Demographical and clinical information of study population
This study contained 434 BC cases and 700 healthy control. All the subjects were Han Chinses women from northwest China. There were no statistically significant differences in age distribution, BMI and menopausal status between the patients and the control group. The detail demographical and clinical information was display in Table 1. BMI was a statistical index to estimate the body fat in people of any age. In this study, BMI was divided into four levels (underweight, normal weight, overweight, and obese) based on Chinese reference standard.
The associations between MEG3 SNPs and BC risk
Three SNP in MEG3 gene (rs3087918, rs11160608 rs7158663) were genotyped in all recruited subjects, and their detected rate were 99.1%, 99.2% and 99.4%, respectively. The genotype distribution of the three polymorphisms in control groups accorded with HWE (rs11160608: PHWE = 0.844; rs3087918: PHWE = 0.968; rs7158663: PHWE = 0.334). We didn’t find statistical significance for rs11160608, rs7158663 and breast cancer (P > 0.05 in all genetic models). Pooled analysis indicated that rs3087918 was related to a decreased risk of breast cancer [GG vs. TT: OR (95%CI) = 0.67(0.45-0.99), P = 0.042; GG vs. TT + TG: OR (95% CI) = 0.69(0.48-0.99), P = 0.046]. The detail results were showed in Table 2.
Stratified Analysis by age, BMI and menopausal status
Then, we conducted stratified analysis based on age, BMI and menopausal status to further explore their effect on relationship between BC susceptibility and the three SNPs in MEG3. BMI was divided into two levels (BMI < 24 kg/m2 and BMI >= 24 kg/m2). No association was found between rs11160608, rs7158663 and breast cancer when stratified by age, BMI and menopausal status (Supplemental Table S2). Rs3087918 was related to a reduced susceptibility for women aged <=49 [GG vs. TT: OR(95%CI) = 0.40(0.22-0.73), P = 0.02] (Table 3).
Relationship between MEG3 rs3087918 and clinical characteristics of BC
To further explore the effect of rs3087918 loci and clinicopathological information on BC susceptibility, correlation analysis was conducted in the cases group defined by age, BMI, menopausal status, tumor size, metastasis, clinical stage, ER/PR status and Her-2. As showed in Table 4, there is a significant association of the GG genotype with tumor size according to the 95%CI (1.01-3.92), while the P value of tumor size is 0.05. In this study, P < 0.05 was considered statistically significant. Thus, we considered there was no association found between GG genotype of rs3087918 and tumor size. This is a controversial result that needs further study to clarify. GG and TG+GG genotypes were associated with the over-expression of Her-2 [GG vs. TT: OR(95%CI) = 2.37(1.24-4.63), P = 0.010; TG + GG vs. TT: OR(95%CI) = 1.50(1.01-2.24), P = 0.045]. We further divided the cases into luminal, Her-2 and triple negative breast cancer (TNBC) groups according to molecular classification. However, we found no association between three SNPs of MEG3 and the different molecular typing states of BC (Table S3).
Haplotype analysis of MEG3 SNPs and associations with the risk of BC
To explore the combined effect the three SNPs in MEG3, we performed haplotype analysis by Haploview. The results of the haploid analysis indicated that TCG haplotype may increase the risk of breast cancer compared with the wild haplotype TAG [OR (95%CI) = 2.97(1.66-5.31), P < 0.001]. Other haplotypes showed no association with BC (Table 5). The order of the three SNPs was rs3087918, rs11160608 rs7158663.
The function prediction of the rs3087918 in MEG3
We used RNAfold (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi) and LncRNASNP2 (http://bioinfo.life.hust.edu.cn/lncRNASNP/) database to predict the potential function of rs3078918. The centroid secondary structure of rs3087918 was shown in Figure 1, we learned that mutant allele “G” would significantly change the centroid secondary structure of MEG3. Moreover, its minimum free energy was change from -28.87 kcal to -26. 90 kcal/mol, which suggests rs3087918 may increase the structural stability of MEG3. The results of LncRNASNP2 indicated that rs3087918 may gain the targets of hsa-miR-1203 to MEG3 (lncRNA ID: NONHSAT039760.2), while loss the target of hsa-miR-139-3p and hsa-miR-5091 to MEG3 (See supplemental Table S3 and Figure S1).