ALEX1在乳腺癌中的作用及其机制研究
发布时间：2018-09-19 14:31
【摘要】：ALEX/ARMCX(Arm proteins lost in epithelial cancers on chromosome X)蛋白是Arm蛋白家族的成员之一,因只具有一或二个Arm repeat结构,不同于经典Arm蛋白具有的6-13个Arm repeat结构,而被单独列为ALEX蛋白家族。其家族成员包括ALEX1、ALEX2和ALEX3。基因表达分析显示ALEX1 mRNA在人类多种正常组织中高表达而在上皮组织来源的癌组织低表达或不表达。ALEX1基因受CREB和Wnt/p-catenin通路调节并能够抑制人直肠癌细胞的克隆形成。这些研究表明ALEX1很可能在上皮来源的肿瘤中发挥抑癌作用。目前,ALEX1在乳腺癌中的表达及其作用和机制仍不清楚,故本论文主要研究ALEX1在乳腺癌组织标本中的表达水平及对乳腺癌细胞增殖、细胞周期、细胞凋亡和侵袭转移等方面的影响及其分子机制。第一部分：ALEX1在乳腺标本中的表达分析目的：检测乳腺癌组织及对应癌旁组织中的ALEX1表达水平,比较两者之间的表达是否存在差异,明确ALEX1与临床病理特征是否具有相关性。方法：应用real-time PCR、免疫组化及western blot方法检测乳腺癌组织及其对应的癌旁组织中ALEX1 mRNA和蛋白表达水平,统计分析免疫组化检测的ALEX1的表达量与病人年龄、肿瘤大小、肿瘤病理分级、临床分期、淋巴结转移和分子分型之间的关系。结果：对62对乳腺癌及对应癌旁临床组织标本进行免疫组化染色,结果显示：ALEX1表达主要定位于胞浆。统计分析表明：乳腺癌组织ALEX1蛋白的表达水平低于乳腺癌旁组织中的表达(p0.01)；进一步对ALEX1的染色评分与乳腺癌患者临床病理特征之间的关系进行分析,发现乳腺癌中ALEX1的表达与病理分级(1 vs.3,p=0.026; 2 vs.3,p=0.045),临床分期(I vs.Ⅲ,p=0.008;Ⅱ vs.Ⅲ, p=0.011),淋巴结转移(p=0.03)和分子分型(Luminal A型vs. HER-2过表达型,p=0.02)有关,而与病人年龄、肿瘤大小和肿瘤类型无关(p0.05)。此外,随机抽取30对乳腺癌与癌旁组织利用real-time PCR进行检测,发现20例乳腺癌组织中ALEX1 mRNA的表达明显低于对应的癌旁组织中的表达(p0.01)；随机抽取6对乳腺癌与癌旁组织采用western blot对ALEX1蛋白进行检测,结果显示：乳腺癌组织ALEX1蛋白表达水平均低于对应的癌旁组织中的蛋白表达。结论：本研究发现ALEX1在乳腺癌组织中的表达明显低于相应的癌旁组织,乳腺癌的病理分级、临床分期越高,ALEX1的表达水平越低；在有淋巴结转移的乳腺癌组织中ALEX1表达水平明显低于无淋巴结转移的乳腺癌组织,而且在乳腺癌的分子分型中乳腺癌HER-2过表达型组织中ALEX1的表达水平低于Luminal A型。第二部分：ALEXl对乳腺癌细胞增殖、细胞周期及凋亡的影响目的：研究过表达／沉默ALEX1对乳腺癌细胞SK-BR3/MCF-7增殖、周期和凋亡的影响。方法：Real-time PCR和western blot方法检测ALEX1在正常乳腺细胞MCF-10A和乳腺癌细胞MCF-7、T47D、SK-BR3、MDA-MB-231中表达情况,免疫荧光细胞化学技术检测ALEX1亚细胞定位。过表达ALEX1慢病毒感染乳腺癌细胞SK-BR3后,荧光显微镜观察重组慢病毒 LV5-ALEX1的感染效率,real-time PCR和western blot方法检测ALEX1 mRNA和蛋白表达水平。利用合成的三条RNAi序列瞬时转染MCF-7后,用real-time PCR和western blot方法检测ALEX1 mRNA和蛋白表达情况。过表达／沉默ALEX1后CCK8(Cell Counting Kit)检测细胞增殖能力,流式细胞术检测细胞周期和凋亡情况。结果：Real-time PCR和western blot结果显示：在乳腺癌各细胞株中ALEX1的表达水平均低于正常乳腺细胞MCF-10A,但ALEX1在MCF-7细胞株中表达水平高于其他乳腺癌细胞株,在SK-BR3中表达水平最低且ALEX1蛋白定位于胞浆中。过表达ALEX1慢病毒感染乳腺癌细胞SK-BR3 72h后,荧光显微镜观察感染效率达90%以上；与对照组LV5-NC (LV5-Negative Control)相比,实验组LV5-ALEX1中ALEX1 mRNA和蛋白的表达明显增高。CCK8结果显示：LV5-ALEX1组感染LV5-ALEX1慢病毒48 h到96 h SK-BR3细胞生长明显受到抑制(p0.05)。Hoechst染色和流式细胞术结果显示LV5-ALEX1组凋亡细胞数量多于LV5-NC (p0.05)。利用小RNA干扰技术瞬时转染MCF-7细胞48 h后,real-time PCR和western blot结果显示三条RNAi序列中其中RNAi-2序列沉默ALEX1效果明显,选择作为后续实验。实验分两组：实验组SiALEX1和阴性对照组SiCon。CCK8结果显示：转染SiALEX148 h到96 h,MCF-7细胞生长强于阴性对照组SiCon(p0.05)。Hoechst染色和流式细胞术结果显示：MCF-7细胞中SiALEX1组的凋亡细胞数量少于SiCon组(p0.05)。流式细胞仪检测细胞周期分布结果显示：过表达／沉默ALEX1对乳腺癌细胞周期变化均没有影响。结论：过表达ALEX1抑制乳腺癌SK-BR3细胞的增殖,诱导细胞凋亡；沉默ALEX1促进乳腺癌MCF-7细胞的增殖,抑制细胞凋亡。第三部分：ALEX1诱导乳腺癌细胞凋亡的机制目的：深入研究ALEX1诱导乳腺癌细胞凋亡的分子机制。方法：Western blot方法检测过表达／沉默ALEX1乳腺癌细胞中凋亡相关蛋白的表达情况。利用生物信息学方法预测调控ALEX1的miRNAs。Western blot方法和双荧光素酶报告实验验证miR-590-5p调控ALEX1。然后分别以MCF-7和SK-BR3乳腺癌细胞为实验模型,在MCF-7细胞中将实验分为NC mimics组和miR-590-5P mimics组;SK-BR3细胞中分为NC inhibitor组和miR-590-5P inhibitor组,利用western blot方法检测ALEX1及凋亡相关蛋白表达情况。接下来,我们利用“功能回复实验”在MCF-7细胞中将实验分为NCmimics+GV230-NC组、GV230-NC+miR-590-5P mimics组、NC mimics+GV230-ALEX1和miR-590-5P mimics+GV230-ALEX1组；在SK-BR3细胞中分为NC inhibitor+SiCon组、SiCon+miR-590-5P inhibitor组、NC inhibitor+SiALEX1和miR-590-5P inhibitor+SiALEX1组,进一步明确ALEX1受miR-590-5P调控诱导乳腺癌细胞凋亡。结果：Western blot结果显示：SK-BR3细胞中过表达ALEX1后,上调Bax、active caspase 9和active caspase 3蛋白表达,下调Bcl-2蛋白表达。MCF-7细胞中沉默ALEX1后,下调Bax、active caspase 9和active caspase 3蛋白表达,上调Bcl-2蛋白表达。Western blot方法和双荧光素酶报告实验验证结果显示：miR-590-5p与ALEX1 3'-UTR结合调控ALEX1mRNA和蛋白水平的表达。在MCF-7细胞中与NC mimics组相比,miR-590-5p mimics组中,ALEX1、Bax、active caspase 9和active caspase 3蛋白表达下调,Bcl-2蛋白表达上调。SK-BR3细胞中miR-590-5P inhibitor组与NC inhibitor对照组相比,ALEX1、Bax. active caspase 9和active caspase 3蛋白表达上调,Bcl-2蛋白表达下调。功能回复实验中显示外源性补充／沉默ALEX1能够逆转上述各凋亡蛋白的表达水平。结论：过表达ALEX1通过内源性凋亡通路诱导SK-BR3细胞凋亡;沉默ALEX1通过内源性凋亡通路抑制MCF-7细胞凋亡。ALEX1是miR-590-5P新的靶基因。ALEX1受miR-590-5P调控通过内源性凋亡通路诱导乳腺癌细胞发生凋亡。第四部分：ALEX1在乳腺癌细胞上皮间质转化中作用机制的初探目的：初步探明ALEX1在乳腺癌细胞上皮间质转化过程中的作用机制。方法：在乳腺癌细胞MDA-MB-231/MCF-7中过表达／沉默ALEX1后,显微镜观察细胞形态变化；细胞划痕愈合和Transwell实验检测乳腺癌细胞迁移侵袭能力；western blot检测上皮间质标志物的表达情况。结果：乳腺癌MDA-MB-231细胞中过表达ALEX1后,细胞形态由长梭形变为多边形；细胞划痕实验结果显示：实验组迁移距离明显小于LV5-NC对照组；Transwell结果显示：细胞穿膜数量明显少于对照组；western blot结果显示：实验组中间质类型标志物N-cadherin和Vimentin的表达消失,Snail-1、Slug未见明显变化,Twist表达水平降低；上皮类型标志物E-cadherin的表达水平升高。乳腺癌MCF-7细胞中沉默ALEX1后,细胞形态由扁圆形变为伸出伪足的多边形；细胞划痕愈合实验结果显示：实验组迁移距离明显大于SiCon对照组；Transwell结果显示：细胞穿膜数量明显多于对照组,western blot结果显示：实验组中上皮类型标志物E-cadherin的表达消失,间质类型标志物N-cadherin、Vimentin和Twist表达水平升高,Snail-1和Slug表达未见明显变化。结论：过表达ALEX1能够减弱MDA-MB-231侵袭迁移能力,诱导MDA-MB-231由间质细胞向上皮细胞转化；沉默ALEX1能够增强MCF-7侵袭迁移能力,诱导MCF-7由上皮细胞向间质细胞转化。我们推测ALEX1的低表达或缺失很可能引起Twist转录因子的表达上调,进而引起E-cad等上皮标志物的表达的下调和间质类型标志物N-cad和Vimentin的表达的上调,导致细胞发生EMT。
[Abstract]:ALEX/ARMCX (Arm proteins lost in epithelial cancers on chromosome X) is one of the members of the Arm protein family. It has only one or two Arm repeat structures, which is different from the 6-13 Arm repeat structures of classical Arm proteins. Its family members include ALEX1, ALEX2 and ALEX3. Gene expression analysis ALEX1 gene is regulated by CREB and Wnt/p-catenin pathway and can inhibit the cloning of human rectal cancer cells. These studies suggest that ALEX1 may play an inhibitory role in epithelial tumors. The expression and mechanism of ALEX1 in breast cancer are still unclear, so this paper mainly studies the expression level of ALEX1 in breast cancer tissue specimens and its effects on proliferation, cell cycle, apoptosis, invasion and metastasis of breast cancer cells and its molecular mechanism. The expression level of ALEX1 in breast cancer tissues and adjacent tissues was measured, and the expression difference between the two tissues was compared to determine the correlation between ALEX1 and clinicopathological features.Methods: The expression of ALEX1 mRNA and protein in breast cancer tissues and adjacent tissues were detected by real-time PCR, immunohistochemistry and Western blot. Results: Immunohistochemical staining of 62 pairs of breast cancer and adjacent clinical tissues showed that the expression of ALEX1 was mainly localized in cytoplasm. Statistical analysis showed that the expression level of ALEX1 protein in breast cancer tissue was lower than that in adjacent tissues (p0.01); furthermore, the relationship between the staining score of ALEX1 and the clinicopathological characteristics of breast cancer patients was analyzed. It was found that the expression of ALEX1 in breast cancer was related to the pathological grade (1 vs. 3, P = 0.026; 2 vs. 3, P = 0.045), clinical stage (I). Vs. III, P = 0.008; II vs. III, P = 0.011; lymph node metastasis (p = 0.03) and molecular typing (Luminal A vs. HER-2 overexpression, P = 0.02), but not related to patient's age, tumor size and tumor type (p0.05). In addition, 30 randomly selected breast cancer and adjacent tissues were detected by real-time PCR. ALEX 1m was found in 20 breast cancer tissues. The expression of ALEX1 protein in breast cancer and adjacent tissues was significantly lower than that in corresponding adjacent tissues (p0.01). Six pairs of breast cancer and adjacent tissues were randomly selected and detected by Western blot. The results showed that the expression of ALEX1 protein in breast cancer tissues was lower than that in corresponding adjacent tissues. The expression of ALEX1 in breast cancer tissues with lymph node metastasis was significantly lower than that in breast cancer tissues without lymph node metastasis. The expression of ALEX1 in breast cancer tissues with lymph node metastasis was significantly lower than that in breast cancer tissues without lymph node metastasis. The expression of ALEX1 in breast cancer cells was lower than that in Luminal A. Part II: Effects of ALEXl on proliferation, cell cycle and apoptosis of breast cancer cells Objective: To investigate the effects of overexpression/silencing of ALEX1 on proliferation, cycle and apoptosis of breast cancer cells SK-BR3/MCF-7. Methods: Real-time PCR and Western blot were used to detect the expression of ALEX1 in normal breast cells MCF-10A. The expression of MCF-7, T47D, SK-BR3, MDA-MB-231 in breast cancer cells and the subcellular localization of ALEX1 were detected by immunofluorescence cytochemistry. The expression of ALEX1 mRNA and protein was detected by real-time PCR and Western blot after transfection of MCF-7 with three synthetic RNAi sequences. Cell Counting Kit (CCK8) was detected after overexpression/silencing of ALEX1. Cell cycle and apoptosis were detected by flow cytometry. The results showed that the expression level of ALEX1 in breast cancer cell lines was lower than that in normal breast cell line MCF-10A, but the expression level of ALEX1 in MCF-7 cell line was higher than that in other breast cancer cell lines. The expression level of ALEX1 in SK-BR3 was the lowest and ALEX1 protein was localized in the cytoplasm. Compared with LV5-Negative Control, the expression of ALEX1 mRNA and protein in LV5-ALEX1 increased significantly. CCK8 results showed that the growth of SK-BR3 cells infected with LV5-ALEX1 lentivirus from 48 h to 96 h was inhibited significantly in LV5-ALEX1 group (p0.05). Hoechst staining and flow cytometry results The number of apoptotic cells in LV5-ALEX1 group was more than that in LV5-NC group (p0.05). After 48 hours of transient transfection of MCF-7 cells with small RNA interference, real-time PCR and Western blot results showed that the silencing effect of ALEX1 in three RNAi sequences was obvious. The experiment was divided into two groups: experimental group SiALEX1 and negative control group SiCon.CCK. The results of Hoechst staining and flow cytometry showed that the number of apoptotic cells in the SiALEX1 group was less than that in the SiCon group (p0.05). The results of flow cytometry showed that the cell cycle distribution in the over-expression/silencing ALEX1 group was fine for breast cancer. Conclusion: Overexpression of ALEX1 inhibits proliferation and induces apoptosis of breast cancer SK-BR3 cells; silencing of ALEX1 promotes proliferation and inhibits apoptosis of breast cancer MCF-7 cells. Part III: Mechanism of ALEX1-induced apoptosis of breast cancer cells Objective: To investigate the molecular mechanism of ALEX1-induced apoptosis of breast cancer cells. Methods: Western blot was used to detect the expression of apoptosis-related proteins in over-expressed/silenced ALEX1 breast cancer cells. Bioinformatics methods were used to predict the regulation of ALEX1 by microRNAs. Western blot and double luciferase reporter assays were used to verify the regulation of ALEX1 by microRNAs-590-5p. Then MCF-7 and SK-BR3 breast cancer cells were used as experimental models respectively. SK-BR3 cells were divided into NC inhibitor group and MIC-590-5P inhibitor group, and ALEX1 and apoptosis-related proteins were detected by Western blot. Next, we divided the experiment into NC MICs + GV230-NC by functional recovery test in MCF-7 cells. Groups GV230-NC+Mi-590-5P mics, NC mimics+GV230-ALEX1 and Mi-590-5P mics+GV230-ALEX1 were divided into NC inhibitor+SiCon group, SiCon+Mi-590-5P inhibitor group, NC inhibitor+SiALEX1 and Mi-590-5P inhibitor+SiALEX1 groups. Western blot analysis showed that after overexpression of ALEX1 in SK-BR3 cells, the expression of Bax, active caspase 9 and active caspase 3 was up-regulated, and the expression of Bcl-2 protein was down-regulated. After silencing ALEX1 in MCF-7 cells, the expression of Bax, active caspase 9 and active caspase 3 was down-regulated, and the expression of Bcl-2 protein was up-regulated. Compared with NC mimics, the expression of ALEX1, Bax, active caspase 9, and active caspase 3 proteins were down-regulated and the expression of Bcl-2 protein was up-regulated in MFC-7 cells and NC inhibiibited cells. Compared with tor control group, the expression of ALEX1, Bax. active caspase 9 and active caspase 3 protein was up-regulated, while the expression of Bcl-2 protein was down-regulated. Inhibiting apoptosis of MCF-7 cells via endogenous apoptosis pathway. ALEX1 is a novel target gene for microarray-590-5P. ALEX1 is regulated by microarray-590-5P and induces apoptosis of breast cancer cells through endogenous apoptosis pathway. Part IV: The role of ALEX1 in epithelial-mesenchymal transformation of breast cancer cells: Preliminary exploration of the mechanism of ALEX1 in breast cancer cells Methods: After overexpression or silencing of ALEX1 in breast cancer cells MDA-MB-231/MCF-7, the morphological changes of cells were observed under microscope; the ability of breast cancer cells to migrate and invade was detected by scratch healing and Transwell assay; and the expression of epithelial-mesenchymal markers was detected by Western blot. After overexpression of ALEX1 in MDA-MB-231 cells, the morphology of cells changed from long shuttle to polygon; the results of cell scratch test showed that the migration distance of experimental group was significantly smaller than that of LV5-NC control group; Transwell results showed that the number of cell membrane penetration was significantly less than that of control group; Western blot results showed that the type of intermediate marker N-cadhe in experimental group. The expression of RIN and Vimentin disappeared, the expression of Snail-1 and Slug did not change significantly, but the expression of Twist decreased. The expression of E-cadherin, a marker of epithelial type, increased. Transwell results showed that the number of cell membrane penetration was significantly higher than that of the control group. Western blot results showed that the expression of E-cadherin, N-cadherin, Vimentin and Twist, the expression of Snail-1 and Slug, the epithelial markers, disappeared, and the expression of N-cadherin, Vimentin and Twist, increased in the experimental group. ALEX1 can attenuate the invasion and migration of MDA-MB-231, induce MDA-MB-231 to transform from mesenchymal cells to epithelial cells; silencing ALEX1 can enhance the invasion and migration of MCF-7, and induce MCF-7 to transform from epithelial cells to mesenchymal cells. We speculate that the low expression or deletion of ALEX1 may induce the up-regulation of Twist transcription factor, and then lead to E-MB-231 transformation from mesenchymal cells. The down-regulation of epithelial markers such as CAD and the up-regulation of interstitial markers N-cad and Vimentin lead to EMT.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
[Abstract]:ALEX/ARMCX (Arm proteins lost in epithelial cancers on chromosome X) is one of the members of the Arm protein family. It has only one or two Arm repeat structures, which is different from the 6-13 Arm repeat structures of classical Arm proteins. Its family members include ALEX1, ALEX2 and ALEX3. Gene expression analysis ALEX1 gene is regulated by CREB and Wnt/p-catenin pathway and can inhibit the cloning of human rectal cancer cells. These studies suggest that ALEX1 may play an inhibitory role in epithelial tumors. The expression and mechanism of ALEX1 in breast cancer are still unclear, so this paper mainly studies the expression level of ALEX1 in breast cancer tissue specimens and its effects on proliferation, cell cycle, apoptosis, invasion and metastasis of breast cancer cells and its molecular mechanism. The expression level of ALEX1 in breast cancer tissues and adjacent tissues was measured, and the expression difference between the two tissues was compared to determine the correlation between ALEX1 and clinicopathological features.Methods: The expression of ALEX1 mRNA and protein in breast cancer tissues and adjacent tissues were detected by real-time PCR, immunohistochemistry and Western blot. Results: Immunohistochemical staining of 62 pairs of breast cancer and adjacent clinical tissues showed that the expression of ALEX1 was mainly localized in cytoplasm. Statistical analysis showed that the expression level of ALEX1 protein in breast cancer tissue was lower than that in adjacent tissues (p0.01); furthermore, the relationship between the staining score of ALEX1 and the clinicopathological characteristics of breast cancer patients was analyzed. It was found that the expression of ALEX1 in breast cancer was related to the pathological grade (1 vs. 3, P = 0.026; 2 vs. 3, P = 0.045), clinical stage (I). Vs. III, P = 0.008; II vs. III, P = 0.011; lymph node metastasis (p = 0.03) and molecular typing (Luminal A vs. HER-2 overexpression, P = 0.02), but not related to patient's age, tumor size and tumor type (p0.05). In addition, 30 randomly selected breast cancer and adjacent tissues were detected by real-time PCR. ALEX 1m was found in 20 breast cancer tissues. The expression of ALEX1 protein in breast cancer and adjacent tissues was significantly lower than that in corresponding adjacent tissues (p0.01). Six pairs of breast cancer and adjacent tissues were randomly selected and detected by Western blot. The results showed that the expression of ALEX1 protein in breast cancer tissues was lower than that in corresponding adjacent tissues. The expression of ALEX1 in breast cancer tissues with lymph node metastasis was significantly lower than that in breast cancer tissues without lymph node metastasis. The expression of ALEX1 in breast cancer tissues with lymph node metastasis was significantly lower than that in breast cancer tissues without lymph node metastasis. The expression of ALEX1 in breast cancer cells was lower than that in Luminal A. Part II: Effects of ALEXl on proliferation, cell cycle and apoptosis of breast cancer cells Objective: To investigate the effects of overexpression/silencing of ALEX1 on proliferation, cycle and apoptosis of breast cancer cells SK-BR3/MCF-7. Methods: Real-time PCR and Western blot were used to detect the expression of ALEX1 in normal breast cells MCF-10A. The expression of MCF-7, T47D, SK-BR3, MDA-MB-231 in breast cancer cells and the subcellular localization of ALEX1 were detected by immunofluorescence cytochemistry. The expression of ALEX1 mRNA and protein was detected by real-time PCR and Western blot after transfection of MCF-7 with three synthetic RNAi sequences. Cell Counting Kit (CCK8) was detected after overexpression/silencing of ALEX1. Cell cycle and apoptosis were detected by flow cytometry. The results showed that the expression level of ALEX1 in breast cancer cell lines was lower than that in normal breast cell line MCF-10A, but the expression level of ALEX1 in MCF-7 cell line was higher than that in other breast cancer cell lines. The expression level of ALEX1 in SK-BR3 was the lowest and ALEX1 protein was localized in the cytoplasm. Compared with LV5-Negative Control, the expression of ALEX1 mRNA and protein in LV5-ALEX1 increased significantly. CCK8 results showed that the growth of SK-BR3 cells infected with LV5-ALEX1 lentivirus from 48 h to 96 h was inhibited significantly in LV5-ALEX1 group (p0.05). Hoechst staining and flow cytometry results The number of apoptotic cells in LV5-ALEX1 group was more than that in LV5-NC group (p0.05). After 48 hours of transient transfection of MCF-7 cells with small RNA interference, real-time PCR and Western blot results showed that the silencing effect of ALEX1 in three RNAi sequences was obvious. The experiment was divided into two groups: experimental group SiALEX1 and negative control group SiCon.CCK. The results of Hoechst staining and flow cytometry showed that the number of apoptotic cells in the SiALEX1 group was less than that in the SiCon group (p0.05). The results of flow cytometry showed that the cell cycle distribution in the over-expression/silencing ALEX1 group was fine for breast cancer. Conclusion: Overexpression of ALEX1 inhibits proliferation and induces apoptosis of breast cancer SK-BR3 cells; silencing of ALEX1 promotes proliferation and inhibits apoptosis of breast cancer MCF-7 cells. Part III: Mechanism of ALEX1-induced apoptosis of breast cancer cells Objective: To investigate the molecular mechanism of ALEX1-induced apoptosis of breast cancer cells. Methods: Western blot was used to detect the expression of apoptosis-related proteins in over-expressed/silenced ALEX1 breast cancer cells. Bioinformatics methods were used to predict the regulation of ALEX1 by microRNAs. Western blot and double luciferase reporter assays were used to verify the regulation of ALEX1 by microRNAs-590-5p. Then MCF-7 and SK-BR3 breast cancer cells were used as experimental models respectively. SK-BR3 cells were divided into NC inhibitor group and MIC-590-5P inhibitor group, and ALEX1 and apoptosis-related proteins were detected by Western blot. Next, we divided the experiment into NC MICs + GV230-NC by functional recovery test in MCF-7 cells. Groups GV230-NC+Mi-590-5P mics, NC mimics+GV230-ALEX1 and Mi-590-5P mics+GV230-ALEX1 were divided into NC inhibitor+SiCon group, SiCon+Mi-590-5P inhibitor group, NC inhibitor+SiALEX1 and Mi-590-5P inhibitor+SiALEX1 groups. Western blot analysis showed that after overexpression of ALEX1 in SK-BR3 cells, the expression of Bax, active caspase 9 and active caspase 3 was up-regulated, and the expression of Bcl-2 protein was down-regulated. After silencing ALEX1 in MCF-7 cells, the expression of Bax, active caspase 9 and active caspase 3 was down-regulated, and the expression of Bcl-2 protein was up-regulated. Compared with NC mimics, the expression of ALEX1, Bax, active caspase 9, and active caspase 3 proteins were down-regulated and the expression of Bcl-2 protein was up-regulated in MFC-7 cells and NC inhibiibited cells. Compared with tor control group, the expression of ALEX1, Bax. active caspase 9 and active caspase 3 protein was up-regulated, while the expression of Bcl-2 protein was down-regulated. Inhibiting apoptosis of MCF-7 cells via endogenous apoptosis pathway. ALEX1 is a novel target gene for microarray-590-5P. ALEX1 is regulated by microarray-590-5P and induces apoptosis of breast cancer cells through endogenous apoptosis pathway. Part IV: The role of ALEX1 in epithelial-mesenchymal transformation of breast cancer cells: Preliminary exploration of the mechanism of ALEX1 in breast cancer cells Methods: After overexpression or silencing of ALEX1 in breast cancer cells MDA-MB-231/MCF-7, the morphological changes of cells were observed under microscope; the ability of breast cancer cells to migrate and invade was detected by scratch healing and Transwell assay; and the expression of epithelial-mesenchymal markers was detected by Western blot. After overexpression of ALEX1 in MDA-MB-231 cells, the morphology of cells changed from long shuttle to polygon; the results of cell scratch test showed that the migration distance of experimental group was significantly smaller than that of LV5-NC control group; Transwell results showed that the number of cell membrane penetration was significantly less than that of control group; Western blot results showed that the type of intermediate marker N-cadhe in experimental group. The expression of RIN and Vimentin disappeared, the expression of Snail-1 and Slug did not change significantly, but the expression of Twist decreased. The expression of E-cadherin, a marker of epithelial type, increased. Transwell results showed that the number of cell membrane penetration was significantly higher than that of the control group. Western blot results showed that the expression of E-cadherin, N-cadherin, Vimentin and Twist, the expression of Snail-1 and Slug, the epithelial markers, disappeared, and the expression of N-cadherin, Vimentin and Twist, increased in the experimental group. ALEX1 can attenuate the invasion and migration of MDA-MB-231, induce MDA-MB-231 to transform from mesenchymal cells to epithelial cells; silencing ALEX1 can enhance the invasion and migration of MCF-7, and induce MCF-7 to transform from epithelial cells to mesenchymal cells. We speculate that the low expression or deletion of ALEX1 may induce the up-regulation of Twist transcription factor, and then lead to E-MB-231 transformation from mesenchymal cells. The down-regulation of epithelial markers such as CAD and the up-regulation of interstitial markers N-cad and Vimentin lead to EMT.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
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