| Background: Breast cancer as one of the most common malignant tumors in women seriously affect patients’ health and survival.To looking for the prognostic risk factors and potential therapeutic targets of breast cancer is one of the hot topics of breast cancer research at present. Breast cancer belongs to hormone-dependent tumor, and estrogen levels in body is the important factor in regulation of breast cancer cell proliferation, and estrogen plays a role mainly through estrogen receptor(ER).Estrogen as a transcription factor transfers to the nucleus after forming complex with ER to make function by adjusting the transcription of downstream target genes.And this mechanism is called the action mode of estrogen genome or nuclear launch of estrogen response. But in recent years, studies have shown that estrogen also exists the non-genome function model which is also known as the membrane start of estrogen response.In this mode, the estrogen combines with the estrogen binding protein distributing in cytoplasm and membrane directly to activate the intracellular signaling pathways rapidly.And these intracellular signaling pathways mainly include ERK/MAPK signaling pathways, Ras- PI3 K pathway, JNK pathway and the second messenger system and so on. Besides, they can make the cells to produce rapid response and promote cell proliferation and malignant transformation.About the composition of estrogen binding protein distributing in the membrane and plasma of cancer cells, it has not been determined. Currently immunohistochemical method is widely used to detect the state of ER of breast cancer cells in clinical. It is widely believed that the ER alpha protein is Located in the nucleus, however the ER in cytoplasm and membrane has little clinical significance and has not been caused widespread attention due to its uncertain content. In the early study,our group discoverd that we can see different degree and different proportion of cytoplasm/membrane coloring by ER alpha immunohistochemical stainning among part of the breast cancer cases.However the composition of these proteins located in cell membrane and/or cytoplasm is unclear and whether the protein has clinical significance is also unclear currently.We hypothesized that the protein may be ER alpha 36, an shear variants of ER alpha 66. At present, the protein’s role in occurrence and progress of breast cancer and its influence on curative effect of related drug have not been clear.Objective:1、To research the chromatic way of ER alpha protein in human breast cancer tissue, determine whether there is a cytoplasmic/membrane positioning and explore the clinical pathology of cytoplasm /membrane ER alpha expressing and its effect on the prognosis of patients.2、To research the relationship between the expression and localization of ER alpha and ER alpha 36 among breast cancer cases, and explore the kind protein of ER alpha in plasma and membrane. 3、To research the intracellular localization of ER alpha protein and the protein expression changes of HER-2、RARα and ki-67 in MCF- 7 cell line after high expression of ER alpha 36 and discusses its possible mechanisms of ERα36 which promotes breast cancer cells to transfer.Method: 1、Immunohistochemical staining method to detect the intracellular expression and localization of ER alpha protein,if different intensity yellow fine granular staining in nucleus or cytoplasm / membrane occurs in smore than one-percent tumor cells,we see it as ER positive nuclear or positive cytoplasm / membrane. Analyze and gather the correlation between ER alpha in cytoplasm / membrane and the patient’s clinicopathologic feature,such as menstruation state, family history, the involvement of nipple big duct, histological grade of breast cancer, clinical stages,T-staging, N-staging, M-staging, recrudesce, HER-2 expression and so on.2、q-PCR to detect the expression quantity of ERα36 m RNA and ERα66 m RNA,analyze the correlation between the two and nuclear localization and cytoplasm/membrane localization of ERα.3、Using nucleoprotein- cytoplasm protein extraction kit from Thermo Fisher to extract nucleoprotein and cytoplasm/ membrane protein respectively,western-blot to detect the expression of ERα36 and ERα66 proteins after getting highly purified nucleoprotein and cytoplasm/membrane protein,then western protein stripe scanning, gray analysis and calculation are needed. 4、Structure p IRES2-EGFP- ERα36 gene overexpression vector,transient transfection to human breast cancer cell line MCF- 7, extract m RNA and protein,detect the expression of ERα36 and ERα66 by q-PCR and western-blot.5、Collect MCF 7 cells after transfection, make cell wax, immunohistochemical method to detect the expression of ERα、HER-2 and ki-67.6、Collect MCF 7 cells after transfection, extract total protein, western-blot method to detect the expression of HER-2 and RARα.Outcome:1 、 Among the 1164 cases, ER alpha positive orientation through immunohistochemical staining was mainly in the nucleus,and some cases could be observed in cytoplasm/membrane. Cytoplasm/ membrane staining could be observed with nuclear staining in the same time(the nuclear, cytoplasm/membrane are both positive).The two could also be observed respectively(positive cytoplasm/membrane,negative nuclear).The cytoplasm/membrane coloring of ERα was related with high N-staging, the score of HER-2, recurrence and metastasis. Furthermore, the patient with positive cytoplasm/membrane of ERα had short progression-free surial.2、There were 18 cases(accounting for 50%) that ERα66 m RNAand/or ERα36 m RNA were detected among 36 samples.In the results,ERα66 m RNA was detected in 10 cases and its positive percentage was 27.8%;ERα36 m RNA was detected in 14 cases and its positive percentage was 38.9%.There were 6 cases in which ERα36 and ERα66 expressed meanwhile, and its percentage of all samples was 16.7%. Cases with ERα66 m RNA positive appeared nuclear coloring by immunohistochemical staining. In the same way,cases with ERα36 m RNA positive appeared cytoplasm/ membrane coloring except one case. There was a significant correlation between the expression of ERα36 m RNA and ERα66 m RNA and immunohistochemical cell subcellular localization(p<0.05). 3、ERα36 and ERα66 were both detected in cases with positive nuclear and positive cytoplasm/membrane by western blot method.However, cases with ERα nuclear positive only expressed ERα66,and cases with ERα cytoplasm/membrane positive only expressed ERα36,and cases with ERαnuclear and cytoplasm/membrane negative expressed no ERα36 and ERα66. 4 、 p IRES2-EGFP-ERα36 eukaryotic expression vector transfered into MCF-7 cells successfully.Its transfection efficiency was about 70% after transfection 72 hours.The results of q PCR detection showed that ERα36 gene overexpressed 129.23 times after ERα36 gene eukaryotic expression vector transfered into MCF-7 cell line;western-blot detected specific ERα36 stripe.And both two kinds of results confirm successful transfection. 5、About the changes of coloring location after transfection in MCF-7 cells, cell cytoplasm and membrane also appeared positive coloring besides nucleus expression. It confirmed that ERα36 expressed primarily in cell cytoplasm or cell membrane; The expression of HER-2 increased, and part of the tumor cell membrane appeared positive staining(1+~2+);Ki- 67 index increased slightly.6、In p IRES2-EGFP- ERα36 expression vector,the expression of HER-2 increased and expression of RARα decreased compared with p IRES2-EGFP- NC after transfection 72 hours.conclusion:1、Human breast cancer estrogen receptor alpha(ERα) protein expressed in nuclear and/or cytoplasm/membrane.Cytoplasm/membrane ERα was mainly for ERα36,and nuclear ERα was mainly for ERα66,with a few of ERα36.2 、 The expression of cytoplasm/membrane ERα in breast cancer cells was correlated to the expression of HER-2 in cancer cells, and it revealed that there was a regulating association between the two.3、The patient with positive breast cancer plasma /membrane ER alpha had high lymph node metastasis rate and short progression-free surial. The expression of cytoplasm/membrane ERα in breast cancer was an independent poor prognosis factor.4、ERα36 promoted the expression of HER-2, while inhibited RARα protein; ERα36 may promote HER-2 expression through down-regulation of RARα protein. |
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