miR-608 rs4919510 C>G polymorphism decreased the risk of breast cancer in an Iranian subpopulation

Hashemi M.*1, Sanaei S.2, Rezaei M.2, Bahari G.2, Hashemi S.M.3, Mashhadi M.A.3, Taheri M.4, Ghavami S.5

Summary. Aim: MicroRNAs (miRNAs) are small noncoding RNAs that function as oncogene or tumor suppressors. The single nucleotide polymorphisms in miRNAs potentially can alter miRNA-binding sites on target genes as well as affecting miRNAs expression. The present study aimed to evaluate the impact of miR-608 rs4919510 C>G variant on breast cancer (BC) risk. Materials and Methods: This case-control study conducted on 160 women with BC and 192 age-matched healthy women. Genotyping of miR-608 rs4919510 was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: Our findings showed that GC genotype significantly decreased the risk of BC (odds ratio (OR) = 0.49, 95% confidence interval (CI) 0.28–0.88, p = 0.018) compared to CC genotype. Furthermore the G allele decreased the risk of BC (OR = 0.53, 95%CI 0.30–0.92, p = 0.024). No significant association was found between miR-609 genotypes and clinicopathological characteristics of BC patients (p >0.05). Conclusion: Our findings indicate that miR-608 polymorphism might be associated with decreased risk of BC in an Iranian subpopulation. Further large-scale studies with different ethnicities are needed to verify our findings.

Submitted: January 07, 2016.
*Correspondence: E-mail: hashemim@zaums.ac.ir
Abbreviation used: BC — breast cancer; CI — confidence interval; miRNAs — microRNAs; mRNAs — messenger RNAs; OR — odds ratio.

Breast cancer (BC) is one of the most prevalent types of cancer in women. It is a major public and global health problem and accounts for 14% of total cancer deaths worldwide annually [1]. Similarly, BC is among the most common malignancies amongst Iranian women [2]. Although causes of BC are not yet completely understood, genetic factors are indicated to play key roles in the pathogenesis and progress of this malignancy [3–8].

MicroRNAs (miRNAs) are a highly conserved class of small (~ 22 nucleotides), endogenous, non-coding RNAs suppressing posttranscriptional gene expression by base pairing with their target messenger RNAs (mRNAs) and inducing either translational repression or mRNA degradation [9–11]. Loss of heterozygosity in the 10q24 locus has been described in numeral of human cancers [12–15]. Hsa-mir-608 lies within an intron of SEMA4G in this region. Several single nucleotide polymorphisms in the sequences of pre-miRNAs such as miR-196a2 (rs11614913 T>C), miR–499 (rs3746444 A>G), and miR-125a (rs12975333 G>T) were associated with significantly increased risks of BC in some but not all studies [7, 16–18].

It has been proposed that rs4919510 C>G variant in miR-608 can alter its binding to target genes [19]. The expected targets of miR-608 include insulin receptor (INSR), interleukin-1 alpha (IL1A), growth hormone receptor (GHR), and TP53 [19].

Several studies examined the impact of miR-608 rs4919510 C>G on the risk of various cancers, but the results were inconsistent [20–26]. To the best of our knowledge, limited studies evaluated association between miR-608 rs4919510 polymorphism and BC risk [21, 27, 28]. So, this case-control study was designed to assess the possible association between miR-608 rs4919510 polymorphism and susceptibility to BC in an Iranian subpopulation.

MATERIALS AND METHODS

This case-control study was done on 160 BC patients and 192 age-matched healthy women with no history of cancer of any type (as the control group) in Zahedan, southeast Iran. Ethical approvals for recruitment were taken from local Ethics Committee of Zahedan University of Medical Sciences, and informed consent was obtained from all patients and healthy individuals. The study design and enrolment procedure were described previously [29–31].

Genotyping of miR-608 rs4919510 C>G was done by PCR–RFLP methods. Briefly, forward and reverse primers were 5´-TCTGGCTAGGTAATGGCTCC-3´ and 5´-GCATCTGTGGCCTTCCATGA-3´, respectively. The PCR product was digested by PvuII restriction enzyme (Fermentas). G allele digested and produced 242-bp and 117-bp pattern, while C allele undigested and produced 359-bp fragment (Figure).

0923 miR 608 rs4919510 C>G polymorphism decreased the risk of breast cancer in an Iranian subpopulation

Figure. Photograph of miR608 rs4919510 C>G polymorphism using PCR-RFLP method. G allele digested by PvuII restriction enzyme and produced 242-bp and 117-bp pattern, while C allele was undigested (359-bp fragment). M — DNA marker; lanes 1 and 3 — CC; lane 2 and 4 — CG

Statistical analysis. Statistical analysis was done using statistical package SPSS 20 software. The categorical and continuous data were analyzed using χ2 and t-test, respectively. The association between genotypes and BC were assessed by computing the odds ratio (OR) and 95% confidence intervals (CI) from logistic regression analyses. A p-value of < 0.05 was considered to be statistically significant.

RESULTS

The study group consisted of 160 BC patients with an average age of 48.5 ± 10.8 years and 192 healthy women with a mean age of 49.5 ± 12.4 years. No significant difference was found between the groups regarding age (p = 0.424).

The genotype and allele frequencies of miR-608 rs4919510 C>G polymorphism in BC patients and healthy women are shown. Our findings showed that rs4919510 variant significantly decreased the risk of BC (OR = 0.49, 95% CI 0.28–0.88, p = 0.018, CG vs CC) (Table 1). Moreover, the G allele significantly decreased the risk of BC (OR = 0.53, 95% CI 0.30–0.92, p = 0.024) compared with C allele. No significant association was found between miR-609 genotypes and clinicopathological characteristics of BC patients (p > 0.05).

Table 1. Association of miR-608 rs4919510 C>G polymorphism and risk of BC

miR608
rs4919510 C>G

Case, n (%)

Control,
n (%)

OR (95% CI)

p

Genotype

CC

140 (87.5)

149 (77.6)

1.00

GC

20 (12.5)

43 (22.4)

0.49 (0.28–0.88)

0.018

GG

0 (0.0)

0 (0.0)

Allele

C

300 (93.8)

341 (88.8)

1.00

G

20 (6.2)

43 (11.2)

0.53 (0.30–0.92)

0.024

Hardy — Weinberg equilibrium (HWE) was assessed by chi-square test. The genotype of miR-608 rs4919510 polymorphism in both controls and cases were in HWE (χ2 = 3.05, p = 0.081 and χ2 = 0.71, p = 0.399, respectively).

As shown in Table 2, miR-608 rs4919510 polymorphism was not associated with clinicopathological characteristics, including age, tumor size, tumor stage, tumor grade, lymph node metastasis, estrogen and progesterone receptors (ER, PgR) status, and human epidermal growth factor receptor 2 (HER2/neu) (p > 0.05).

Table 2. Association between miR-608 rs4919510 C>G variant and clinicopathological characteristics of BC patients

Variables

miR-608 rs4919510

p

CC

CG

Age

0.943

≤ 50

82

12

> 50

58

8

Tumor size, cm

0.467

≤ 2

49

9

> 2

86

11

Lymph node metastasis status

0.793

Yes

83

13

No

39

5

Grade

0.811

I

23

4

II

71

10

III + IV

19

4

Stage

0.994

I

24

3

II

55

8

III

39

6

IV

21

3

Histology

0.611

Ductal carcinoma

91

12

Others

43

8

ER

0.799

Positive

82

12

Negative

50

6

PgR

0.311

Positive

78

8

Negative

54

10

HER2/neu

0.244

Positive

74

7

Negative

65

12

DISCUSSION

In the present study we inspected the impact of miR-608 rs4919510 variant on the risk of BC risk in a sample of Iranian population. Our data showed that rs4919510 polymorphism significantly decreased the risk of BC in our population. We found no significant association between miR-608 rs4919510 genotypes and clinicopathological characteristics of BC patients (p > 0.05).

Huang et al. [27] identified that the mir-608 rs4919510 was not associated with BC risk, but CG/GG genotypes were specifically associated with increased risk of HER2-positive BC (OR = 1.97, 95% CI 1.34–22.90). Variant G allele was the risk allele with OR of 1.62 (95% CI 1.23–22.15). They found that patients carrying GG-genotype also had larger HER2-positive tumors. They concluded that rs4919510 polymorphism in mature miR-608 may influence HER2-positive BC risk and proliferation of tumor. Jiao et al. [21] have found no significant association between miR-608 rs4919510 polymorphism and BC survival in Chinese population. Dai et al. [28] observed that miR-608 rs4919510 was not associated with BC risk.

In summary, our findings proposed that miR-608 rs4919510 polymorphism decreased the risk of BC in a sample of Iranian population. However, we found no significant association between this variant and the clinicopathological characteristics of BC patients. Population based studies with larger sample sizes with different ethnicities and long-term follow-up are required to confirm our finding.

Acknowledgement

This paper was funded by a research grant from the Deputy for Research, Zahedan University of Medical Sciences.

Conflicting Interests

The Authors declare that there is no conflict of interest to disclose.

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