| Breast cancer is the most frequently diagnosed cancer that account for 15%of all new cancers among women in China,and is the leading cause of cancer death in women younger than 45 years.Over the last decades,genome-wide association studies(GWAS)have linked 3800 SNPs to 427 diseases and traits,only 7%of the SNPs are located in protein-coding regions,but 93%are located in noncoding regions.LincRNAs are noncoding transcripts longer than 200 nucleotides,originating from the region between two protein-coding genes and have been described as the largest subclass in the noncoding transcriptome in human.LincRNAs are emerging as key regulators of diverse biological and cellular processes through mechanisms such as transcriptional and post-transcriptional processing.Recently,increasing number of researches identified that genetic variation in lincRNAs may confer susceptibility to cancer by causing aberrant expressions and dysregulations in a cell-type or tissue-specific manner.MethodsWe carried out a two-stage case-control study including 2881 breast cancer cases and 3220 controls.In stage I,we genotyped 17 independent(r~2<0.5)SNPs of 6tumor-related lincRNAs by using the TaqMan platform.In stage II,SNPs potentially associated with breast cancer risk were replicated in an independent population.Quantitative real-time PCR was used to measure H19 level in tissues from 213 breast cancer patients with different genotypes,and H19 expression level in the adjancent normal tisues was compared to the breast cancer tissue.Based on this clue,we carried out the next stage of the study of functional mechanisms of the SNPs of H19 to investigate whether H19 plays an important role in the development of breast cancer and acts as an oncogene or tumor repressor.Wether H19 act as itself or via encode miR-675 to induce breast cancer.Which of the target genes is controlled by miR-675?Does the SNP of the H19 affect the expression of H19 or miR-675?Results1.We identified 2 SNPs significantly associated with breast cancer risk in stage I(P<0.05),but not significantly replicated in stage II.We combined the data from stage I and stage II,and found that,compared with the rs2071095 CC genotype, AA and CA+AA genotypes were associated with significantly decreased breast cancer risk(OR=0.82,95%CI:0.68-0.98;OR=0.87,95%CI:0.79-0.97,respectively).Stratified analyses showed that rs2071095 was associated with breast cancer risk in estrogen receptor(ER)positive patients(P=0.002),but not in ER negative ones(P=0.332).2.We further analyzed the effects of the rs2071095 genotypes on the risk of breast cancer among different subgroups of demographic characteristics in stage I+II:. For AA genotype,decreased risks of breast cancer were more likely to be evident in subgroups of younger patients(age≤55 years,OR=0.86,95%CI:0.68-0.98;), non-smoking women(OR=0.88,95%CI:0.74-0.98),premenopausal women (OR=0.76,95%CI:0.57-0.96),non-history of benign breast disease(OR=0.83,95%CI:0.67-0.98)and non-family history of cancer(OR=0.82,95%CI:0.65-0.97).The interactions between genotype and demographic characteristics were not significant in stage I,stage II and stage I+II.3.We found no significant statistics relationship between SNPs and prognosis of breast cancer(Log-rank test,OS:P=0.334;cancer-specific overall survival time:P=0.359;DFS:P=0.739).Multi-factor cox regression analysis identified no significant statistics relationship between H19(rs2071095)and prognosis of breast cancer(OS:CA vs CC,P=0.842;AA vs CC,P=0.360;cancer-specific overall survival time:CA vs CC,P=0.476;AA vs CC,P=0.913;DFS:AA vs CC,P=0.594;AA vs CC,P=0.498).4.We further explored the H19 expression by rs2071095 genotypes among 213 breast cancer tissues.Rs2071095 CA and AA genotypes were found to be significantly associated with decreased expression of H19 in breast cancer patients compared with the CC genotype(P=0.027 and P<0.001,respectively).5.According to the transcription factor prediction website,transcription factor BCL6B,TAL1,ASCL2 and SP1 may bind to rs2071095 loci.Luciferase assay and EMSA indicated that there was no difference in the transcriptional factor binding ability to the H19 promoter between AA and CC.6.The expression of H19 in breast cancer tissue samples was positively correlated with miR-675-5p/3p expression and was verified in TCGA data.The expression of miR-675-5p/3p was up-regulated after the expression of H19 in the cell line.The expression of H19 in cancer tissues is lower than that of adjacent tissues, and H19 overexpression can inhibit the ability of breast cancer cell migration, and vice versa.The effect of H19 on cell proliferation,invasion and cell cycle was not obvious.MiR-675-5p/3p could inhibit cell migration and inhibit cell proliferation by blocking cells in G1 phase and promoting apoptosis.MiR-675-3p could target IGF1R to reduce the expression of its protein level,and thus play a role of tumor suppressor.7.Compared with H19 lower expression,ER+ratio in the H19 higher expression of breast cancer tissues was high.The expression level of H19 and miR-675-5p/3p was not statistically significant for the prognosis of breast cancer.ConclusionThis study found that H19(rs2071095)A allele relative to C allele can reduce the risk of breast cancer,and A allele can significantly reduce the H19 gene expression level in patients with breast cancer,H19(rs2071095)genotypes did not affect the ability of protein combined with H19 promoter region,rs2071095 may not be a functional SNP.The expression of H19 in cancer tissues was lower than that of the adjacent tissues,and the high proportion of the breast cancer tissues with higher H19expression was higher.H19 can inhibit cell migration.H19 overexpression could increase the expression of miR-675-5p/3p,and the expression of H19 in breast cancer tissue samples was positively correlated with mir-675-5p/3p expression.MiR-675-5p/3p could inhibit cell migration and inhibit cell proliferation by blocking cells in G1 phase and promoting apoptosis.MiR-675-3p can target IGF1R and down-regulate the expression of its protein level and play a role of tumor suppressor. |
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