performance of PCR-RFLP applied to analysis the genotyping of CXCR4 gene for rs2228014 SNP. BccI as restriction enzyme was utilized to digest the PCR products. The BccI restriction enzyme was applied to identify the CCATCN sequence. The results supposed to be producing Homozygous C/C would give double bands at 204 and105 bp, the C/T heterozygous genotype would yield three bands of 309, 105, and 204 bp, while the T/T state would produce only band at 309 bp, as it is not digested. The result of PCR-RFLP illustrate that the homozygous genotype T/T only was presented. All samples produced 309 bp band after using BccI enzyme and this indicated that the enzyme wasn't working correctly.

Fig. 3-5: PCR–RFLP products upon 2% the gel of agarose at 70 V. for one hour/Amplification of CXCR4 gene for patient and control group

These findings are represented negative results of utilizing RFLP, which may be contributed to the restriction enzymes efficiency that might became inactive or denatured due to improper storing or handing processes from outside the country.

3.4 Genotype Sequencing

The regions of the CXCL12 and CXCR4 genes were screened by direct sequencing for 200 samples (100 samples for CXCL12 and 100 samples for CXCR4). The sequence of the samples was done by NICEM Company, South Korea. All results (200 samples) were directly compared with human reference CXCR4 and CXCL12 sequence compared with data in NCBI-NIH (Blast), available in http://www.ncbi.nlm. The comparison between subjects and reference sequence were done by BioEdit program and amino acids translations were determined on https://web.expasy.org/translate/

3.4.1 Genotypic Analysis (GG, AA and AG) and Allelic (G/A) allocation of rs1801157

The homozygous AA genotype frequency was 6 (10.0%) in patients treatment and 3 (7.5%) in control treatment; while the incidence of heterozygous AG in patients treatment was 26 (43.3%) and 20 (50.0%) for healthy group. Meanwhile, homozygous GG genotype frequency tended to be non-significant increase in patients’ treatment 28 (46.6%) as compared to controls 17 (42.50%). Likewise, the incidence of allele G was tend to be nonsignificant in patients treatment (OR 1.04) with Etiological Fraction (EF: 0.02) and Prevention Fraction (PF: 0.3). It can be put in consideration that breast cancer G allele has 95% Confidence Interval (C.I: 0.57-1.9) as shown in table (3-1) and fig. (3-6).

Table (3-1): Observed and expected genotype and allele frequencies of CXCL12

(rs1801157) in breast cancer patients and controls

Fig 3-6: NCBI Reference SNP of rs-1801157

As reviewing of table (3-8) it is obvious that the frequency of genotypic for co-dominant pattern of A/G was detect to be non- significant (OR: 0.7; 95% CI [0.35-1.69]). The G/G genotypes were a little more predominant in breast tumor group than control group. That can be obvious where the incidence of G/G was higher 82 (68.3%) in patients than the control group where it was 38 (31.6%). Whereas the recessive AA genotype showed nonsignificant correlations.

                Studies have explored that the expression of elevated level of CXCL12 run the breast tumor to metastasis [9,10]. Few reports presented that SNP rs1801157 may be a prognostication indicator of breast tumor lymphocytic metastatic cell [11,12]

[13] reported that the study’s data indicate that the CXCL12 rs1801157 GA polymorphism may affect the development of chronic lymphocytic leukemia, disease progression as well as response to treatment.

[14] conducted a meta-analysis and suggested that the polymorphism of rs1801157 CXCL12 coding gene did not considerably confer the Colorectal Cancer (CRC) risk among Caucasians while it might have this conferring among other populations.

In the present study, incidence of SNP rs1801157 was researched among (60) breast cancer patients and (40) healthy females of Iraqi parents. The allelic analyzing of distribution displayed non-significant difference among the breast tumor treatment and control group. These are supported by former meta-analysis investigated at CXCL12 rs1801157 and the breast tumor risk and concluded a positive association [15]

The findings suggested that allele G is perhaps associated with the breast tumor risk, which came in accordance with the study of [16] where it proposed that genotype GG is probably increasing the breast cancer risk in Pakistani cohorts. This is dissimilar to many of reports shown that allele A to be a risk element of breast tumor [17]

Further researches have illustrated that CXCL12 genetic polymorphisms participating different environmental elements in the growth of tumor pathogenesis. Chemokines are well-known of promoting inflammation and play a task in the cancer development through altering the tumor microsurrounding and metastasis to additional body parts such as brain, lung, liver or bones [17-20] 

polymorphism has significant associations with breast tumor, cancer of urinary tract, hematologic malignancy and lung carcinoma as predispositions were found in various genetic patterns.

Studies have exposed that the homozygous AA genotype of CXCL12 rs1801157 was associated with the elevated levels of the expression of protein, a higher risk of tumor, and poorer clinical consequence in many malignancies [21]

Studies implied that there was no meaningful dissimilarities about the distribution of CXCL12 genotypes: GG, AA, and AG in patients with the bladder transitional cell carcinoma and healthy people [22,23]

discovered that the incidence of breast tumor is correlated with the mutation of gene and the low CXCL12 rs1801157  expression. Also, the study of that applied PCR-RFLP analysis technique in the Polish ethnicity explained that polymorphisms of

CXCR4/CXCL12 may be linked to the occurrence and development of Inflammatory Bowel Disease and its activation may be the defense response of chronic persistent inflammation. 

1. Breast Cancer and CXCR4/CXCL12 Genes
   The study highlights the role of CXCR4 and CXCL12 gene polymorphisms in breast cancer development. The chemokine receptor CXCR4 and its ligand CXCL12 are critical in cancer cell migration, metastasis, and proliferation, making them potential biomarkers for breast cancer risk and progression.

2. Genetic Polymorphisms and Breast Cancer Risk

• The rs1801157 (CXCL12) polymorphism showed a non-significant difference in genotype and allele distribution between breast cancer patients and the control group. However, some previous studies suggest that allele G may be associated with an increased risk of breast cancer.

• The rs2228014 (CXCR4) polymorphism was also analyzed, but PCR-RFLP results were inconclusive due to potential inefficiencies in restriction enzyme activity.

• PCR and sequencing techniques successfully amplified and analyzed the CXCL12 and CXCR4 genes. However, the study faced limitations due to enzyme inefficiency in the RFLP analysis, which may have affected the accuracy of genotype identification.

• Direct sequencing provided more reliable results, confirming the presence of specific genetic variations in both patient and control groups.

• While no significant association was found between CXCL12 rs1801157 and breast cancer risk in this Iraqi cohort, previous studies suggest a potential role in other populations.

• CXCR4 and CXCL12 may still serve as potential targets for breast cancer prognosis and therapy, emphasizing the need for further studies on larger and more diverse populations.

• Future studies should include larger sample sizes and more precise genotyping techniques to confirm the findings.

• Functional studies are needed to assess the impact of CXCR4/CXCL12 polymorphisms on breast cancer progression and metastasis.

• Investigating additional genetic and environmental factors contributing to breast cancer risk will provide a more comprehensive understanding of disease mechanisms.

1. Molecular Techniques and Study Limitations

2. Clinical Implications

3. Future Recommendations

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the Kut University College, Iraq.

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