The Association of pre-miR27a Gene Polymorphism and Clinicopathological Data in Thai Breast Cancer Patients

Background: MiR27a plays an important role in carcinogenesis, cell proliferation, apoptosis, invasion, migration and angiogenesis. Several studies have identified an important role of pre-miR27a (rs895819) A>G polymorphism in several types of cancer. This research aims to investigate the association of pre-miR27a (rs895819) A>G and breast cancer susceptibility, clinicopathological data and survival. Blood DNA samples of 143 Thai breast cancer patients and 100 healthy Thai women were studied for pre-miR27a (rs895819) A>G polymorphism using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). Results: The results revealed that the frequency of pre-miR27a (rs895819) A>G genotypes was not statistically significant different between breast cancer patient and normal control subjects. The rs895819 A>G genotype was significantly associated with clinicopathological parameter of grade III differentiation (P = 0.006), progesterone receptor (P = 0.011) and triple negative (P = 0.031) in breast cancer patients, but not with breast cancer susceptibility. Conclusion: The pre-miR27a (rs895819) A>G genotype was significantly associated with poorly differentiated, progesterone receptor and triple-negative in breast cancer patients. Therefore, pre-miR27a (rs895819) A>G may be used as a biomarker for poor prognosis.


Introduction
Breast cancer is the most common cancer of women worldwide. In Thailand, the tumor in 40.3% of breast cancer patients spread to lymph node and 23.4% of cases have distant metastasis (Hospital based cancer registry, Medical record and database cancer unit, Medical Digital Division, National cancer Institute, Thailand, 2020). The etiology of breast cancer derived from environmental factors and genetic susceptibility. Researches on candidate genes have been investigated in order to understand breast cancer pathogenesis, with an emerging interest in epigenetics and gene regulation.
MicroRNAs (MiRNAs) are one of the most important mechanism of gene regulation. MiRNAs are non-coding single stranded RNA of 22 nucleotides that regulate gene expression by blocking translation of mRNA. MiRNAs play an important role in many molecular pathway and biological process including cell proliferation, cell cycle control, apoptosis, cell differentiation and metabolism (Jansson and Lund, 2012). A single miRNA can target hundreds of mRNA and about 50% of miRNA genes

RESEARCH ARTICLE
The Association of pre-miR27a Gene Polymorphism and Clinicopathological Data in Thai Breast Cancer Patients are located in cancer related chromosomal region. MiRNA may participate in carcinogenesis, progression and prognosis of human cancer. MiRNA polymorphism has been reported to affect miRNA processing and miRNA-mRNA interaction. Aberrant expression of miRNA may affect the etiology, diagnosis and prognosis of cancer (Ma et al., 2013). Single nucleotide polymorphism (SNPs) in gene coding for pre-miRNA can be used as biomarkers for breast cancer susceptibility and prognosis .
There is evidence that miR27a plays an important role in carcinogenesis, cell proliferation, apoptosis, invasion, migration and angiogenesis. Moreover, miR27a has clinical significance in drug sensitivity, treatment and prognosis of cancer (Li et al., 2019). Recently, many studies have identified an important role of pre-miR27a (rs895819) A>G polymorphism in several types of cancer. In Thailand, most of the breast cancer patients are diagnosed in late stage and the disease progression into the stage of poor prognosis. Therefore, the purpose of this research is to investigate the association of pre-miR27a (rs895819) A>G and breast cancer susceptibility, prognostic factors and survival.

Specimen
The retrospective blood samples comprised of 143 breast cancer patients and 100 normal individuals were collected from National Cancer Institute, Thailand. All cases had Thai ethnicity with age ranged from 26 -77 years (Mean ± SD) (51.43 ± 10.56). Clinicopathological data of breast cancer cases included histological grading, tumor size, tumor staging, lymph node involvement, distant metastasis. Immunohistochemistry result of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and triple negative status of ER-negative, PR-negative and HER2-negative were collected from pathology laboratory of National Cancer Institute of Thailand. The study was approved by the Ethics committee of the National Cancer Institute and inform consent was obtained from all the participants (Code number 031_2020RB_OUT67).

DNA extraction.
Genomic DNA in 100 healthy controls and 143 breast cancer patients was isolated from EDTA blood using High Pure PCR Template Preparation kit (Roche Molecular Diagnostics, Mannheim, Germany). The DNA purity and concentration was determined by using spectrophotometer measurement of absorbance at 260 and 280 nm. The concentration of DNA was 50 ng in total volume PCR reaction of 25 μL. The purity of DNA calculated by absorbance of 260/280 ratio is 1.8-2.0

Genotyping of pre-miR27a (rs895819) using PCR-RFLP
Genotyping of pre-miR27a (rs895819) was investigated in genomic DNA using polymerase chain restriction length polymorphism (PCR-RFLP) as described previously (Sun et al., 2010). The 182-bp DNA fragment containing the polymorphic site was amplified using 5´-GAA CTT AGC CAC TGT GAA CAC CAC TTG G-3´, and 5´-TTG CTT CCT GTC ACA AAT CAC ATT G-3´ as forward and reverse primer, respectively. The PCR cycling conditions were the initial denaturation of 95 o C for 5 min followed by 35 cycles of 95 0 C for 35 sec, annealing at 60 0 C for 30 sec, and 72 0 C for 30 sec, with a final elongation at 72 0 C for 6 min. The PCR products were identified on electrophoresis on 2% agarose gel. The PCR product sizes, after restriction digested with DraII (New England Bio-Labs, USA) at 37 0 C overnight, were assessed on electrophoresis on 2.5% agarose gel and stained with NEOgreen (GELLGENTEK Co., Ltd. Korea). The restriction fragments included a single 182 bp fragment for the GG genotype which is homozygous polymorphism, two fragments of 155 and 27 bp for the AA genotype which is wild type; and three fragments of 182, 155 and 27 bp for the AG genotype which is heterozygous polymorphism.
The quality control of genotyping was done by DNA sequencing in 10% of cases and the result of sequencing is the same as the result of PCR-RFLP.

Statistical analysis
The statistics were based on two tailed probabilities and a p-value of <0.05 was considered statistically significant. The differences of age between 143 breast cancer patients and 100 control subjects were assessed by Student's T-test. The allele frequencies of pre-miR27a (rs895819) genotypes were calculated from Hardy-Weinberg equilibrium. The association between pre-miR27a (rs895819) genotypes and risk of breast cancer susceptibility was assessed by Pearson Chi-square and odds ratios (OR) with 95% confidence intervals (CI). Carriers of the genotype AA comprised of the reference group. The association between pre-miR27a (rs895819) genotype frequencies and clinicopathological data in breast cancer cases was evaluated by Pearson Chi-square test. The association between pre-miR27a (rs895819) genotype frequencies and overall survival was investigated by Kaplan-Meir method and log rank test. The Cox regression method was utilized to assess the prognostic effect for pre-miR27a (rs895819) A>G on breast cancer patient survival. The P <0.05 was considered a significant association.

Results
Pre-miR27a (rs895819) genotypes were analyzed by restriction digestion with DraII enzymes in 143 breast cancer cases and 100 normal controls. The age range of all cases were from 26 to 77 years age (Median age, 52 years). There was no statistically significant difference with mean age between breast cancer patients (51.43+4.56) and normal control. (51.39+4.55), (P = 0.975).

Association of pre-miR27a (rs895819) genotypes and clinicopathological data
The association of pre-miR27a (rs895819) genotypes and clinicopathological data of 143 cases of breast cancer cases was assessed by Pearson Chi-square. It was found that miR27a (rs895819) variant genotype (AG and GG genotype) was significantly associated with poorly differentiated breast cancer (Grade III), (P = 0.006). Moreover, miR27a (rs895819) polymorphism (AG and GG genotype) was statistically associated with PR positive (P = 0.011) and triple negative breast cancer cases (P = 0.031) ( Table 2).

Survival of breast cancer patients
Overall survival analysis was determined by Kaplan-Meir survival curve and Log rank test which was used to compare the survival time between pre-miR27a (rs895819) variant genotype (AG and GG genotype) and AA genotype of breast cancer patients.
It was found that there was no statistically significant difference of overall survival time in pre-miR27a (rs895819) variant genotype (AG and GG genotype) and AA genotype of breast cancer patients, (P= 0.942) as shown in Figure 1. Furthermore, the multivariate Cox regression method of prognostic marker for survival of breast cancer patients revealed that pre-miR27a (rs895819) A>G genotype was not an independent prognostic factor

Discussion
MicroRNAs are small non-coding RNA, which play an important role in controlling cancer development by regulating target genes at post transcriptional level. (Bartel, 2004). MicroRNA27 family composes of miR27a and miR27b. MicroRNA27a play an important role in tumor development by controlling genes involved in cell proliferation, apoptosis, differentiation, cell cycle regulation and chemotherapeutic resistance. MiR27a has been first found in breast cancer as an oncogenic function because high miR27a expression increased the percentage of cells in G2/M stage. (Mertens et al., 2007). MiR27a has rendered not only oncogenic function in breast cancer, lung cancer, hepatocellular carcinoma, prostate cancer, but also acts as tumor suppressor gene in gastric cancer, bladder cancer and esophageal squamous cell carcinoma (Li et al., 2019). Moreover, miR27a regulates the tumor immune response and epithelial mesenchymal transition (Zhang et al., 2019).
The presence of SNPs in the miR gene may involve in its expression process in cancers. There are two sites of polymorphism at pre-miR27a region of miR27a gene including rs895819 and rs11671784 located at positions 40 and 36 related to the first nucleotide (Gong et al., 2015). The pre-miR27a (rs895819) A>G polymorphism has been first identified. This mutant genotype increased the production of mature miR27a (Li et al., 2019). It was reported that pre-miR27a (rs895819) polymorphism is significantly associated with increased risk of lung cancer susceptibility but not in gastric cancer and esophageal cancer (Chen et al., 2017, Shankaran et al., 2020. The inconsistent results on rs895819 polymorphism in colorectal cancer have been reported (Chen et al., 2017, Zhang et al., 2020, Shankaran et al., 2020. The present case-control study found that pre-miR27a (rs895819) AG and GG genotype was not associated with breast cancer susceptibility. (P = 0.701). This present finding concurred with the study of Liu et al., 2021, who investigated the meta-analysis on the association of pre-miR27a (rs895819) polymorphism and breast cancer susceptibility. The results showed that no significant association between pre-miR27a (rs895819) polymorphism and breast cancer susceptibility in Asian or Chinese subpopulations. Nevertheless, they found that among Caucasians, the wild-type AA genotype at rs895819 may confer increased susceptibility to breast cancer, while the G-allele and AG genotype may be the protective factors. A study by Zhang et al. demonstrated that the G-allele exhibited decreased in risk of breast cancer in the younger Chinese women ).
Inconsistent results on the possible relationship between pre-miR27a (rs895819) polymorphism and clinicopathological parameters have been reported in Chinese population. One report showed that no significant association was observed between pre-miR27a (rs895819) polymorphism and clinicopathological parameters, including pathologic grade, TNM stage, estrogen and progesterone receptors, HER-2 status (Qi et al, 2015). In contrast with findings of another study done by Zhang et al. found significant association between rs895819 polymorphism and histological grade and estrogen status in older age group with moderately associated with progesterone status . In this study, we found that pre-miR27a (rs895819)A>G is statistically significant associated with poorly differentiation (P =0.006), progesterone receptor (P= 0.011) and triple negative cases (P = 0.031) (Table2). Consistent with the prognostic indicator for breast cancer observed by Zhang et al. , our result also indicated that rs895819 polymorphisms was not associated with overall survival of breast cancer cases. As positive for triple hormone receptor negative status and low grade tumor were both associated with rs895819 polymorphisms, therefore, pre-miR27a rs895819 A>G may be served as a candidate biomarker of poor prognosis for breast cancer in Thai population.

Author Contribution Statement
All authors contributed equally in this study. OR P-value