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乳腺癌组织中FANCF相关基因的表达及其与顺铂耐药的作用机制研究
中文摘要

【目的】 研究非化疗组和化疗组不同亚型乳腺癌组织中FANCF相关基因mRNA的表达情况,寻找与乳腺癌密切相关的FA通路基因;在此基础上进一步研究顺铂耐药的癌组织中FANCF的表达情况;通过建立三阴型乳腺癌细胞MDA-MB-231顺铂耐药细胞模型,验证FANCF耐药前后的差异表达,并通过RNAi干扰技术敲低FANCF的表达,探讨FANCF在乳腺癌顺铂耐药中的作用及其机制。 【方法】 收集手术切除的女性乳腺癌患者乳腺癌组织和正常乳腺组织标本共93例,其中非化疗组的三阴型乳腺癌、Luminal型乳腺癌、HER2阳性型乳腺癌各20例,化疗组相对应的三型乳腺癌分别为11、13和9例。采用实时荧光定量RT-PCR法检测乳腺癌组织和正常乳腺组织中的FANCF相关基因FANCA、FANCD2、 FANCF、FANCI、BRCA1和BRCA2 mRNA的表达水平。收集顺铂敏感和顺铂耐药的乳腺癌复发转移灶组织石蜡切片共27例,其中顺铂敏感14例、顺铂耐药13例,采用免疫组化方法检测FANCF在其中的表达。以体外药物浓度递增法建立三阴型乳腺癌细胞MDA-MB-231顺铂耐药细胞模型。CCK8法检测细胞的顺铂抑制活性,实时荧光定量RT-PCR法检测诱导顺铂耐药前后FANCF的mRNA表达,Western blotting法验证差异蛋白的表达。通过RNAi干扰技术敲低MDA-MB-231敏感细胞和顺铂耐药细胞中FANCF的表达,在mRNA和蛋白水平进行敲低效果验证。以CCK8法检测顺铂对两种细胞的IC₅₀,流式细胞仪分析细胞凋亡的情况。 【结果】 1、FANCA、FANCF、FANCD2、FANCI、BRCA1和BRCA2在93例非化疗组和化疗组患者组织样本的mRNA表达:非化疗组三阴型、Luminal型、HER2型乳腺癌组织中FANCF基因mRNA相对表达量均低于相应正常乳腺组织(P <0.01),HER2型乳腺癌组织中BRCA1、BRCA2、FANCI和FANCD2基因mRNA相对表达量高于对应的正常乳腺组织且均高于三阴型和Luminal型乳腺癌组织中的mRNA表达量,三阴型乳腺癌组织中FANCD2和FANCI基因的mRNA相对表达量高于相应的正常乳腺组织,Luminal型乳腺癌组织中FANCA基因mRNA表达量高于HER2型和三阴型(P<0.05);化疗组患者的组织样本中三阴型乳腺癌组织中除FANCF外其余5个基因表达量均非常低,Luminal型和HER2型乳腺癌组织中BRCA1、FANCI的表达均显著高于正常乳腺组织,Luminal型乳腺癌患者BRCA1、BRCA2、FANCA、FANCI基因mRNA表达量均高于三阴型和HER2型(P均<0.05);化疗组三种亚型乳腺癌组织中FANCF的表达水平高于非化疗组(P<0.05),且TNBC组差异最显著,三阴型和HER2型乳腺癌组织的BRCA1、BRCA2、FANCD2、FANCI基因mRNA表达量显著低于对应非化疗组患者的乳腺癌组织的表达量(P< 0.05);基因相关性分析结果显示FANCF的表达与其余5个基因的表达水平呈现显著负相关,FANCA、FANCD2、FANCI、BRCA2、BRCA1 5个基因之间的表达水平呈显著正相关。 2、免疫组化检测发现FANCF在顺铂耐药的乳腺癌复发转移灶中的表达显著高于顺铂敏感的乳腺癌复发转移组织(P<0.05)。 3、体外顺铂浓度递增法诱导MDA-MB-231细胞顺铂耐药3个月后的耐药指数为13.5,G0/G1期细胞增多、S期和G2/M期细胞减少。在顺铂耐药的乳腺癌细胞中FANCF的mRNA和蛋白表达水平显著升高(P<0.05)。 4、使用RNAi干扰技术敲低FANCF后,MDA-MB-231和MDA-MB-231/Pt细胞凋亡增加、细胞对顺铂的药物敏感性显著升高,且以MDA-MB-231/Pt细胞明显。 【结论】 1、FANCF基因在非化疗组乳腺癌组织中相对表达量均低于相应正常乳腺组织,而在化疗组乳腺癌组织中FANCF基因mRNA的表达上调,提示这与化疗药物的使用有一定关系;FANCF mRNA在非化疗组和化疗组乳腺癌组织中的表达存在差异,尤其在TNBC中的表达差异最为显著;三种不同亚型乳腺癌组织中FANCF相关基因的表达差异性较大,新辅助化疗可在一定程度上影响BRCA1、BRCA2、FANCD2、FANCI的表达。 2、FANCF在顺铂耐药的乳腺癌复发转移组织中的表达显著增加,提示FANCF可能与乳腺癌顺铂耐药有关。 3、在三阴型乳腺癌细胞MDA-MB-231顺铂耐药细胞模型中,顺铂耐药细胞MDA-MB-231/Pt较敏感细胞MDA-MB-231中FANCF的mRNA和蛋白表达水平均显著升高,推测三阴型乳腺癌细胞MDA-MB-231顺铂耐药性的产生可能与FANCF基因的表达上调有关。 4、体外合成FANCF-siRNA,敲低MDA-MB-231和MDA-MB-231/Pt细胞的FANCF后,细胞对顺铂的敏感性均增加,细胞凋亡率亦显著增加,且以MDA-MB-231/Pt细胞更为明显,提示FANCF可能通过抗凋亡的作用参与三阴型乳腺癌细胞MDA-MB-231顺铂耐药的发生,FANCF有望成为乳腺癌顺铂耐药的潜在治疗靶点。 关键词 乳腺肿瘤;FANCF;三阴型乳腺癌;顺铂;耐药

英文摘要

Objectives: The mRNA expression profiles of FANCF related genes (FANCA, FANCF, FANCD2, FANCI, BRCA1/FANCS and BRCA2/FANCD1) in different subtypes of breast cancer in non-chemotherapy group and neoadjuvant chemotherapy group were examined in order to find which of these genes are closely related to breast cancer. The expression of FANCF in cisplatin-resistant breast carcinoma was further investigated. In addition, the differential expression of FANCF was verified in an induced MDA-MB-231 cisplatin-resistant cell model before and after drug resistance, and the role and mechanism of FANCF in cisplatin-resistant breast cancer were investigated by RNAi knockdown technology. Methods: Ninety-three breast cancer tissues from invasive ductal carcinoma and normal breast tissues were collected during surgical resection. Among them, 20 cases each of triple-negative breast cancer, Luminal breast cancer, and HER2 over-expression breast cancer underwent surgical operation. The cases of these three types of breast cancer who underwent neoadjuvant chemotherapy were 11,13 and 9 cases, respectively. The mRNA expression levels of the FANCF related genes FANCA、 FANCD2、 FANCF、 FANCI、 BRCA1 and BRCA2 in breast samples were detected by means of real-time quantitative PCR (RT-PCR). Tissues from twenty-seven patients of relapsed breast cancer who were treated by chemotherapy based on cisplatin were collected, and the expression of FANCF was examined by using immunohistochemistry in cisplatin-sensitive and cisplatin-resistant relapsed tissues embeded in paraffin. Cisplatin-resistant MDA-MB-231 cell line was established by means of long-term cisplatin-exposed cultures. The sensitivity of the cells to cisplatin was determined by the CCK8 assay. The cell cycle distribution was examined by flow cytometry after exposure to cisplatin. The mRNA and protein expression of FANCF gene was examined using qRT-PCR and western blotting, respectively. The expression of FANCF in the cells was reduced by RNAi interference technology and the effect of the RNAi was verified. The sensitivity of the two cells to cisplatin was examined by CCK8 assay, and the apoptosis of the cells was analyzed by flow cytometry. Results: 1、 The mRNA expression levels of FANCA 、 FANCF 、 FANCD2、 FANCI、 BRCA1 and BRCA2 were detected in both cancer tissues and normal breast tissues. In non-chemotherapy group, the mRNA expression of FANCF gene in breast cancer tissues of the three subtypes was lower than that in normal breast tissues (P< 0.01) . The expression of BRCA1, BRCA2, FANCD2 and FANCI was significant higher in HER2 over-expression breast cancer tissues than normal breast tissues and also higher when compared with those in the other two subtypes cancer tissues. The expression of FANCD2 and FANCI in cancer tissues was higher than that of normal breast tissues in TNBC. The expression level of FANCA in Luminal breast cancer tissues is higher than that in HER2 over-expression type and triple-negative type breast cancer tissues (P<0.05). The expression levels of the other five genes except FANCF were found to be low in neoadjuvant chemotherapeutic triple-negative breast cancer tissues. In neoadjuvant chemotherapy group, the expression levels of BRCA1 and FANCI of Luminal and HER2 subtype in breast cancer tissue are higher than those in normal breast tissue. The expression levels of BRCA1 BRCA2、 FANCA and FANCI in Luminal breast cancer tissues were found to be higher than those in HER2 over-expression type and triple-negative type breast cancer tissues (P<0.05). The expression of FANCF was higher in neochemotherapy group than that in non-chemotherapy group (P< 0.05 ) and the difference between the TNBC group was the most significant. The mRNA expression levels of BRCA1 BRCA2n FANCD2、 FANCI gene in breast cancer tissues of TNBC and HER2 subtypes who underwent neochemotherapy were lower than those in untreated group (P﹤0.05). The results of gene correlation analysis showed that the expression of FANCF was negatively correlated with the other five gene expression levels, and the expression level of FANCA、 FANCD2、 FANCI、 BRCA2 and BRCA1 was positively correlated with each other. 2、 Twenty-seven samples of relapsed breast cancer were examined by immunohistochemical method and the result showed that the expression of FANCF was higher in cisplatin-resistant tissue than that in cisplatin-sensitive breast cancer tissue (P<0.05) . 3、 MDA-MB-231 cell had a 13.5-fold resistance to cisplatin after being induced chemoresistance by means of longterm exposure to cisplatin. Cell cycle analysis indicated that the cisplatin treatment significantly induced G0/G1 arrest and the number of cells in phase S and G2/M decreased after exposure to cisplatin. The mRNA and protein expression levels of FANCF were significantly higher in cisplatin-resistant cells (P<0.05) . 4、 SiRNA-mediated interference of FANCF not only increased sensitivity of both MDA-MB-231 and MDA-MB-231/Pt to cisplatin but also able to upregulate apoptosis of both cells in response to cisplatin. Conclusions: 1、 The mRNA expression level of FANCF gene in breast cancer tissues in non-chemotherapy group was lower than that in corresponding normal breast tissue and the mRNA expression level of FANCF in neoadjuvant chemotherapy group were upregulated, suggesting that the use of chemotherapeutic drugs might play a role in the expression of FANCF. The mRNA expression levels of FANCF between the non-chemotherapy group and the neochemotherapy group were different, and the difference between the TNBC group of genes was the most significant. The mRNA expression levels of FANCF related genes in three different subtypes of breast cancer tissues differ significantly. To a certain extent, neoadjuvant chemotherapy can affect the expression of BRCA1, BRCA2, FANCD2 and FANCI genes. 2、 The finding that the expression level of FANCF was significantly increased in cisplatin-resistant relapsed cancer tissue indicated that FANCF might play a role in cisplatin-resistance of breast cancer. 3、 MDA-MB-231 cells were induced to become cisplatin-resistant in vitro by means of incremental increase of cisplatin concentrations and the cisplatin-resistant cell line MDA-MB-231/Pt was successfully established. The expression levels of FANCF mRNA and protein in MDA-MB-231/Pt cells were significantly higher than those in MDA-MB-231 cells which suggested that the upregulation of FANCF expression in TNBC cells might be related to cisplatin-resistance. 4、 When FANCF gene was knockdown by FANCF-siRNA, the sensitivity to cisplatin and the induction of apoptosis of MDA-MB-231/Pt and MDA-MB-231 cells were significantly increased, which suggested that FANCF gene might play an important role in cisplatin-resistance of TNBC cell MDA-MB-231 and indicated that FANCF might be a novel target to develop treatment for breast cancers. Key words: breast neoplasma; FANCF; TNBC; cisplatin; chemoresistance

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