【作者】 吴艳萍；

【导师】 朱铁年；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2010， 博士

【摘要】 目的:乳腺癌是严重威胁女性健康的第一位恶性肿瘤。随着经济发展和人民生活水平的提高,乳腺癌的发病率呈现上升的趋势,肿瘤复发和远处转移是乳腺癌患者主要的死亡原因。乳腺癌的发生和发展机制至今仍未完全阐明,临床尚缺乏有效的治疗措施。表皮生长因子受体(epidermal growth factor receptor,EGFR)家族包括erbB-1/EGFR、erbB-2/HER2/neu、erbB-3和erbB-4四大成员,属于Ⅰ型酪氨酸激酶受体,酪氨酸磷酸化可使受体活化,控制着细胞的增殖、分化、移动和存活,受体的表达失调与肿瘤的发生相关。HER2是细胞来源的癌基因,在正常情况下处于非活化状态,参与细胞生长、分化的调节,在乳腺癌的研究中,HER2的扩增和过度表达研究最为深入,见于30%的乳腺癌,与乳腺癌的浸润和扩散有关,曲妥珠单抗(Trastuzumab、Herceptin、赫赛汀)是针对HER2强阳性表达乳腺癌首选的生物靶向治疗药物。但临床有19%乳腺癌的HER2、雌激素受体(estrogen receptor, ER)和孕激素受体(progesterone receptor, PR)均为阴性,且易发生复发和转移,预后较差,目前仍无有效的治疗靶点。研究发现EGFR蛋白在此类乳腺癌呈现过表达,且乳腺癌的表型与EGFR蛋白、淋巴结转移相关。EGFR是细胞膜的跨膜受体,与细胞的增殖、分化、运动、存活有关。正常细胞表达适量的EGFR,用于维持细胞的生命活动,但其过表达或活性增强可持续启动细胞增生信号的传递系统,致细胞过度增殖和表型恶化。EGFR在超过35 %～50 %的乳腺癌组织中表达。EGFR的表达与乳腺癌患者的预后有关,其表达越多则乳腺癌的预后越差,有淋巴结转移的乳腺癌的EGFR表达明显高于无淋巴结转移者。EGFR酪氨酸的磷酸化是EGFR信号转导过程中的关键分子事件,EGFR的磷酸化程度决定着肿瘤的预后,EGFR磷酸化程度高,肿瘤的预后差。影响EGFR磷酸化的原因目前不清楚,因此本研究重点探讨影响EGFR磷酸化的分子,为寻找治疗肿瘤的新靶点奠定基础。细丝蛋白A(Filamin A,FLNa)又称为Filamin-1或ABP-280,是细胞内一种肌动蛋白结合蛋白,同时又是脚手架蛋白,在细胞浆内广泛分布,也可以跨膜分布或转位至细胞核。FLNa蛋白是以“V”形的同型二聚体存在,每个FLNa分子内含一个肌动蛋白结合区(actin-binding domain,ABD)、24个重复单位及两个绞链区。特殊结构决定其具有强大的功能:FLNa与肌动蛋白丝结合,有助于细胞运动,与细胞增殖、迁徙、血管形成和器官发生有关;FLNa为脚手架蛋白,将细胞内多种蛋白分子与细胞骨架连接,并募集细胞膜的共刺激分子,参与细胞内的多种重要功能的信号转导和细胞对外界刺激的反应。许多研究证实FLNa在恶性肿瘤细胞的表达增多。如肺癌细胞内FLNa的表达上调,与肺癌的迁徙、粘附和侵袭相关;前列腺癌细胞的胞浆内FLNa表达较正常前列腺组织显著增高,且有转移的前列腺癌FLNa的表达高于无转移者;乳腺癌细胞内也存在FLNa高表达。通过免疫组化、流式细胞技术已观察到FLNa的表达量与乳腺癌的恶性度和转移相关,FLNa的表达随乳腺癌恶性度的增高而增加,有淋巴结转移的乳腺癌的FLNa表达明显高于无淋巴结转移者。Fiori和Zhu等对不同转移能力的黑色素瘤细胞系研究发现FLNa可影响EGFR的磷酸化(见第二部分文献2)。但是,FLNa是否也影响乳腺癌细胞的EGFR的磷酸化目前尚未见报道。为此,我们拟利用人乳腺癌细胞系,通过转染FLNa的siRNA降低FLNa表达、转染FLNa全长基因增加FLNa的表达后,观察EGFR的磷酸化水平及与EGFR磷酸化的相关的信号转导分子的变化;经免疫共沉淀(co-Immunoprecipitation,IP)证实FLNa与EGFR分子间的相互关系;利用MTT检测FLNa的表达对乳腺癌细胞增殖的影响。从组织、细胞和分子水平综合分析FLNa对EGFR磷酸化的调控,探讨FLNa调控EGFR磷酸化在乳腺癌发病中的作用,为有效治疗乳腺癌提供新靶点。第一部分细丝蛋白A在浸润性乳腺癌组织中的表达及与临床病理特征的相关性方法:采用链霉抗生物素蛋白-过氧化物酶(SP)免疫组织化学方法和流式细胞分析技术,检测46例分化程度不同的浸润性乳腺癌细胞中FLNa和增殖细胞核抗原(pro-liferating cell nuclear antigen, PCNA)的表达情况,以正常乳腺组织或乳腺良性增生为阴性对照。结果:1. FLNa在乳腺癌组织中的表达免疫组化结果:FLNa在正常乳腺上皮细胞的胞浆内少量表达(Fig. 1-1);FLNa在浸润性乳腺癌细胞阳性表达的程度较强,且随组织分化程度的降低而增高(Fig.1-2～4、Table 1-1),差异有统计学意义(P<0.05);有淋巴结转移乳腺癌细胞的FLNa的表达明显强于无转移组(Table 1-1),差异有统计学意义(P<0.05)。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。流式细胞检测结果: FLNa在正常乳腺组织的表达量较少;在低分化乳腺癌细胞中FLNa的表达(1.22±0.13)明显高于中高分化的乳腺癌细胞(1.10±0.11),差异有统计学意义(P<0.05),见Fig.1-6、Table 1-2。无淋巴结转移者(1.21±0.11)明显高于有淋巴结转移者(1.10±0.10),差异有统计学意义(P<0.05),见Table 1-2。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。2. PCNA在乳腺癌组织中的表达免疫组化结果:PCNA在浸润性乳腺癌细胞核呈阳性表达,PCNA的表达随乳腺癌分化程度的降低而增强,在低分化浸润性乳腺癌细胞的表达呈强阳性,见Fig.1-5。流式细胞检测结果:低分化浸润性乳腺癌细胞中PCNA的表达(1.65±0.22)明显高于中高分化乳腺癌(1.35±0.15),差异有统计学意义(P<0.05),无淋巴结转移的乳腺癌细胞PCNA的表达(1.42±0.15)明显低于有淋巴结转移者(1.56±0.16),差异有统计学意义(P<0.05),见Fig.1-7、Table 1-3。PCNA的表达与FLNa呈正相关(P<0.05)。第二部分FLNa对乳腺癌细胞表皮生长因子受体活化的影响方法:1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响用含10%胎牛血清的低糖DMEM培养乳腺癌MDA-MB-231细胞。实验分组:FLNa-siRNA转染组;通用HK对照转染组。每组分别经EGF(20nM)刺激后0min、5min和10min后收集细胞,提取蛋白,Western-blot检测各时间点FLNa、EGFR、磷酸化EGFR (p-EGFR)、ERK (extracellular signal-regulated kinase)和磷酸化ERK(p-ERK)蛋白的表达。2. FLNa过表达对乳腺癌细胞EGFR活化的影响细胞培养同方法1。实验分组:人FLNa-full(hfilamin A/pREP4质粒,内含FLNa全长基因)转染组;空质粒对照pcDNA3.1转染组。每组分别经EGF(20nM)刺激0min、5min和10min后收集细胞,提取蛋白, Western-blot检测各时间点FLNa、EGFR、p-EGFR、ERK和p-ERK蛋白的表达。3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR的活化影响细胞培养同方法1。用含10%胎牛血清的DMEM培养液调细胞浓度为3.5×105/ml,接种至60mm培养皿。实验分组:转染FLNa-siRNA组;转染通用HK对照组。每组分别经EGF(20nM)刺激0min和10min后,收集细胞,提取蛋白,对提取的蛋白分别用抗EGFR抗体和抗磷酸化酪氨酸的4G10抗体免疫共沉淀,Western-blot分别检测沉淀蛋白内p-EGFR和EGFR的水平。结果:1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响1.1 FLNa蛋白的表达:FLNa-siRNA转染组FLNa的表达(1.40±0.10、1.27±0.06)较HK对照组(3.97±0.29、3.33±0.06)明显减少,差异有统计学意义(均P<0.01),见Fig.2-1、Table 2-1。1.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-siRNA转染组的EGFR磷酸化水平(0.73±0.01、0.37±0.05)明显低于HK对照组(1.08±0.01、1.30±0.06),差异有统计学意义(均P<0.01),见Fig.2-2、Table 2-2。1.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa的siRNA转染组ERK磷酸化水平(0.17±0.00、0.12±0.04)明显低于HK对照组(0.32±0.02、0.54±0.04),差异有统计学意义(均P<0.01),见Fig.2-3、Table 2-3。2. FLNa过表达对乳腺癌细胞EGFR活化的影响2.1 FLNa的表达:FLNa-full质粒转染组FLNa的表达(5.93±0.38、6.07±0.32)明显高于空质粒pcDNA3.1对照组组(4.50±0.20、4.60±0.17),差异有统计学意义(均P<0.01),见Fig.2-4、Table 2-4。2.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组EGFR的磷酸化水平(1.53±0.11、0.86±0.14)明显高于空质粒pcDNA3.1对照组(0.57±0.01、0.44±0.02),差异有统计学意义(分别P<0.01,P<0.05),见Fig.2-5、Table 2-5。2.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组ERK的磷酸化水平(1.83±0.07、1.06±0.07)明显高于空质粒pcDNA3.1对照组(0.74±0.01、0.59±0.03),差异有统计学意义(均P<0.01),见Fig.2-6、Table 2-6。3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR活化的影响3.1 FLNa-siRNA转染后FLNa的表达:FLNa-siRNA转染组FLNa的表达(3.53±1.08、4.33±1.33)较HK对照组(7.23±2.20、7.53±1.86)组明显减少,差异有统计学意义(均P<0.01)。见Fig.2-7、Table 2-7。3.2经抗EGFR抗体免疫沉淀后的EGFR磷酸化的水平: EGF刺激10min后,FLNa-siRNA转染组的EGFR磷酸化水平(1.09±0.09)明显低于HK对照组(2.32±0.22),差异有显著性(P<0.01)。见Fig.2-8、Table 2-8。3.3经抗酪氨酸磷酸化4G10抗体免疫沉淀后EGFR的表达: EGF刺激10min时,FLNa-siRNA转染组的EGFR表达(0.42±0.01)明显低于HK对照组(1.07±0.01),差异有显著性(P<0.01),见Fig.2-9、Table 2-9。第三部分细丝蛋白A对乳腺癌细胞增殖的影响方法:细胞培养见第二部分的方法1。用含10%胎牛血清的低糖DMEM培养液调细胞浓度为9×103/ml,按100μL /孔体积接种于96孔板。实验分组:FLNa-siRNA转染组和HK对照组;FLNa-full质粒转染组和空质粒pcDNA3.1对照组。分别用不同浓度的EGF(4nM、20nM、100nM)刺激过夜,用MTT法检测细胞的增殖水平。结果:1.FLNa沉默后MDA-MB-231细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-siRNA转染组细胞的增殖水平随刺激浓度的增加而增高, FLNa沉默后细胞的增殖水平(1.28±0.09、1.46±0.02、1.58±0.06)均低于对照组(1.55±0.12、1.62±0.06、1.68±0.12),差异有显著性(p<0.01),见Fig.3-1、Table 3-1。2. FLNa过表达后MDA-MB-231的细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-full质粒转染组细胞的增殖水平随刺激浓度的增加而增高,但FLNa-full质粒转染组细胞的增殖水平(1.14±0.08、1.28±0.11、1.51±0.03)高于对照组(1.09±0.17、1.21±0.17、1.25±0.05),差异有显著性(P<0.01),见Fig.3-2、Table 3-2。结论:1. FLNa在正常乳腺组织少量表达;FLNa的表达随浸润性乳腺癌分化程度的降低而增高,且与淋巴结转移有关;FLNa的表达与乳腺癌细胞的增殖能力呈正相关。2. FLNa的表达可调控乳腺癌细胞EGFR的磷酸化,磷酸化的EGFR又通过MAPK增殖信号传导通路活化ERK,影响乳腺癌细胞的增殖。3.沉默FLNa的表达导致乳腺癌细胞的增殖水平降低;FLNa的过表达则使乳腺癌细胞的增殖水平升高,表明FLNa可通过调控EGFR的活化影响乳腺癌的发生和发展。更多还原

【Abstract】 Objective:Breast Carcinoma is the leading fetal cancer that affects the health of women now. With the development of economy and the improve- ment of peoples’life, the mortality of breast carcinoma is going up. Breast carcinoma recurrence and metastasis are the main death causes of these patients. However, the mechanism of occurrence and development remains obscure. Patients suffering from breast cancer have a poor prognosis because of deficiency in effective treatment measures in clinical practices.EGFR family encompasses members of erbB-1/EGFR, erbB-2/ HER2/ neu, erbB-3 and erbB-4. They belong to tyrosine kinase receptor which can be activated by tyrosine phosphorylation. HER2 is unactived oncogene in normal correlating with regulating to growth and differentiation. Amplification and overexpression of HER2 have been examined in a deep-going way and associated with invasion and diffusion. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, Nineteen percent of breast carcinoma which has no EGFR, ER and PR, is prone to recurrence and metastasis. Total survival rate is lower than that of other breast carcino ma.These patients have worse prognisis. But effective targeted therapy has not been emerged now. Studies have shown that overexpression of EGFR exists in this kind of breast carcinoma whose phenotype is significantly correlated with EGFR and lymphnode metastasis.EGFR distributes in the way of transmembrane controlling proliferation, differentiation, motility and survival. Appropriate amount expression in normal tissue is necessary for normal vital movement.Overexpression or strong activity of EGFR can promote proliferation system. The excessive proliferation and malignant phenotype will be emerged. Overexpression of EGFR has been in 35%～50 % breast carcinoma and correlates with prognosis. The expression of EGFR in metastasis is significantly higher than that of non-metastasis one. No recurrence and high survival rate result from negative expression of EGFR. Tyrosine phosphorylation is the key molecular event in the EGFR signaling pathway. The level of EGFR phosphorylation decides the prognosis of breast carcinoma. Higher phosphorylation of EGFR will lead to poor prognosis. However, the mechanisms accounting for the upregulation of EGFR activity are largely unknown. So searching the factors that affect EGFR activity is the key purpose and provides foundation for new target in tumor.Filamins A (Filamin-1, FLNa) are large actin-binding and scaffolding proteins expressed widely in cytoplasm. FLNa can distribute in the way of transmembrane or localize to the nucleus. Vertebrate filamins are elongated dimeric V-shaped proteins with two large polypeptide chains. Each monomeric chain of filamins consists of an N-terminal actin-binding domain (ABD), a long rod-like domain of twenty-four repeated and two hinges. FLNa is endued with powerful functions because of special structure. FLNa stabilizes cortical three-dimensional F-actin networks, anchors cytoskeleton to cellular membranes by binding to transmembrane receptors and integrate cell architecture and functions signaling, which is essential for cell motility correlating with proliferation, migration, organ development and potential role in oncogenesis;Acting as cross-linking proteins, Filamins not only link various signaling proteins to the cytoskeleton,but also collect costimulus molecule of cytomembrane. So they can take part in many important signaling transduction and make cell response to external stimulus in time;Overexpression of FLNa has been confirmed in many malignancy tumors. It correlates with migration, adhesion and invasion in lung and prostate carcinoma. High expression of FLNa exists in breast carcinoma, too. The expression of FLNa correlating with the malignancy degree and metastasis has been proved with immunohistochemistry and Flow cytometry. Fiori and Zhu et al have shown that FLNa can regulate the activity of EGFR in melanoma cells and the expressions of FLNa and EGFR in melanoma with highly metastatic potentiality are higher than that of lower one. Up to now, FLNa influencing EGFR phosphorylation or not has not been reported.Thus, the phosphorylation level of EGFR and correlated signaling molecules in breast carcinoma cells were observed after FLNa siRNA and full-lengh FLNa transfection, then the relationship between FLNa and EGFR phosphorylation as well as the effection of FLNa on proliferation were detected by Immunoprecipitation and MTT, respectively. Analysis was performed aggregately about the role of FLNa regulating to EGFR plosphorylation in breast carcinoma genesis from the levels of tissue, cell and molecule. In this study, the putative connection between FLNa and EGFR plosphorylation was explored and would provide a new target for effective treatment of breast carcinoma.Part one The expression of filaminA in invasive breast carcinoma and its relationship with clinicopathological featuresMethods:Streptavidin-biotin-peroxidase (SP) Immunohistochemistry and Flow Cytometry were applied to detect the expression of FLNa and PCNA in 46 cases invasive breast cancer with different differentiation, using normal breast tissue or benign breast hyperplasia as negative control.Results:1. FLNa expression in breast cancer cells1.1 FLNa expression with immunohistochemical detection in breast cancer cells:FLNa expression was mainly distributed in the cytoplasm of mammary epithelial cells, staining shallow was regarded as negative in normal breast tissue (Fig.1-1); however, FLNa positive expression was gradually increased with the reducing of differentiation in invasive breast cancer. There were significant differences between the results of different differentiations (P <0.05); FLNa expression in the metastasis group was significantly stronger than that of non-metastasis one, the differences between them was statistically significant (P <0.05). FLNa has nothing to do with the histologic types in invasive breast cancer cells. (Fig.1-2～4 and Table1-1)1.2 FLNa expression with Flow cytometry in breast cancer cells:There was little expression of FLNa in normal breast tissue. It was gradually increasing with the reducing of differentiation. The highest expres- sion was in poorly differentiated breast cancer. The expression of FLNa in poorly differentiated breast carcinomas (1.22±0.13) was higher than that of well differentiated ones (1.10±0.11); Metastasis group (1.21±0.11) was higher than that of non-metastasis group (1.11±0.10), too. It has nothing to do with the histological types. There was a statistical difference between two groups (P <0.05). (Fig.1-6 and Table1-2)2. PCNA expression in breast cancer cells2.1 The results of PCNA with immunohistochemical detection in breast cancer cells:The positive expression of PCNA in nucleus of breast cancer cell was very similar to the expression tendency of FLNa. PCNA expression had also changed with the differentiation. PCNA expression showed strong positive expression in poorly differentiated invasive breast cancer. (Fig.1-5)2.2 The results of PCNA with Flow Cytometry in breast cancer cellsThe expression of PCNA increased with differentiation decreased. The expression of PCNA in poorly differentiated breast carcinoma (1.65±0.22) was significantly higher than that of well-differentiated ones (1.35±0.15); Metastasis group (1.56±0.16) was higher than that of non-metastasis group (1.42±0.15), too. There was a statistical difference between two groups (P <0.05). (Fig.1-7 and Table 1-3). The expression of PCNA was correlated positively with that of FLNa (P <0.05).Part two Effect of FLNa on the EGFR phosphorylation in breast carcinoma Methods:1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.Human breast carcinoma MDA-MB-231 cells were cultured at 37℃, 5%CO2 in DMEM media containing 10% fetal calf serum and divided into two groups: FLNa-siRNA group and HK group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were assessed by western-blot at the end.2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.MDA-MB-231 cells were cultured and divided into two groups: FLNa-full group and pcDNA3.1 group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were examined by western-blot at last.3. FLNa regulating to EGFR phosphorylation was demonstrated by co- immunoprecipitation in human breast carcinoma.MDA-MB-231 cells were cultured and divided into two groups: FLNa-siRNA group and HK group.Each group was stimulated for 0min and 10min by 20nM EGF. Then 4G10 or EGFR protein immuneprecipitation was performed. The expressions levels of EGFR and p-EGFR were assessed by western-blot at the end.Results:1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.(1)The expression of FLNaAfter FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (1.40±0.10, 1.27±0.06) were lower than that of control group of HK. (3.97±0.29, 3.33±0.06). There was a statistical difference between two groups(P<0.01).(Fig. 2-1, Table 2-1)(2)The expression of phosphorylated EGFRAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-siRNA group (0.73±0.01, 0.37±0.05) were lower than that of control group of HK (1.08±0.01, 1.30±0.06). There was statistical difference between two groups(P<0.01). (Fig. 2-2, Table 2-2)(3) The expression of phosphorylated ERK After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-siRNA group (0.17±0.00, 0.12±0.04) were lower than that of control group of HK (0.32±0.02, 0.54±0.04). There was a statistical difference between two groups(P<0.01). (Fig. 2-3, Table 2-3).2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.(1)The expression of FLNaAfter FLNa-full transfection, the expressions of FLNa in FLNa-full group (5.93±0.38, 6.07±0.32) were higher than that of control group of pcDNA3.1 (4.50±0.20, 4.60±0.17). There was a statistical difference between two groups (P<0.01). (Fig. 2-4, Table 2-4).(2)The expression of phosphorylated EGFRAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-full group ( 1.53±0.11,0.86±0.14) were higher than that of control group of pcDNA3.1(0.57±0.01, 0.44±0.02).There was statistical difference between two groups(P<0.01, P<0.05). (Fig. 2-5, Table 2-5).(3) The expression of phosphorylated ERK after FLNa-full transfectionAfter stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-full group (1.83±0.07, 1.06±0.07) were higher than that of control group of pcDNA3.1 (0.74±0.01, 0.59±0.03). There was statistical difference between two groups(P<0.01). (Fig. 2-6, Table 2-6). 3. FLNa regulating to EGFR phosphorylation was proved by Immunoprecipi- tation in human breast carcinoma.3.1The expressions of FLNa with Western blot detection after FLNa-siRNA transfectionAfter FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (3.53±1.08,4.33±1.33) were lower than that of control group of HK (7.23±2.20,7.53±1.86). There was statistical difference between two groups(P<0.01). (Fig. 2-7, Table 2-7)3.2 The expression of phosphorylated EGFR after EGFR IP: After stimulation of EGF at 10min,the expression of phosphorylated EGFR in FLNa-siRNA group (1.09±0.09) was lower than that of control group of HK (2.32±0.22).There was statistical difference between two groups(P<0.01). (Fig. 2-8, Table 2-8)3.3 The expression of EGFR after 4G10 IP:After stimulation of EGF at 10min,the expression of EGFR in FLNa- siRNA group(0.42±0.01) was lower than that of control group of HK (1.07±0.01). There was statistical difference between two groups(P<0.01). (Fig. 2-9, Table 2-9)Part Three Effect of FLNa on proliferation of breast carcinoma Methods:MDA-MB-231 cells were cultured and transferred cells into 96 holes plate. MDA-MB-231 cells were divided into two groups: FLNa-siRNA and HK-siRNA transfection group;FLNa-full and pcDNA3.1 plasmid transfection group.EGF stimulating was done with concentration of 4nM, 20nM and 100nM on the two groups overnight. The MDA-MB-231 cells proliferation was examined by MTT after FLNa-siRNA and FLNa-full plasmid trans- fection.Results:1. MTT detection after FLNa-siRNA transfectionUnder the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-siRNA group (1.28±0.09, 1.46±0.02, 1.58±0.06 was lower than that of control group of HK(1.55±0.12, 1.62±0.06, 1.68±0.12).There was statistical difference between two groups (P<0.01).The proliferation level of two groups was increasing following concentration of EGF.2. MTT detection after FLNa-full transfectionUnder the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-full group (1.14±0.08, 1.28±0.11,1.51±0.03) were higher than that of control group of pcDNA3.1 (1.09±0.17, 1.21±0.17, 1.25±0.05). There was statistical difference between two groups(P<0.01). The proliferation level of two groups was rising following concentration of EGF.Conclusion:1. The expression of FLNa is little in normal breast tissue. It is gradually increasing with the reducing of differentiation and correlated with lymphnode metastasis in breast carcinoma. The expression of FLNa is correlated positive- ly with proliferation of breast carcinoma.2. The expression of FLNa can regulate EGFR phosphorylation which active the molecule of ERK through MAPK signaling pathway and effect the proliferation of breast carcinoma.3. FLNa silence can lead to the proliferation level of breast carcinoma decreasing, while the overexpression of FLNa can lift the the proliferation level of breast carcinoma.The results show that FLNa can regulate phos- phorylation of EGFR and effect genesis and development of breast carcinoma.更多还原