| Breast cancer is the most frequently diagnosed malignant tumor and the first leading cause of cancer death among females. The etiology of breast cancer is complex and multifactorial. It is generally considered that genetic, environmental, and reproductive factors all contribute to the development of BC. Long non-coding RNAs(lnc RNAs) are defined as transcribed RNA molecules that longer than 200 nucleotides, lacking open reading frame of significant length(less than 100 amino acids), and having no obvious protein-coding capacity. Several lnc RNAs have been reported to be associated with tumorigenesis and cancer progression. Up-regulated expression of HOTAIR is a powerful indicator of poor prognosis for several cancers, including breast cancer. H19 is a maternally expressed imprinted gene transcribing a long non-coding RNA, and it could act as an oncogene for breast cancer. The level of SRA expression is increased in breast tumors and the expression of SRA correlate with estrogen receptor(ER) and progesterone receptor(PR) levels, which may alter ER/PR action and promote tumorigenesis. As a new generation of biomarker, lnc RNAs single nucleotide polymorphism may change the lnc RNA structure, affect the stability of the lnc RNA, influence the level of lnc RNA expression and affect cancer susceptibility. However, to date, no research has been executed to evaluate the lnc RNAs polymorphism and the risk of breast cancer. On the basis of the above description, it is valuable to investigate the role of lnc RNAs genetic variations in the development of breast cancer, and found a useful marker to predict breast cancer risk. ObjectiveThe aim of our study was to evaluate the association between severe haplotype tagging SNPs(ht SNPs)(HOTAIR s1899663C>A、rs4759314G>A and rs920778G>A; H19 rs3741219T>C and rs217727C>T; SRA rs10463297 T>C and rs801460G>A)and breast cancer risk in Chinese women, and examine the gene–reproductive factor interaction on breast cancer susceptibility. We aimed to find several breast cancer susceptive genes, provide cancer biomarker for breast cancer early detection and early diagnosis, and provide the theoretical basis for breast cancer risk assessment. And in order to provide the theoretical basis for the regulatory mechanism of lnc RNAs polymorphisms, we further investigated the correlations between lnc RNAs polymorphisms and lnc RNAs m RNA expression level. Method(1) Based on several human lnc RNA databases, haplotype-tagging SNPs selection was conducted with the Haploview software 4.2.(2) Our case-control study included 502 newly diagnosed breast cancer patients from hospital and 504 age frequency matched healthy controls from community in Henan Han population, China. All subjects were genotyped for HOTAIR rs1899663 and rs4759314, H19 rs3741219, and SRA rs10463297 by polymerase chain reaction-restriction fragment-length polymorphism(PCR-RFLP).While the genotyping of HOTAIR rs920778, H19 rs217727, SRA rs801460 polymorphisms were determined by created restriction site PCR(CRS-RFLP) assays. By using SPSS 21.0, Student’s t test and Chi-squared(c2) test were used to compare the differences in the distributions of demographic characteristics, reproductive variables, as well as the SNPs genotype frequencies between BC cases and controls. Associations between case-control status and each SNP were also evaluated with an unconditional logistic regression model, adjusted for demographic characteristics and reproductive variables by using SAS 9.1. Furthermore, the false-positive report probability(FPRP) was used to test false positive associations between lnc RNA SNPs and breast cancer for all significant genetic effects observed in our study. Hardy–Weinberg equilibrium(HWE) for the genotype distribution of each SNP was tested by the Hardy–Weinberg software to compare the observed genotype frequencies with the expected ones among the cancer-free control subjects. The online SHEsis(http://analysis.bio-x.cn/my Analysis.php) was taken for Haplotype Analysis. Multifactor Dimensionality Reduction(MDR) method was used to detect the gene–reproductive factors interaction. A two-sided P value less than 0.05 was considered as the significant level.(3) The relative levels of SRA m RNA were examined using SYBR-Green real-time quantitative PCR method to explore the effects of different genotypes of rs10463297 on the SRA m RNA expression. Results(1) There were negative associations between GT+TT genotypes of rs1899663 and BC risk in subjects age at menarche>14(OR: 0.42, 95%CI: 0.21-0.82) and no. of pregnancy>2(OR: 0.65, 95%CI: 0.49-0.95). For the age at menopause≤50, a significantly lower risk of BC was found in individuals with at least one G allele(AG, GG) of rs4759314 than in individuals with homozygous A alleles(OR:0.97, 95%CI: 0.84-0.99). In this population-based case-control study, we found a significant positive association between individuals carrying T rs920778 and BC risk(OR: 1.41, 95%CI: 1.13-1.75). The haplotype analysis further demonstrated the Grs1899663 Ars4759314Trs920778 haplotypes(OR: 1.22, 95%CI: 1.01-1.45) increased BC susceptibility in this Chinese population.(2) No significant association between H19 polymorphisms(rs3741219 and rs217727) and BC risk was observed in our study. However, in further stratified analyses, CT+TT genotypes of rs217727 had a significantly lower risk of breast cancer among women with No. of pregnancy>2(OR: 0.79, 95%CI: 0.55-0.97). CT genotype of rs217727 was significantly associated with ER positivity(OR: 2.19, 95 % CI: 1.07-4.45) and HER-2 positivity(OR: 1.34, 95 % CI: 1.05-2.12).(3) TC genotype of SRA rs10463297 polymorphism was associated with significantly increased risks of BC in both the codominant(TC vs. TT, OR: 1.43, 95 %CI: 1.02-2.00) and recessive(TC+CC vs. TT, OR: 1.39, 95 %CI: 1.01-1.92) inheritance genetic models. On analyzing the data in SRA polymorphism with logistic regression, TC+CC genotype of rs10463297 was associated with the risk of BC in women of age>50(ORadjusted: 1.79, 95% CI: 1.05-3.05). No significant association was observed between PR, HER-2 status and the genetic variants. However, rs10463297 TC, TC + CC and rs801460 GA, AA, GA+AA genotype were significantly associated with ER positivity. SRA m RNA expression levels were significantly higher for the rs10463297 TC(2.09 ± 0.41), CC(2.42 ± 0.51) and TC + CC genotypes(2.20 ± 0.47) than the rs10463297 TT genotype(1.45 ± 0.34)(P = 0.002, 0.001 and 0.002, respectively). A significant increased SRA m RNA expression towards was found for the effect of the C allele(Ptrend =0.001).(4) The gene–reproductive factors interaction analyze shown that carrying rs920778 T allele, carrying rs10463297 C allele, post-menopausal and having family history of BC in fist-degree relatives had 1.58-fold breast cancer risk(OR:1.58, 95 % CI:1.23-2.03) compared to the all the other groups. Conclusion(1) HOTAIR rs920778 T allele, SRA rs10463297 TC and TC+CC genotypes, and could increase the BC risk. H19 rs217727 CT genotype could decrease the BC risk.(2) HOTAIR Grs1899663 A rs4759314Trs920778 haplotype could increase the BC risk. H19 rs217727 CT genotype and HOTAIR Trs1899663 A rs4759314Crs920778 could decrease the BC risk.(3) H19 rs217727 CT genotype was associated with breast cancer HER-2 and ER positive. SRA rs10463297 TC and TC+CC genotype, rs801460 GA, AA and GA+AA genotype was associated with breast cancer ER positive.(4) SRA rs10463297 polymorphisms could increase SRA m RNA expression.(5) rs920778, rs10463297, post-menopausal and having family history of BC in fist-degree relatives had significant gene–reproductive interaction on breast cancer risk. |
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