Introduction

Breast cancer is the most common type of cancer and is responsible for the deaths of most women in the world [1]. Recent studies have shown that functional polymorphisms in lncRNA, as a non-coding RNA, may be associated with a higher risk of various cancers [10, 11]. Genetic variants at miRNA target sites within lncRNAs can also be associated with cancer risk [11, 12]. Based on studies of five functional polymorphisms in the lincRNA-NR_024015 exon, it has been shown that rs8506 polymorphism in this lncRNA is associated with the risk of gastric cancer, and C-to-T transition within this region destroys the miR-526b binding site and increases the risk of gastric cancer [16]. In another study, the effect of rs8506 polymorphism in increasing the risk of esophageal squamous cell carcinoma was reported [17]. However, no study has been done on its association with breast cancer. In this regard, this study aimed to examine the role of rs8506G polymorphism in developing sporadic breast cancer.

Materials and Methods

In this case-control study, blood samples of people with breast cancer (n=120) and healthy peers (n=120) were used. A written consent was obtained from the participants and their demographic and clinical data were recorded by a questionnaire. Genomic DNA was extracted using the phenol-chloroform method and its quality and quantity were investigated. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the ‍genotype of blood samples. To determine the PCR product genotype, it was affected by the BanII enzyme for cutting; if there is no nucleotide change in the position of study variant, there will be a cutting site for the enzyme, and two bands of 108 and 311 bp will be observed on the gel, but if there is a change in the sequencing, there will be no cutting position and the 419 bp band will be observed (Figure 1).

The allele frequency of polymorphism for Hardy-Weinberg equilibrium was performed using the online statistical database and their validation [19], and chi-square test was used to examine the difference in the allele and genotype frequencies between patients and healthy peers. ANOVA test was used to calculate the risk and then the relationship between the various factors and genotypes. All statistical test were conducted in SPSS V. 20 software by considering the significance level of P<0.05.

Results

The studied samples had sporadic cancer and the age of the patients ranged from 26 to 77 years (Mean±SD=51.8±11 years) (Table 1).

The frequency of heterozygous (CT) genotype was higher in cancer patients than in healthy individuals (36% vs. 24%). For homozygous (TT), it was two-fold higher in patients than in healthy peers. In the dominant genetic model, there was a significant association between the polymorphism and breast cancer in the presence of two TT and CT genotypes in the population (P=0.027). Examination of other models also revealed that the presence of TT genotype in the recessive model doubled the risk of disease (OR=2.01). In the presence of T allele genotype n both co-dominant and dominant models, the risk of disease was observed to be 2.43 and 1.84, respectively. Given the calculated risk, allele change increases the risk of disease, and this change was statistically significant (P=0.031). All of these results confirm the role of rs8506G polymorphism in the susceptibility to breast cancer. In examining the relationship between clinopathological factors and disease risk, no significant relationship was observed between this polymorphism and those factors. Table 2 shows the study of clinopathological factors in the dominant genetic model.

Discussion

The expression of target genes is regulated by miRNAs, and any change in miRNA binding site affects gene expression [20]. The present study found that a nucleotide change in lincRNA-NR_024015 exon, which destroys the miR-526b binding site and subsequently removes the inhibitory effect of miRNA, increases the risk of breast cancer. There is ample evidence that lncRNAs are important in carcinogenesis, including breast cancer. HOTAIR rs920778 polymorphism, as a lncRNA, is associated with increased expression of this factor and the susceptibility to breast cancer. Therefore, genetic alteration of the lncRNA sequence increases malignancy and invasion of breast cancer cells [24]. 

Hahn et al. in a study on the effect of rs8506 polymorphism in the lincRNA-NR_024015 exon on esophageal squamous cell carcinoma found that the presence of this polymorphism disrupted the miR-526b binding site. This is accompanied by an increase in expression in the lincRNA-NR_024015 exon followed by an increase in malignancy [17]. Given that miR-526b expression is reduced in various cancers [25], the presence of such variant inhibits its binding to the lncRNA, thus increasing the risk of tumorigenesis and cancer progression for individuals with the TT genotype. It was found that in cancer patients, the expression level of lincRNA-NR_024015 increases and this mechanism (the effect of genetic variant in sequencing), intensifies the increase in expression.

Conclusion

The presence of a genetic variant in lincRNA-NR_024015 can be a risk factor for breast cancer; the allele C to T change in the lincRNA-NR_024015 disrupts the miR-526b binding site in this sequence and thus, by eliminating the inhibitory effect, increases the expression of this lncRNA.

Ethical Considerations

Compliance with ethical guidelines

This study was obtained ethical approval from the Research Ethics Committee of Shahrekord University of Medical Sciences (Code: 91-0215). All ethical principles were considered in this study including obtaining informed consent from participants, confidentiality of their information, and explaining study process to them. 

Funding

The present paper was extracted from the MSc. thesis first author, Department of Genetics, Faculty of Basic Sciences, Shahrekord University.

Authors' contributions

Experiments and initial draft preparation: Fatemeh Tavakoli; Data analysis, editing and review: Somayeh Reiisi

Conflicts of interest

The authors declare no conflict of interest

Acknowledgements

This study was extracted from the master thesis of first author. The authors would like to thank the Deputy for Research of Shahrekord University of Medical Sciences for their financial support.