Expressions Of EphA2, EphrinA1, ERα, ERβ, And PR In Breast Carcinomas And Experimental Study Of Targeted Silencing Of EphA2 By RNA Interference

Breast carcinoma is one of the most common malignant tumors and has become the number one killer among women cancers.which greatly affects human health and life.It is estimated that 1.2 million of female will be diagnosed of breast carcinoma, with 0.5 million death annually in the world. The incidence and mortality of breast cancer are increasing, with an annual rate of approximately 0.2～0.3%.And more and more young women were affected by this disease.Classical clinical pathological factors such as age, stage of disease, volume, lymphatic spread and microvascular intensity have been confirmed to have a close relationship with prognosis.And disease recurrence and metastasis in early stage are key factors affecting the outcome of initial therapy. Identification of sensitive biomarkers for recurrence and metastatic potential and their regulatory genes in breast cancer cells, therefore, is important for both early clinical detection and effective prophylactic therapy.As the rapid development of immunological and molecular biological technologies in the past few decades, the clinical and pathological significance of many biomarkers such as c-er-B-2,ER, PR, uPA,VEGF, BRCA, muc-1,CD44, maspin, mdrl,etc,have been studied in breast cancer at cellular and molecular levels. Among all these biomarkers, c-erbB-2(HER-2), ER and PR are widely studied and considered to be of important prognostic values.But using c-erbB-2,ER and PR as diagnosis and prognostic markers are still not satisfactory enough. However, up to now, it is still mandatory to find better survival prognostic markers to better understand the biological behavior of breast carcinoma, supply with better clinical management of breast carcinoma.Recently, more and more investigators have focused on the roles of Eph family in tumours, which extends a new wide area for deep researches of the potential mechanisms of signal transduction in carcinomas.The Eph (erythropoitin producing hepatocellular carcinoma) receptor family is the largest subfamily of receptor protein tyrosine kinases (RTK),consisting of at least sixteen distinct receptors and nine membrane-bound ligands, known as Ephrins(Eph family receptor interacting proteins).Signal conduction mediated by Eph receptors and their ligands Ephrins is important to cell-cell repulsion and adhesion, which is also the base of the formation, patterning and plasticity of tissue. Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network, patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfinding, and vascular formation. Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.Recently, gene targeting experiments and angiogenesis assay in vitro and vivo indicates that the genes for Eph receptors and ephrins have been recognized to be differentially expressed in various human tumors.The aberrant expression is associatied with the altered tumor behavior, such as increased invasiveness, increased metas;tatic potential,prominent vascularization, and thus a poor prognosis.EphA2 was a number of Eph subfamily sifted from human horny cDNA library by Linsberg in 1990,it was the first gene discovered with tyrosine kinase active in Eph subfamily. EphA2 is a transmembrane receptor tyrosine kinase that is found at low levels on adult epithelial cells and enriched within sites of cell-cell adhesion and has been implicated in neuronal development, as well as regulating cell migration and adhesion.Compared with other members of Eph family, EphA2 has been shown frequently overexpressed in multiple types of carcinoma tissues and cell lines, such as, mammary cancer, malignant melanoma, prostatic carcinoma and esphageal carcinomas, et al.Many investigations suggest that EphA2 has a important role in the regulation of angiopoiesis, cell proliferation, invasion and metastasis of carcinoma. It has been proved that the high level of EphA2 can induce the maglignant transformation of mammary epithelial cells in vitro and vivo and it can also promote the invasion and metastasis of the carcinoma. Interestingly, the test of targeting inhibition of EphA2 protein in vitro have shown that using anti-EphA2 antibody can down-regulates expression of EphA2 and inhibits tumour cell growth, and thus lower the malignant degree of the tumor.EphrinAl (B16) is the major and also the first ligand discovered for EphA2 and was originally cloned from human umbilical vein endothelial cells as a factor induced by tumor necrosis factor-a(TNF-α).Ligand binding contributes to EphA2 autophosphorylation. However, little is known about its expression pattern in tumours. EphA2 and EphrinAl co-overexpressed in many malignant carcinomas and it is reported that the expression of EphA2 correlates with EphrinAl expression. Some study have reported that activation of endogenous EphA2 kinase stimulated by EphrinAl ligand can suppresse integrin-regulated cell adhesion, decreasing cell-cell attachment, endows tumour cells with more aggressive ability. Meanwhile,they found that cell adhesion mediated by integrin is mainly determined by density of EphrinAl ligand. All of these studies indicated that EphA2 and EphrinAl may be involved in the process of tumorigenesis and development of tumours.Recent research reported that the overexpression of EphA2 is related to histological grade, lymph node metastases, and poor prognosis, Suggesting its potential prognostic value in tumor treatment. But recent investigations have indicated that the relationships between overexpression of EphA2 and all sorts of clinicopathological features were different in different typies of tumours.For example, high levels of EphA2 protein in lung cancer predict a high risk of brain metastasis of patients, however, high levels of EphA2 protein did not relate to metastasis in prostatic carcinomas.However, few reports concerned with the expressions of EphA2 and EphrinAl in breast carcinoma in literature in our country is available.Known and suspected causes for oncogenesis and development of breast cancer include high estrogen level and low progestin level.Metabolic disorder of steroid hormone promotes the development of some kinds of breast cancer, but the involved regulation mechanism remains as yet unclear. Evidence is accumulating that the changes of EphA2 in breast carcinoma interact with ER. But there is little literature about the combined detection of the protein of EphA2,EphrinAl,ERα, ERβand PR in breast carcinoma.Neo-adjuvant chemotherapy is a method of therapy for non-metastatic cancer that involves systemic treatment with the application of cytotoxic drugs before the local treatment.Currently, the new adjuvant chemotherapy has become the standard treatment for locally advanced breast cancer. The results of the international multi-center randomized clinical trial on neo-adjuvant chemotherapy for breast cancer showed that:Neo-adjuvant chemotherapy is as useful as adjuvant chemotherapy for malignant tumors.The patients with breast cancer that are sensitive to neo-adjuvant chemotherapy have a better prognosis than those who are not sensitive to it. And, good results with the application of the therapy in patients with locally advanced breast carcinoma have already been obtained.With the neo-adjuvant chemotherapy treatment, many pathological and biological indicators of breast carcinoma have changed, and the expression of many genes were altered too.To find out the molecular markers which can predict the efficacy of neo-adjuvant chemotherapy and help to select effective endocrine therapeutic regimen, chemotherapy plan, and to determine the prognosis of breast carcinoma has become a hot spot in the current study. While recent researches on expression of EphA2 and ER, PR in breast carcinoma before and after neoadjuvant chemotherapy is few overseas, and less in China.Using immunohistochemistry technology to detect the protein expression of EphA2, EphrinAl,ERα, ERβand PR in breast carcinomas, and then studying their relation to clinicopathological factors;The expressions of ER,PR and EphA2 were detected by immunohistochemical method in breast cancer tissues before and after Neo-adjuvant Chemotherapy. We systematically analyzed the expression patterns of EphA2 and EphrinAl in a series of breast carcinomas by RT-PCR technology, explored the relationship between mRNA expressions of EphA2 and EphrinAl and clinicopathological features;In addition, suppression of the expression of EphA2 in MCF-7 cells by using retrovirus-mediated RNAi method was also conducted, In order to find the effective indicators that can help to select effective strategy for treatment and predict prognosis, and to explore the mechanism and prognostic value of EphA2 in breast carcinomas, and to provide new ideas and theoretical basis for gene therapy for breast cancer.This study is divided into four parts as listed below:Part One:Protein Expressions of EphA2, EphrinA1, ERα, ERβ, and PR in Breast Carcinomas and Their Significance Objectives:To study the protein expressions of EphA2,EphrinAl, ERα, ERβ,and PR in breast carcinomas and to explore their significance.Methods1.Using Immunohistochemistry Sp method to detect the protein expressions of EphA2, EphrinAl, ERα, ERβ, and PR in tumors from 130 breast carcinomas. Then studying their relationship to clinicopathological factors.2.The SPSS version 13.0 Statistical package was used for the statistical analysis. We also usedχ2-test(chi-square), t-test(studentt), ANVOA method, and other correlated tests.values of<0.05 were considered statistically significant.Results1.The distribution of EphA2,EphrinAl,ERa, ERP and PR was identical, EphA2 and EphrinAl are mainly located in the cytoplasm of tumor cells and endothelial cells and less in the cytoplasm of inflammatory cells. Positive expressions of ERα, ERβand PR were observed in the nucleus of the tumor cells.2.Among the 130 breast carcinoma cases,94 cases for EphA2 protein were positive,total positive rate was 72.31%;For EphrinAl protein expression,77 cases were positive, total positive rate was 59.23%;For ERαprotein expression,82 cases were positive, total positive rate was 63.08%;For ERβprotein expression,69 cases were positive, total positive rate was 53.08%;67 cases for PR protein expression were positive,total positive rate was 53.08%.3.The positive expression of EphA2 protein were not associated with age, tumor size and pathological type(P>0.05);But with clinical stage,lymph node metastasis and histological grade(P<0.05).The expression of EphA2 protein of the group with late clinical stage,lymph node metastasis, and high histological grade was respectively higher than the group with early clinical stage, negative lymph node metastasis and low histological grade.4.The positive expression of EphrinAl protein were not related to age, tumor size, pathological type and lymph node metastasis(P>0.05),but significantly correlated with clinical stage and histological grade(P<0.05).The expression of EphrinA1 protein of the group with late clinical stage, high histological grade was respectively higher than the group with early clinical stage and low histological grade.5.The protein expressions of ERa and PR were not associated with age,tumor size,pathological type, clinical stage, lymph node metastasis and histological grade(P >0.05).6.The positive expression of ERβprotein were not associated with age, tumor size, pathological type, clinical stage,and lymph node metastasis(P>0.05), but with histological grade(P<0.05).The expression of ERβprotein of the group with high histological grade was higher than the group with low histological grade.7.The positive staining of EphA2 and EphrinAl protein co-located in roughly the same tumor areas and vascular endothelial cells.Their distribution are fundamentally unanimous and their expression were positively correlated (P<0.05).8.Their were no significant correlations between the positive expression of EphA2 protein and the positive expression of PR protein(P>0.05).The exprssion of EphA2 protein was significantly correlated with the expression of ER in breast carcinoma(P<0.05),the positive rate of EphA2 protein expression decreased when the positive rate of ERa protein increased.The positive rate of EphA2 protein in ERa protein-negative group was significantly higher than ERa protein-positive group.The exprssion of EphA2 protein was significantly correlated with the expression of ERβin breast carcinoma(P<0.05),the positive rate of EphA2 protein expression increased when the positive rate of ERβprotein increased. Conclusions1.Through the study, we found that EphA2,EphrinA1,ERα, ERβand PR expressed in breast cancer, it is indicated that they may be involved in carcinogenesis, development procedures of breast carcinoma.2.The positive expression of EphA2 protein were associated with clinical stage, lymph node metastasis and histological grade.The protein expression of EphrinA1 were related to clinical stage and histological grade.The positive expression of ERβprotein were associated with histological grade.These indicated that they may have important potential prognosis value and may be used as new markers to the prognosis evaluation of breast carcinoma. The overexpression of EphA2 is associated with lymph node metastases, indicating the possibility that it may be involved in metastasis of breast carcinoma.3.Consistent pattern of protein expressions of EphA2 and EphrinAl in breast carcinoma reveals EphrinAl is one of the major ligands which interacted with EphA2 receptor, and EphA2 and EphrinAl could coordinate with each other in signal conduction in breast carcinoma. EphA2 and EphrinAl colocalized in tumour cells and vascular endothelial cells in breast carcinomas, suggesting that EphA2 and EphrinAl may may be invovled in tumour angiogenesis, and therefore could be attractive new targets for breast cancer therapy.4.Protein expression of ERP and ERa in breast carcinomas suggesting that breast cancer is a kind of hormone-dependent tumor, and we can give directions to clinical endocrine treatment in accordance with the receptor content.5.There were significantly correlations between EphA2 and EphrinA1,and hinted the expression of EphrinAl was induced by EphA2,EphrinA1 acted on its receptor EphA2 in an autocrine and/or paracrine manner to promote the growth of tumor cells and proliferation of endothelial cells. 6.The protein exprssion of EphA2 were significantly negatively correlated with the expression of ERa protein, and positively correlated with ERβprotein in breast carcinoma(all P<0.05),It old us that EphA2 may be negative regulated by ERa, and may be positively regulated by ERβ.7. Detecting of the expression of EphA2 and EphrinA1 may be valuable for evaluating the prognosis of breast carcinoma.Part two:Expressions of ER,PR and EphA2 in Breast Carcinomas with Neoadjuvant Chemotherapy and Their SignificanceObjectivesTo study the influence of neo-adjuvant chemotherapy on expression of ER,PR and EphA2 in breast cancer and their clinical sigficance.MethodsThe expressions of ER,PR and EphA2 were detected by SP immunohistochemical method in breast cancer tissues of 52 patients before and after Neoadjuvant Chemotherapy.ResultsThe expressions of ER,PR and EphA2 were significantly related to treatment response(P<0.05).The positive rate of EphA2 in breast cancer tissues after neoadjuvant chemotherapy was significantly lower than that before the treatment(P<0.05),but The difference of ER,PR before and after Neoadjuvant chemotherapy were insignificant(P>0.05).ConclusionsNeoadjuvant chemotherapy could inhibit tumor proliferation and induce tumor cell apoptosis,and significantly decrease the expression of EphA2,but it could not reduce the expression of ER and PR significantly. Part Three:The expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinomas and their significanceObjectives:To study the expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma and their significanceMethods1.Frozen tissues from 40 cases in which the tumours showed different grades of EphA2 and EphrinAl protein expressions were used for extraction of mRNA.2.The levels of EphA2 and EphrinA 1 mRNA were detected by RT-PCR and compared if the levels of mRNA correspond to their levels of protein and clinicopathological features.3.Statistical analysis:All the data were analyzed by the SPSS version 13.0 statistical package.We have used t-test(student t),One-way ANVOA and Nonparametric test(Kruskal-Wallis test method).P values of<0.05 were considered statistically significant.Results:1.The positive expressions of EphA2 mRNA were associated with histological grade,lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).2.The positive expressions of EphrinAl mRNA were associated with histological grade, lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).3.All the 40 breast carcinoma specimens with different grade EphA2 protein expression have expressed EphA2 mRNA.The mRNA expression levels in those 10 cases with negative protein expression (0.34±0.14) were obviously lower than those 30 cases with 1～3 grade protein expression(protein gradel:1.33±0.25,grade 2: 1.55±0.11,grade 3:1.71±0.19,each compare with grade 0:all P<0.05).Among the 22 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphA2mRNA expression did not fully correspond to its protein expression in breast carcinoma.4. All the 40 breast carcinoma specimens with different grade EphrinAl protein expression have expressed EphrinAl mRNA.The mRNA expression levels in those 7 cases with negative protein expression(0.41±0.11)were obviously lower than those 33 cases with 1～3 grade protein expression(protein gradel:1.37±0.38, grade 2: 1.59±0.28,grade 3:1.67±0.19, each compare with grade 0:all P<0.05).Among the 21 cases with protein expression form grade 1 to 2,There were no significant difference in their mRNA expression levels(P>005).Among the 24 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphrinA1 mRNA expression did not fully correspond to its protein expression in breast carcinoma.Conclusions1.The positive expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma were associated with histological grade, lymph node metastasis and clinical stage(P<0.05).This indicated that the positive expressions of EphA2 mRNA and EphrinAl mRNA were related to the malignant biological properties of breast carcinoma. And indicating that they may become new markers to the evaluation of the prognosis of breast carcinoma.2.To some extent, the positive expression of EphA2mRNA and EphrinA1 mRNA in breast carcinoma were associated with the expression of EphA2 protein and EphrinAl protein respectively, indicating that they were both regulated at transcriptional level. 3.The positive expression of EphA2mRNA and EphrinAlmRNA in breast carcinoma were partly different from the expression of EphA2 protein and EphrinAl protein respectively, indicating that there may be some post-transcriptional and translational regulation mechanisms, some accumulation obstacles and some degradation pathway disorders.Part four:Study of targeted silencing of EphA2 in breast cancer cell line MCF-7 by RNA interferenceObjectivesTo investigate the effects of retrovirus-mediated siRNA targeting EphA2 on the breast cancer cell line MCF-7, in order to provide the possibility of theoretical basis of gene therapy in vitro for breast cancer.Methods1.Based on the human EphA2mRNA gene sequencing and according to the design principle, two complementary oligodeoxyribonucleotides encoding EphA2 short hairpin RNA were designed and synthesized with the software online.2.Then these two complementary oligodeoxyribonucleotides were annealed and ligated into a linear vector RNAi-READY-pSIREN-RetroQ to construct the recombinant plasmid pSIREN-RetroQ-EphA2.The control vector (pSIREN-RetroQ-C) was constructed at the same time which contain a free correlation sequence.3.The ligation mixtures were transformed into competent E.coli DH5a. And the recombinant plasmids pSIREN-RetroQ-EphA2 were extracted from small-scale bacterial cultures by Alkaline Lysis, Subsequently the plasmids were digested and then identified by DNA sequence analysis.4.The vectors based RNAi were transfected into packing cell line PA317,which was selected by puromycin later. 5.MCF-7 cells were transfected by recombinant plasmids pSIREN-RetroQ-EphA2 and stable transfectants were isolated by puromycin.6.The EphA2 protein in transfected MCF-7 cells was detected by Western blots.Results1.The pSIREN-RetroQ-EphA2 vector was confirmed by restriction endonuclease digestion and It was verified by DNA sequencing that the insert sequence was successfully cloned into the vector.2.Compared with cells transfected with empty retroviru vector and untransfected cells, the level of EphA2 protein expression was greatly decreased in MCF-7 cells transfected by the recombinant retroviru vector by Western blots techniques.Conclusions1.We selected effective EphA2 siRNA sequence from EphA2 mRNA sequence and designed and synthesized the hairpin structure of double complementary chains of oligonucleotides,The recombinant retro viral vectors were constructed successfully and retroviru has been successfully transfected into MCF-7 cells line and it works well.2.EphA2 protein expression in MCF-7 cell line is successfully suppressed by using the recombinant retroviru-mediated RNAi technique, indicating that this may contributes to the study of the changes of malignant biological activity of breast cancer cell lines by transfection of siRNA retrviral vectors and provide a new solution for EphA2-targeting therapeutic intervention of breast carcinoma.