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  • br rs genotype distribution was tested for HardyeWeinberg

    2019-10-07


    rs145204276 genotype distribution was tested for HardyeWeinberg equilibrium (HWE) using the Pearson c2 test. Differences of rs145204276 genotype frequencies between cases and controls were assessed using the c2 test. Results are presented as odds ratios (ORs) with 95% confidence intervals (CIs) and P values after adjustment for age and age at menarche. Multiple testing was performed by adjusting the significance level of 0.0125 (0.05/4) using Bonferroni correction. The difference of lncRNA GAS5 expression between the 2 groups was compared using the ManneWhitney U test. The relative level of luciferase activity among groups was compared using 1-way analysis of variance.
    Abbreviation: bp ¼ Resiniferatoxin pairs.
    GAS5 Indel Polymorphism and Risk of Breast Cancer
    Table 2 Association Between rs145204276 in the Promoter of GAS5 and Risk of Breast Cancer (DNA From Blood Samples)
    Controls (n [ 602), Breast Cancer Adjusted OR
    Abbreviations: Del ¼ deletion; Ins ¼ insertion; OR ¼ odds ratio. aOdds ratios were adjusted for age and age at menarche.
    The rs145204276 Ins/Del and Del/Del Genotypes Were Associated With Increased Levels of lncRNA GAS5
    We performed qPCR on RNA isolated from tissues of 69 BC patients and corresponding nontumor normal tissues. As shown in Figure 2A, lower levels of lncRNA GAS5 were observed in BC patients compared with normal controls (P < .05). The expression data of lncRNA GAS5 were also analyzed with regard to the rs145204276 ins/del polymorphism. Compared with rs145204276
    ins/ins genotype carriers, individuals with rs145204276 ins/del and del/del genotypes expressed significantly higher levels of lncRNA GAS5 in BC patients (Figure 2B) and in normal controls (Figure 2C; P < .05).
    The rs145204276 Del Allele Increased Transcriptional Activity
    Investigation of putative transcriptional factor for rs145204276 was performed using TRANSFAC software. As shown in Figure 3A, in silico analysis showed that the rs145204276 del allele but not ins allele contains the binding sequence (GCGCGAGGAA) of tran-scriptional factor specificity protein 1 (SP1). To determine whether rs145204276 results in the change of transcriptional activity, we cloned the promoter region of lncRNA GAS5 containing rs145204276 del or ins allele and transfected into HEK293, MCF7, MDA-MB-231, T-47D, and A549 cells. As shown in Figure 3B, a significant increase of luciferase activity was observed in cells transfected with rs145204276 del construct compared with cells transfected with rs145204276 ins construct (P < .01).
    Discussion
    In this study, we designed a hospital-based case-control study to explore the relationship between rs145204276 in the promoter of lncRNA GAS5 and BC risk in a Chinese Han population. We presented, to our knowledge, the first evidence that the rs145204276 del allele is associated with a reduced risk of BC whether the samples came from blood or tumor tissues. The expression levels of GAS5 were significantly increased in carriers with rs145204276 ins/del and del/del genotypes. Through functional analysis using dual luciferase reporter assay, we confirmed
    Table 3 Association Between rs145204276 in the Promoter of GAS5 and Risk of Breast Cancer (DNA From Tissues of Breast Cancer)
    Controls (n [ 602), Breast Cancer Adjusted OR
    Abbreviations: Del ¼ deletion; Ins ¼ insertion; OR ¼ odds ratio. aOdds ratios were adjusted for age and age at menarche.
    Yiyin Tang et al
    Table 4 Subgroup Analysis Between rs145204276 in the Promoter of GAS5 and Risk of Breast Cancer
    Case I
    Case I vs. Controls
    Case II vs. Controls
    Controls,
    Adjusted OR