| Background:Breast in females does not only involve in the physiological functions like development of secondary sexual characteristics and involves in the feeding mechanism (breast feeding), but it is also a forming embody feminity, formation of body curves. Commonly the physical and psychological stress and pain is expressed in females regarding excess development and dysplasia of breast. Although the surgical treatment of breast dysplasia gradually improve, the causes of the disease are few studied and the pathogenesis is still not clear. Various hormones and humoral factors involve in the breast development, and estrogen is the main sexual hormone in female and play an important role. Most of the research on hypermastia are concentrated in estrogen and estrogen receptor(ER), but the results and opinions are varying.The variation of estrogen and its receptors will cause the malformations and abnormal development of breast. Estrogen as the growth factor promotes the breast cells and cancer cell proliferations by means of ER. The levels of estrogen are found normal in vast number of patients with gigantomastia. Gigantomastia may associated with increased ER, but micromastia may associated with depressed ER or reduced sensitivity. Some scholars believe that gigantomastia has not a directly association with ER. ER regulates a series of genes expression and mediate intracellular signal transduction. ER gene polymorphisms may alter gene transcriptional regulation, and then impact the physiological function of estrogen.ER gene includes two subtypes, they are ERa and ERβ.They have different expression and play different role in breast tissue. Many studies have demonstrated that the Eragene play the more important role. One report demonstrated that ER gene polymorphisms may effecting sensibility of estrogen, that may be related to breast cancer, ER a gene XbaI polymorphism is related to cyclomastopathy, however, ER a gene PvuII gene polymorphism is not related to it. Therefore, it need to further investigate the biological mechanism of ER and whether ER play a key role in gigantomastia and micromastia.Objective:To investigate the relationship between polymorphism of ERaand gigantomastia, and the relevance of gene differential expression in Chinese, in order to supply theory foundation for the diagnosis, therapy and prevention of gigantomastia and hypogenetic micromastia.Methods:1) Select the patients admitted in August to October2011,3female patients with breast hypertrophy, and3female patients after breast fibroadenoma excision for the study. Surgery to take the distance of the breast tissue and fibroadenoma of the breast in patients with breast hypertrophy patients abandoned the lesions3cm in breast tissue, as the experimental group and control group, the application Illumina chips to carry out genome-wide scan to find differentially expressed genes. Furthermore, analyzing the level of ERa gene, Gonadotropin-releasing hormone(GnRH), Luteinizing hormone(LH), Follicle stimulating hormone(FSH) in hypothalamus-pituitary-sex gland axle and other related genes expression.2.) A control case study was conducted in our hospital during2009.12-2011.6. The women admitted to us for abnormal galactophore dysplasia and gigantomastia, and the healthy check-up, who aged18to45yeas old. They were divided into3groupes.(1)Macromastia group (28cases):patients with the weight of each paredmastectomy>350g;(2)Micromastia group (70cases):The breast augmentation by incision of areola and armpit. The preoperative measurment of the total weight of breast tissue<200g;(3)Normal group(69cases):Healthy women whose breast size and form are normal.Collected peripheral blood samples from the objectives, then extract genomic DNA of3groups and investigate the polymorphism of ERa of XbaI and PvuII by PCR-reconstruction enzyme studies. The analysis were done by statical mean and (RELP)analysis.3) Using reporter gene:the sequences which have enhancer and promoter function in the first intron in the above three groups were cloned into the pGL3-Basic reporter vector, and restructuring the reporter gene plasmid within the reference plasmid and pRL-TK was transfected into breast cancer cells (MCF-7) cells relative luciferase activity after trarisfection was detected by chemiluminescence analyzer, analysis of the first intron polymorphism of the three groups by differences in the levels of estrogen gene transcription. Using dual-luciferase reporter gene technology to regulate transcription of the gene mutation.Results:1) Differentially expressed genes in hypermastia.This study analyzed and selected2808differentially expressed mRNA include1815up-regulated mRNA and993down-regulated mRNA,223differentially expressed miRNAs.123(55%) of differentially expressed genes were up-regulated miRNAs, and other100(45%) miRNAs were down-regulated. Further study found that these genes may play an important role in the occurrence of breast hypertrophy. By means of enrichment analysis of two types of genes corresponding gene ontology term, found that up-regulated genes enriched in chemokine signaling pathway, Fc gamma R-mediated phagocytosis, GnRH signaling pathway, leukocyte transendothelial migration and vascular smooth muscle contraction and so on. However, the down-regulated genes enriched in oxidative phosphorylation, proteasome. cardiac muscle contraction,citrate cycle (TCA cycle). mRNA-mediated pathway and ubiquitin mediated proteolysis pathway.2) The relationship between ERaPvu Ⅱ, Xba Ⅰ polymorphism and hypermastia and micromastiaThe analytical results of ERa gene at Pvu Ⅱ restriction sites as follows:breast hypertrophy group:the incidence of polymorphisms were25%,57.14%and17.86%of PP, Pp and PP-type Respectively; Normal control group:type of PP, Pp and PP-type10.14%,68.12%and21.74%respectively. The difference was not statistically significant (P>0.05). Micromastia group:pp-type40%,10.14%of the normal control group, the difference was statistically significant (P<0.0001); Pp, 47.14%,68.12%of the normal control group, the difference was statistically significant (P=0.0124).The analytical results of ERa gene Xba I restriction enzyme polymorphism as follows:the results of breast hypertrophy group:xx, Xx and XX were25%,64%and10%; normal control group:xx, Xx and XX were63%,10%and26%,in the two groups the difference was statistically significant (P<0.0001). Micromastia group:xx, Xx and XX were77%,21%and0.10%respectively; Normal control group:xx, Xx and XX respectively63%,10%and26%, further analysis of the alleles constitute of the two groups, the difference was statistically significant (P<0.0001).The relative susceptibility analysis of Pvu II restriction enzyme polymorphism: Micromastia group:P/p-two allele of the OR value is0.4540(95%CI:.2093～.8326), which may be p gene is a small breast disease susceptibility gene. Recessive genotype pp and P carriers OR value is5.9048(95%CI:2.3622-14.7604), which accounted for the relationship between Small breast disease and the pp genotype.The relative susceptibility analysis of Xba I restriction enzyme polymorphism: breast hypertrophy group:X/x alleles OR value is1.6570(95%CI:0.095～1.2634), which may be the X gene is the disease easy to sense gene; Recessive genotype xx and X carriers OR value is0.1894(95%CI:0.0706～0.5078), heterozygous and pure and sub-genotype OR value is15.9429((95%CI:5.3110～47.8580), breast hypertrophy associated with Xx genotype. Micromastia group:X/x two-allele OR value is0.3054(95%CI:.1639～0.5688),which means that x gene may be susceptibility gene of the disease; Recessive genotypes xx and X carriers, the OR value is1.9176(95%CI:.9121～4.0317) suggesting that small breast disease associated with the xx genotype.3) The dual luciferase reporter gene activity assay.The relative luciferase activity of normal control group, hypermastia and micromastia were2.66±1.41,3.13±1.17and1.97±0.79respectively. Compared With normal control group, the relative activity luciferase enzyme of hypermastia group was significantly elevated (P<0.05), and the relative activity luciferase enzyme of micromastia group was significantly depressed(P<0.01). Through analyzing the relative luciferase activity and XbaI polymorphism in each group, we find that the transcriptional expression activity of ER gene that showing xx genetype was significant lower than xX and XX genetypes, whereas, between the micromastia and normal control, the individual that showing xx genetype, its transcription expression activity of ER gene significant lower in the former. In hypermastia, the transcription expression activity of ER a gene that showing XX genetype was high relative to the normal control, and there has no significant in the xX genetype and xx genetype compared with normal control. Combined with the second part of the results, the XX genetype individuals account for10.1%(3/28) in hypermastia. It supposed that the reason of hypermastia was not the transcriptional expression activity of ER a gene with XX genetype.Between normal control and micromastia groups, the transcriptional expression activity of ER gene was lower in pp genetype polymorphism than other polymorphisms(pP or PP). In PP polymorphism, the activity was high in hypermastia but in normal control. It demonstrate that the expression of ERa gene was up-regulated in PP polymorphism in hypermastia. In pp and pP polymorphisms of ER a gene, the activity was no significant compared to the normal control. Combined with the second part, PP polymorphisms account for17%(5/28) in hypermastia. It suggested that although PP genetype of ER gene Pvu Ⅱ have a relationship with the up-regulated transcriptional expression activity of ER a gene, it may not the main cause of the hypermastia formation.Conclusion:1) The scan results of gene chip technology in hypermastia group and normal control group show that ER a gene was up-regulated in hypermastia and enriched in GnRH signaling pathway which was related in the pituitary gland-gonad-estrogen axis. This axis is closely related to breast development. These indicate that up-regulate of ER a gene is the one reason of hypermastia formation. Meanwhile, there have some other genes different expressed, so it demonstrated that hypermastia may be a complex biological network involved multiple genes and multiple pathways.2) The occurrence of micromastia was related to recessive genotype pp and xx, whereas hypermastia was associated with heterozygous genotype Xx. The differences in breast size and shape are not determined by differential expression of one gene.3) The polymorphism of the first intron in ER a gene may result in different transcriptional of ER gene. The transcriptional expression activity of ER in hypermastia group was significant higher than normal control, whereas, the micromastia was lower than normal control. Combined the polymorphism of ER a gene in hypermastia and micromastia, it supposed that the low level transcriptional expression of the genetype xx and pp may be a part reason of the micromastia. Although PP genetype has a relationship with the transcriptional expression level of ER a gene, it may not the main reason of the hypermastia formation.However, the cases that we investigated are less, it need to increase more samples and in-depth studies to get a more definite conclusion. |
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