TGFβ1对三阴性乳腺癌肝转移及干细胞特性的作用研究
发布时间：2018-07-17 17:54
【摘要】：第一部分TGFβ1对三阴性乳腺癌肝脏转移的作用研究研究背景:我们课题组前期的研究发现TGFβ1在三阴性乳腺癌中特异性的高表达,但是这种高表达是否参与对三阴性乳腺癌的调控研究仍较少。本实验,我们主要在体探讨了 TGFβ1对三阴性乳腺癌体内成瘤、局部侵袭及肝脏转移能力的影响。研究方法:将对照组及经TGFβ1反复处理的三阴性乳腺癌MDA细胞注入3-4周大的雌性裸鼠乳腺脂肪垫中建立乳腺癌原位模型。观察原位肿瘤的生长情况及肝脏转移情况。4-5周左右处死小鼠,HE染色观察对照组及TGFβ1组乳腺癌原位肿瘤及肝脏转移灶。利用免疫组化染色对照组及TGFβ1组原位及肝脏肿瘤灶Ki67、α-MSA及KRT8等相关乳腺癌免疫组化指标,以判断转移灶来源。研究结果:与对照组相比,TGFβ1处理组三阴性乳腺癌细胞裸鼠原位成瘤能力增加,HE染色显示TGFβ1预处理增加了三阴性乳腺癌局部侵袭能力。同时,肝脏大体观及HE染色结果显示TGFβ1预处理增加了三阴性乳腺癌肝转移的能力。免疫组化结果显示肝脏转移灶来源于原位肿瘤灶。研究结论:我们的结果提示TGFβ1预处理能够增加三阴性乳腺癌体内成瘤、局部侵袭及肝脏转移能力。第二部分TGFβ1对三阴性乳腺癌干细胞特性的作用研究研究背景:研究普遍认为干细胞是肿瘤转移的根源。近年来研究发现,TGFβ配体和它的信号元件在人乳腺癌干细胞的调控中起到了极其重要的作用。我们的前期研究也发现TGFβ1在三阴性乳腺癌细胞中特异性表达升高,高度提示TGFβ1可能参与三阴性乳腺癌干细胞的调控。因此,本实验中,我们主要探讨TGFβ1对三阴性乳腺癌干细胞及其特性的影响。研究方法:首先,利用流式细胞术分析TGFβ1对乳腺癌干细胞样CD44+/CD24-表型的作用;其次,通过RT-PCR检测乳腺癌细胞腺上皮标志物(KRT8、KRT18)和间质/肌上皮标志物(KRT14、ACTA2)的表达,从而研究TGFβ1对乳腺癌干细胞样间质细胞表型的作用影响;然后,RT-PCR检测TGFβ1对乳腺癌干细胞球的干性相关转录因子OCT4、SOX2、NANOG表达的作用;之后,成球实验及成球效率评估TGFβ1对乳腺癌干细胞自我更新能力的影响;随后,利用TGFβ1受体阻断剂研究TGFβ1对乳腺癌干细胞自我更新能力的影响是否通过其受体发挥作用的;最后,3D培养检测TGFβ1对乳腺癌干细胞球侵袭能力的作用。研究结果:我们的结果显示:TGFβ1增加三阴性乳腺癌中CD44high/CD24-/low干细胞群比例;TGFβ1增加三阴性乳腺癌干细胞样间质细胞表型;TGFβ1促进干细胞成球并增加干性标志物的表达;TGFβ1增强三阴性乳腺癌干细胞的自我更新能力;TGFβ1增强三阴性乳腺癌干细胞的自我更新能力能够被其受体抑制剂阻断;TGFβ1增强三阴性乳腺癌干细胞的侵袭能力。研究结论:我们的结果提示TGFβ1能够增强三阴性乳腺癌的自我更新能力等干细胞特性。第三部分CDK4参与TGFβ1对三阴性乳腺癌干细胞的调控研究研究背景:细胞周期蛋白依赖性激酶4(CDK4)为细胞周期蛋白D1(cyclinD1)的配体,通常与cyclinD1结合并被激活来发挥细胞周期调节的作用。研究发现CDK4促进胚胎干细胞、造血干细胞、神经干细胞及乳腺前提细胞增殖并抑制其分化。提示CDK4可能也在乳腺癌干细胞的增殖及转移中发挥了重要的作用。前期我们的研究也发现TGFβ1可以提高三阴性乳腺癌中CyclinD1/CDK4的表达,猜测CDK4可能在TGFβ1参与的三阴性乳腺癌干细胞调控中发挥重要作用。因此,本部分实验主要探讨CDK4是否作为TGFβ1的下游底物,介导了其对三阴性乳腺癌干细胞的调控。研究方法:流式细胞术检测对照组、CDK4蛋白沉默组及CDK4激酶抑制剂组SUM159中CD44+/CD24-干细胞比例;成球实验及成球效率检测不同组(对照组、CDK4蛋白沉默组、CDK4激酶抑制剂组、TGFβ1组、CDK4激酶抑制剂+TGFβ1组)SUM159干细胞自我更新能力,RT-PCR检测不同组导管上皮细胞标志物(KRT8,KRT18)及间质细胞标志物(ACTA2,KRT14)的表达。免疫共沉淀(Co-IP)及Western blot技术检测对照组及TGFβ1组CyclinD1、CDK4、p-RB等蛋白的表达。研究结果:沉默CDK4蛋白或使用CDK4激酶抑制剂能够降低三阴性乳腺癌中CD44+/CD24-干细胞比例及自我更新能力,抑制三阴性乳腺癌干细胞样间质细胞表型。TGFβ1上调CDK4蛋白表达及激酶活性,CDK4激酶抑制剂可以阻断TGFβ1增强三阴性乳腺癌干细胞自我更新能力及维持其间质样表型的作用。研究结论:这些结果提示CDK4蛋白本身在维持三阴性乳腺癌干细胞自我更新及间质样表型中发挥重要的作用,TGFβ1可能是通过上调CDK4蛋白及其激酶活性来维持三阴性乳腺癌干细胞自我更新能力及干细胞样间质细胞表型的。第四部分肿瘤干细胞与三阴性乳腺癌肝转移及预后相关分析研究背景:近年来,很多临床数据库的开发与建立为多种不同来源的肿瘤研究提供了有效的生物信息学分析。因此,本部分研究主要利用Kaplan Meier Plotter、TCGA Cancer Browser以及GOBO geneset数据库对三阴性乳腺癌中肿瘤标志物,TGFβ1和CDK4做相关生物信息学分析。研究方法:首先利用Kaplan Meier Plotter对CD44及CD24的基因表达与249例三阴性乳腺癌患者的预后进行生物信息学分析。然后利用UCSC Cancer Browser对TCGA数据库1215例乳腺癌患者中三阴性乳腺癌肝转移患者进行CD44及CD24的基因表达的生物信息学分析。之后,利用GOBO基因分析工具对51株已报道的三阴性乳腺癌细胞系中CD44及CD24的基因表达进行分析。最后,利用GOBO基因数据库分析TGFβ1及CDK4在三阴性乳腺癌中的表达。研究结果:生物信息学分析提示CD44高表达/CD24低表达患者无复发生存率(RFS)较低,且三阴性乳腺癌伴肝转移患者肿瘤干细胞表面标志物CD44高表达,而CD24低表达。TGFβ1、CDK4在三阴性乳腺癌细胞系中的表达较高。研究结论:TCGA及GOBO geneset等数据库生物信息学分析提示三阴性乳腺癌中CD44high/CD24low/-干细胞含量较高,且TGFβ1及CDK4可能与三阴性乳腺癌干细胞调控相关,与我们的研究结果TGFβ1/CDK4参与三阴性乳腺癌干细胞调控一致。
[Abstract]:The first part of the study on the role of TGF beta 1 on liver metastasis of three negative breast cancer: our previous study found that TGF beta 1 was highly expressed in three negative breast cancer, but whether this high expression is involved in the regulation of three negative breast cancer is still less. In this experiment, we mainly discussed the TGF beta 1 to three yin in vivo. The effect of the tumor formation, local invasion and liver metastasis in the body of sexual breast cancer. Methods: the control group and the three negative breast cancer cells treated with TGF beta 1 were injected into the breast fat pad of the female nude mice for 3-4 weeks, and the in situ model of breast cancer was established. The growth and liver metastasis of the tumor in situ were observed for about.4-5 weeks. Mice, HE staining and control group and TGF beta 1 group of breast cancer in situ tumor and liver metastases were observed. Immuno histochemical staining control group and TGF beta 1 group in situ and liver tumor Ki67, alpha -MSA and KRT8 related immunohistochemical indexes of breast cancer were used to determine the origin of metastatic foci. The results were compared with those in the group of TGF beta 1, three negative breast cancer cells The tumor ability in nude mice was increased in situ. HE staining showed that TGF beta 1 preconditioning increased the local invasiveness of three negative breast cancers. Meanwhile, the liver gross and HE staining results showed that TGF beta 1 preconditioning increased the ability of three negative breast cancer to liver metastasis. The results suggest that TGF beta 1 preconditioning can increase the tumor formation, local invasion and liver metastasis in three negative breast cancers. Second the research background of the role of TGF beta 1 on the characteristics of three negative breast cancer stem cells: the research is generally believed that stem cells are the root of tumor metastasis. In recent years, the research has found that TGF beta ligand and its signal components are in human The regulation of breast cancer stem cells plays an extremely important role. Our previous study also found that the specific expression of TGF beta 1 increased in three negative breast cancer cells. It is highly suggestive that TGF beta 1 may be involved in the regulation of three negative breast cancer stem cells. Therefore, in this experiment, we mainly discuss the TGF beta 1 against three negative breast cancer stem cells and their specificity. Study methods: first, flow cytometry was used to analyze the effect of TGF beta 1 on the CD44+/CD24- phenotype of breast cancer stem cells. Secondly, the expression of KRT8 (KRT18) and interstitial / myoepithelial markers (KRT14, ACTA2) of breast cancer cells were detected by RT-PCR, and the phenotype of TGF beta 1 on the stem cell like cell phenotype of breast cancer was studied. The effect of TGF beta 1 on the expression of OCT4, SOX2, NANOG in the stem cells of breast cancer stem cells was detected by RT-PCR. After that, the effect of TGF beta 1 on the self-renewal capacity of breast cancer stem cells was evaluated by the spherocification test and the ball efficiency. Then, the self-renewal of TGF beta 1 on breast cancer stem cells was studied by TGF beta 1 receptor blocker. The effect of the effect of capacity through its receptor; finally, 3D culture was used to detect the effect of TGF beta 1 on the invasive ability of breast cancer stem cells. Results: Our results showed that TGF beta 1 increased the proportion of CD44high/CD24-/low stem cells in three negative breast cancers; TGF beta 1 increased the phenotype of three negative breast cancer stem cell like mesenchymal cells; TGF beta 1 Promote the formation of stem cells and increase the expression of dry markers; TGF beta 1 enhanced the self-renewal capacity of three negative breast cancer stem cells; TGF beta 1 enhanced the self-renewal ability of three negative breast cancer stem cells to be blocked by its receptor inhibitors; TGF beta 1 enhanced the invasive ability of three negative breast cancer stem cells. Conclusions: Our results suggest TGF beta 1 can enhance the self renewal capacity of three negative breast cancer. Third part of CDK4 participates in the research background of TGF beta 1 on three negative breast cancer stem cells: cell cyclin dependent kinase 4 (CDK4) is a ligand of cyclin D1 (cyclinD1), which is usually combined with cyclinD1 and is activated to play cell cycle. The study found that CDK4 promotes embryonic stem cells, hematopoietic stem cells, neural stem cells, and mammary cells to proliferate and inhibit their differentiation. It suggests that CDK4 may also play an important role in the proliferation and metastasis of breast cancer stem cells. Our previous study also found that TGF beta 1 could improve the CyclinD1/C of breast cancer in negative breast cancer. The expression of DK4 suggests that CDK4 may play an important role in the regulation of three negative breast cancer stem cells involved in TGF beta 1. Therefore, this part of this experiment mainly discusses whether CDK4 is a downstream substrate of TGF beta 1 and mediates its regulation of three negative breast cancer stem cells. The proportion of CD44+/CD24- stem cells in the enzyme inhibitor group SUM159, the ball forming experiment and the detection of the ball forming efficiency in different groups (control group, CDK4 protein silencing group, CDK4 kinase inhibitor group, TGF beta 1 group, CDK4 kinase inhibitor +TGF beta 1 group) SUM159 stem cells self-renewal capacity, RT-PCR detection of different groups of ductal epithelial cell markers (KRT8, KRT18) and interstitial cell markers Expression of ACTA2 (KRT14). Immunocoprecipitation (Co-IP) and Western blot techniques for the detection of CyclinD1, CDK4, p-RB and other proteins in the TGF beta 1 groups. The results of the study: silencing CDK4 protein or using CDK4 kinase inhibitors can reduce the ratio of CD44+/CD24- stem cells and self renewal in three negative breast cancers and inhibit three negative breast cancer The phenotype of stem cell like mesenchymal cells.TGF beta 1 up-regulated the expression of CDK4 protein and kinase activity. CDK4 kinase inhibitor can block the role of TGF beta 1 to enhance the self-renewal capacity of three negative breast cancer stem cells and maintain its interstitial like phenotype. These results suggest that the CDK4 protein itself maintains the self renewal of the three negative breast cancer stem cells. An important role in interstitial like phenotype, TGF beta 1 may be to maintain the self-renewal capacity of three negative breast cancer stem cells and the phenotype of stem like mesenchymal cells by up regulation of CDK4 protein and its kinase activity. Fourth part of tumor stem cells and the correlation analysis of liver metastasis and prognosis in three negative breast cancer: many clinical trials in recent years The development and establishment of the database provide an effective bioinformatics analysis for a variety of different sources of cancer research. Therefore, this part of this study mainly uses Kaplan Meier Plotter, TCGA Cancer Browser and GOBO geneset database to analyze the tumor markers in three negative breast cancers, TGF beta 1 and CDK4, and the related bioinformatics analysis. Method: bioinformatics analysis was performed on the gene expression of CD44 and CD24 with Kaplan Meier Plotter and the prognosis of 249 cases of three negative breast cancer. Then, UCSC Cancer Browser was used to analyze the gene expression of CD44 and CD24 in three negative breast cancer patients with breast cancer in 1215 cases of breast cancer. Then, the GOBO gene analysis tool was used to analyze the expression of CD44 and CD24 in 51 reported three negative breast cancer cell lines. Finally, the expression of TGF beta 1 and CDK4 in three negative breast cancer was analyzed by GOBO gene database. The results of bioinformatics analysis suggested that the CD44 high expression of low expression of /CD24 in patients with high expression of /CD24 had no recurrence. The rate (RFS) was low, and the expression of the surface marker of the tumor stem cells was high in the three negative breast cancer patients with liver metastasis, while the expression of.TGF beta 1 was low in CD24, and the expression of CDK4 in the three negative breast cancer cell lines was higher. Conclusion: TCGA and GOBO geneset and other database bioinformatics analysis showed that CD44high/CD24low/- stem cells contained in three negative breast cancer contained CD44high/CD24low/- stem cells. High volume, and TGF beta 1 and CDK4 may be related to the regulation of three negative breast cancer stem cells. It is consistent with our findings that TGF beta 1/CDK4 participates in the regulation of three negative breast cancer stem cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
本文编号：2130468
[Abstract]:The first part of the study on the role of TGF beta 1 on liver metastasis of three negative breast cancer: our previous study found that TGF beta 1 was highly expressed in three negative breast cancer, but whether this high expression is involved in the regulation of three negative breast cancer is still less. In this experiment, we mainly discussed the TGF beta 1 to three yin in vivo. The effect of the tumor formation, local invasion and liver metastasis in the body of sexual breast cancer. Methods: the control group and the three negative breast cancer cells treated with TGF beta 1 were injected into the breast fat pad of the female nude mice for 3-4 weeks, and the in situ model of breast cancer was established. The growth and liver metastasis of the tumor in situ were observed for about.4-5 weeks. Mice, HE staining and control group and TGF beta 1 group of breast cancer in situ tumor and liver metastases were observed. Immuno histochemical staining control group and TGF beta 1 group in situ and liver tumor Ki67, alpha -MSA and KRT8 related immunohistochemical indexes of breast cancer were used to determine the origin of metastatic foci. The results were compared with those in the group of TGF beta 1, three negative breast cancer cells The tumor ability in nude mice was increased in situ. HE staining showed that TGF beta 1 preconditioning increased the local invasiveness of three negative breast cancers. Meanwhile, the liver gross and HE staining results showed that TGF beta 1 preconditioning increased the ability of three negative breast cancer to liver metastasis. The results suggest that TGF beta 1 preconditioning can increase the tumor formation, local invasion and liver metastasis in three negative breast cancers. Second the research background of the role of TGF beta 1 on the characteristics of three negative breast cancer stem cells: the research is generally believed that stem cells are the root of tumor metastasis. In recent years, the research has found that TGF beta ligand and its signal components are in human The regulation of breast cancer stem cells plays an extremely important role. Our previous study also found that the specific expression of TGF beta 1 increased in three negative breast cancer cells. It is highly suggestive that TGF beta 1 may be involved in the regulation of three negative breast cancer stem cells. Therefore, in this experiment, we mainly discuss the TGF beta 1 against three negative breast cancer stem cells and their specificity. Study methods: first, flow cytometry was used to analyze the effect of TGF beta 1 on the CD44+/CD24- phenotype of breast cancer stem cells. Secondly, the expression of KRT8 (KRT18) and interstitial / myoepithelial markers (KRT14, ACTA2) of breast cancer cells were detected by RT-PCR, and the phenotype of TGF beta 1 on the stem cell like cell phenotype of breast cancer was studied. The effect of TGF beta 1 on the expression of OCT4, SOX2, NANOG in the stem cells of breast cancer stem cells was detected by RT-PCR. After that, the effect of TGF beta 1 on the self-renewal capacity of breast cancer stem cells was evaluated by the spherocification test and the ball efficiency. Then, the self-renewal of TGF beta 1 on breast cancer stem cells was studied by TGF beta 1 receptor blocker. The effect of the effect of capacity through its receptor; finally, 3D culture was used to detect the effect of TGF beta 1 on the invasive ability of breast cancer stem cells. Results: Our results showed that TGF beta 1 increased the proportion of CD44high/CD24-/low stem cells in three negative breast cancers; TGF beta 1 increased the phenotype of three negative breast cancer stem cell like mesenchymal cells; TGF beta 1 Promote the formation of stem cells and increase the expression of dry markers; TGF beta 1 enhanced the self-renewal capacity of three negative breast cancer stem cells; TGF beta 1 enhanced the self-renewal ability of three negative breast cancer stem cells to be blocked by its receptor inhibitors; TGF beta 1 enhanced the invasive ability of three negative breast cancer stem cells. Conclusions: Our results suggest TGF beta 1 can enhance the self renewal capacity of three negative breast cancer. Third part of CDK4 participates in the research background of TGF beta 1 on three negative breast cancer stem cells: cell cyclin dependent kinase 4 (CDK4) is a ligand of cyclin D1 (cyclinD1), which is usually combined with cyclinD1 and is activated to play cell cycle. The study found that CDK4 promotes embryonic stem cells, hematopoietic stem cells, neural stem cells, and mammary cells to proliferate and inhibit their differentiation. It suggests that CDK4 may also play an important role in the proliferation and metastasis of breast cancer stem cells. Our previous study also found that TGF beta 1 could improve the CyclinD1/C of breast cancer in negative breast cancer. The expression of DK4 suggests that CDK4 may play an important role in the regulation of three negative breast cancer stem cells involved in TGF beta 1. Therefore, this part of this experiment mainly discusses whether CDK4 is a downstream substrate of TGF beta 1 and mediates its regulation of three negative breast cancer stem cells. The proportion of CD44+/CD24- stem cells in the enzyme inhibitor group SUM159, the ball forming experiment and the detection of the ball forming efficiency in different groups (control group, CDK4 protein silencing group, CDK4 kinase inhibitor group, TGF beta 1 group, CDK4 kinase inhibitor +TGF beta 1 group) SUM159 stem cells self-renewal capacity, RT-PCR detection of different groups of ductal epithelial cell markers (KRT8, KRT18) and interstitial cell markers Expression of ACTA2 (KRT14). Immunocoprecipitation (Co-IP) and Western blot techniques for the detection of CyclinD1, CDK4, p-RB and other proteins in the TGF beta 1 groups. The results of the study: silencing CDK4 protein or using CDK4 kinase inhibitors can reduce the ratio of CD44+/CD24- stem cells and self renewal in three negative breast cancers and inhibit three negative breast cancer The phenotype of stem cell like mesenchymal cells.TGF beta 1 up-regulated the expression of CDK4 protein and kinase activity. CDK4 kinase inhibitor can block the role of TGF beta 1 to enhance the self-renewal capacity of three negative breast cancer stem cells and maintain its interstitial like phenotype. These results suggest that the CDK4 protein itself maintains the self renewal of the three negative breast cancer stem cells. An important role in interstitial like phenotype, TGF beta 1 may be to maintain the self-renewal capacity of three negative breast cancer stem cells and the phenotype of stem like mesenchymal cells by up regulation of CDK4 protein and its kinase activity. Fourth part of tumor stem cells and the correlation analysis of liver metastasis and prognosis in three negative breast cancer: many clinical trials in recent years The development and establishment of the database provide an effective bioinformatics analysis for a variety of different sources of cancer research. Therefore, this part of this study mainly uses Kaplan Meier Plotter, TCGA Cancer Browser and GOBO geneset database to analyze the tumor markers in three negative breast cancers, TGF beta 1 and CDK4, and the related bioinformatics analysis. Method: bioinformatics analysis was performed on the gene expression of CD44 and CD24 with Kaplan Meier Plotter and the prognosis of 249 cases of three negative breast cancer. Then, UCSC Cancer Browser was used to analyze the gene expression of CD44 and CD24 in three negative breast cancer patients with breast cancer in 1215 cases of breast cancer. Then, the GOBO gene analysis tool was used to analyze the expression of CD44 and CD24 in 51 reported three negative breast cancer cell lines. Finally, the expression of TGF beta 1 and CDK4 in three negative breast cancer was analyzed by GOBO gene database. The results of bioinformatics analysis suggested that the CD44 high expression of low expression of /CD24 in patients with high expression of /CD24 had no recurrence. The rate (RFS) was low, and the expression of the surface marker of the tumor stem cells was high in the three negative breast cancer patients with liver metastasis, while the expression of.TGF beta 1 was low in CD24, and the expression of CDK4 in the three negative breast cancer cell lines was higher. Conclusion: TCGA and GOBO geneset and other database bioinformatics analysis showed that CD44high/CD24low/- stem cells contained in three negative breast cancer contained CD44high/CD24low/- stem cells. High volume, and TGF beta 1 and CDK4 may be related to the regulation of three negative breast cancer stem cells. It is consistent with our findings that TGF beta 1/CDK4 participates in the regulation of three negative breast cancer stem cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
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