STAT-3信号通路在27-羟基胆固醇促进乳腺癌细胞侵袭转移中的作用
发布时间：2018-07-28 11:43
【摘要】：21世纪乳腺癌已经成为危害女性健康的重要因素,在女性肿瘤中占有相当重要的地位。然而肿瘤细胞的侵袭转移是乳腺癌发展过程和致死过程中的重要机制之一。肿瘤细胞的侵袭转移过程复杂多样,例如癌细胞的上皮间质转化(epithelial-mesenchymal transition,EMT)以及基质金属蛋白酶(matrix metalloproteases,MMPs)降解细胞外基质等等。众多文献报道细胞上皮间质转化可以使肿瘤细胞由多边形、方形变为长梭形伴有上皮指标E-钙黏蛋白(E-cadherin)等下降、间质指标波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)和E盒结合锌指蛋白1(ZEB1)等上升而具有更强的运动迁移能力,是癌症进展的一个标志;已有研究表明 RECK(the reversion-inducing cysteine-rich protein with kazal motifs)蛋白可以通过下调MMP2和MMP9的表达进而抑制肿瘤细胞的侵袭转移,因此在肿瘤发展过程中RECK表达的下调成为肿瘤恶转的一个标志。转录激活因子-3(signal transducers and activators of transcription-3,STAT-3)的活化在肿瘤侵袭转移过程中也发挥重要作用,可以激活下游侵袭转移指标的表达。27-羟基胆固醇(27-hydroxyl cholesterol,27HC)是人体内胆固醇的代谢产物,由细胞色素P450酶CYP27A1代谢产生,并由CYP7B1分解。高胆固醇血症患者体内27HC水平明显升高;妇女尤其是绝经后妇女肥胖导致胆固醇水平升高进而催生27HC引发各种包括肿瘤在内的疾病,严重危害人类健康。2013年Science报道,27HC可与ER受体结合促进肿瘤细胞增殖及与LXR受体结合促进肿瘤细胞侵袭转移,但是目前关于27HC能否通过下调肿瘤抑制基因RECK的表达从而影响STAT-3信号通路促进肿瘤细胞的侵袭转移尚未有报道。目的:本研究以人ER阳性乳腺癌细胞系MCF7、T47D和人三阴乳腺癌细胞系MDA-MB-231为研究对象,探讨27HC促进乳腺癌细胞侵袭转移的机制及STAT-3在其中的作用,从而为肿瘤的预防及治疗提供更多的实验数据,打开新的思路。方法:检测27HC对细胞活力的影响:CCK8实验;检测27HC对乳腺癌细胞的侵袭转移能力的影响:细胞划痕实验,预铺matrigel胶和未铺matrigel的Transwell实验;检测MMP2和MMP9的活性:明胶酶谱实验;检测目的基因的mRNA表达水平:RT-PCR和实时定量PCR(qRT-PCR)实验;检测基因的蛋白水平:蛋白质免疫印迹实验;si-RNA敲除RECK和STAT-3:细胞转染实验;检测ROS:活性氧检测实验。结果:(1)27HC能够促进乳腺癌MCF7和T47D细胞的侵袭迁移能力:首先,CCK8实验和划痕愈合实验结果发现4.0μM是处理乳腺癌细胞72小时不影响细胞活力的最高浓度;采用预铺胶和未铺胶的Transwell实验证实27HC能够在不影响细胞活力的前提下,上调乳腺癌细胞的侵袭迁移能力;RT-PCR和qRT-PCR实验结果显示27HC可以呈剂量依赖性地上调乳腺癌细胞内MMP9的mRNA,但并不影响MMP2的mRNA水平;明胶酶谱实验结果显示27HC可以上调MMP9的活性,但并不影响MMP2的活性。另外,27HC处理后,Western Blot实验发现,乳腺癌细胞内EMT相关指标E-cadherin下降,Vimentin和ZEB1上升。(2)27HC下调RECK的蛋白水平和mRNA水平并上调MMP9的表达和活性:在分子机制探索研究中,我们发现经过27HC处理后乳腺癌细胞的REECK的蛋白水平和mRNA均呈现下调趋势,采用RECK siRNA干扰RECK蛋白表达后,乳腺癌细胞内MMP9表达、活性及细胞侵袭能力上升,但是MMP2的表达和活性不变。(3)27HC可以通过ERα非依赖性途径激活STAT-3信号通路进而促进乳腺癌细胞的侵袭迁移能力:另外,27HC处理ER阳性乳腺癌细胞MCF7和T47D及ER阴性乳腺癌细胞MDA-MB-231后,p-STAT-3上升,STAT3信号通路被激活,GREB1的表达也上升,说明27HC也激活了经典的ER信号通路。应用siSTAT-3敲除STAT-3之后,27HC所引起的乳腺癌侵袭转移能力增强有所下降,其下游MMP9下降,E-cadherin上升,Vimentin和ZEB1下降。表明STAT-3信号通路在27HC诱导的乳腺癌侵袭转移过程中发挥重要作用。(4)27HC通过激活细胞内ROS水平并促进乳腺癌细胞侵袭迁移能力:27HC和阳性参照H2O2处理ER阳性乳腺癌细胞MCF7和T47D后,加入碧云天ROS检测试剂,并应用荧光显微镜拍照,结果显示27HC可以促进ROS产生和蓄积,引起细胞氧化应激。(5)27HC通过ROS引起甲基化导致RECK的表达下调进而激活STAT-3:27HC,甲基转移酶抑制剂(AZA),甲基供体(SAM)和阳性参照H2O2处理ER阳性乳腺癌细胞MCF7后,发现联合AZA处理后,27HC所引起的RECK下调有所逆转,SAM处理后RECK的表达呈现下降趋势。采用RECK siRNA干扰RECK蛋白表达后乳腺癌细胞内p-STAT-3水平下降,STAT-3信号通路被阻断。所以27HC通过ROS甲基化抑制RECK的表达进而激活STAT-3最终正向调控乳腺癌细胞的侵袭转移能力。另外采用STAT-3 siRNA干扰STAT-3后乳腺癌细胞内RECK的mRNA水平上调。提示在这一过程中RECK与STAT-3信号通路存在相互作用关系。结论:在人乳腺癌细胞中,27HC通过激活ROS,甲基化下调RECK的mRNA和蛋白水平进而激活STAT-3信号通路,升高MMP9的转录水平和活性以及导致EMT相关分子指标E-cadherin下降、Vimentin和ZEB1上升,使乳腺癌细胞降解细胞外基质的能力和运动迁移的能力上升,最终促进其侵袭转移能力的上升。
[Abstract]:In twenty-first Century, breast cancer has become an important factor endangering women's health and plays an important role in female tumors. However, the invasion and metastasis of tumor cells is one of the most important mechanisms in the development and death process of breast cancer. The invasion and metastasis of tumor cells are complex and varied, such as the epithelial transformation of cancer cells (epithelial -mesenchymal transition, EMT) and matrix metalloproteinases (matrix metalloproteases, MMPs) degradation of extracellular matrix and so on. Many literature reports that cell epithelial mesenchymal transformation can make tumor cells from polygon, square into long spindle with epithelial index E- calcium mucin (E-cadherin) and so on, interstitial index vimentin (Vimentin), N- calcium mucin (N-cadherin) and E box combined with zinc finger protein 1 (ZEB1) increase and have a stronger movement ability. It is a sign of cancer progression. The existing studies have shown that RECK (the reversion-inducing cysteine-rich protein with Kazal motifs) can inhibit the invasion of tumor cells by down regulation and expression. The downregulation of RECK expression in the process of tumor development has become a sign of tumor metastasis. The activation of the transcription activating factor -3 (signal transducers and activators of transcription-3, STAT-3) also plays an important role in the process of tumor invasion and metastasis, and can activate the expression of.27- hydroxyl cholesterol (27). -hydroxyl cholesterol, 27HC, a metabolite of cholesterol in the human body, is produced by the metabolism of cytochrome P450 enzyme CYP27A1, and is decomposed by CYP7B1. The level of 27HC in patients with hypercholesterolemia is significantly elevated; women, especially postmenopausal women, are obese and lead to higher levels of cholesterol, causing 27HC to cause a variety of diseases, including tumors. Science reports that serious harm to human health in.2013, 27HC can combine with ER receptor to promote tumor cell proliferation and LXR receptor binding to promote tumor cell invasion and metastasis. However, there has been no report on whether 27HC can affect the invasion and metastasis of tumor cells by decreasing the expression of the tumor suppressor gene RECK and thus affecting the invasion and metastasis of the tumor cells. In this study, ER positive breast cancer cell line MCF7, T47D and human three shade breast cancer cell line MDA-MB-231 were studied in this study. The mechanism of 27HC to promote invasion and metastasis of breast cancer cells and the role of STAT-3 in the breast cancer cells were discussed, so as to provide more experimental data for the prevention and treatment of cancer and to open new ideas. Methods: detection of 27HC to cell survival. Influence of force: CCK8 experiment; detection of the influence of 27HC on the invasion and metastasis of breast cancer cells: cell scratch test, prelaying Matrigel glue and unpaved Matrigel Transwell test; detection of MMP2 and MMP9 activity: gelatinase assay; detection of mRNA expression level of target genes: RT-PCR and real-time quantitative PCR (qRT-PCR) experiment; detection of gene eggs White level: protein immunoblotting test, si-RNA knockout RECK and STAT-3: cell transfection experiments, and detection of ROS: reactive oxygen test. Results: (1) 27HC can promote the invasion and migration of MCF7 and T47D cells in breast cancer: first, the results of CCK8 and scratch healing experiments found that 4 mu of breast cancer cells did not affect cell viability for 72 hours. The highest concentration of Transwell showed that 27HC could increase the invasion and migration ability of breast cancer cells without affecting cell viability, and the results of RT-PCR and qRT-PCR showed that 27HC could increase the mRNA of MMP9 in breast cancer cells in a dose-dependent manner, but did not affect the mRNA level of MMP2, gelatin. The results of enzyme spectrum test showed that 27HC could increase the activity of MMP9, but did not affect the activity of MMP2. In addition, after 27HC treatment, Western Blot experiment found that EMT related index E-cadherin decreased and Vimentin and ZEB1 increased in breast cancer cells. (2) 27HC down RECK protein levels and levels and up regulation of expression and activity: molecular mechanism exploration In the study, we found that the REECK protein level and mRNA of breast cancer cells were down downward after 27HC treatment. After RECK siRNA interference RECK protein expression, MMP9 expression, activity and cell invasiveness increased in breast cancer cells, but MMP2 expression and activity did not change. (3) 27HC can activate STAT through ER alpha non dependent pathway -3 signaling pathway further promotes the invasion and migration of breast cancer cells. In addition, after 27HC treatment of ER positive breast cancer cells MCF7 and T47D and ER negative breast cancer cells MDA-MB-231, p-STAT-3 rises, STAT3 signaling pathway is activated and GREB1 expression rises, indicating that 27HC also activates the classical ER signal pathway. The enhanced invasion and metastasis of breast cancer caused by 27HC decreased, its downstream MMP9 decreased, E-cadherin increased, and Vimentin and ZEB1 decreased. It showed that STAT-3 signaling pathway played an important role in the invasion and metastasis of breast cancer induced by 27HC. (4) 27HC can promote the invasion and migration of breast cancer cells by activating the ROS level in the cells and promoting the invasion and migration of breast cancer cells: 27HC and After positive reference to H2O2 to treat ER positive breast cancer cells MCF7 and T47D, ROS detection reagent was added to the blue cloud sky ROS, and the fluorescence microscope was photographed. The results showed that 27HC could promote the production and accumulation of ROS and induce cell oxidative stress. (5) 27HC through ROS caused methylation to reduce RECK and activate STAT-3:27HC, methyltransferase inhibitor ( AZA), after the treatment of ER positive breast cancer cells MCF7 by methyl donor (SAM) and positive reference H2O2, it was found that the RECK down regulation caused by 27HC was reversed after the combined AZA treatment. The expression of RECK decreased after SAM treatment. The level of mammary cancer cells decreased and the signaling pathway was blocked after the RECK siRNA interference protein was expressed. ROS methylation inhibits the expression of RECK and then activates STAT-3 eventually regulating the invasion and metastasis of breast cancer cells. In addition, the mRNA level of RECK in breast cancer cells is up regulated by STAT-3 siRNA interfering STAT-3. It is suggested that there is a interaction between RECK and STAT-3 signaling pathway in this process. Conclusion: in human breast cancer cells are fine. In the cell, 27HC activates ROS, reduces the mRNA and protein level of RECK and activates the STAT-3 signaling pathway, increases the transcriptional level and activity of MMP9, and leads to the decrease of the EMT related molecular index E-cadherin, Vimentin and ZEB1 increase, which makes the ability of the breast cancer cells to degrade the extracellular matrix and the ability to move the movement, and eventually promotes it. An increase in the ability to invasion and metastasis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2150025
[Abstract]:In twenty-first Century, breast cancer has become an important factor endangering women's health and plays an important role in female tumors. However, the invasion and metastasis of tumor cells is one of the most important mechanisms in the development and death process of breast cancer. The invasion and metastasis of tumor cells are complex and varied, such as the epithelial transformation of cancer cells (epithelial -mesenchymal transition, EMT) and matrix metalloproteinases (matrix metalloproteases, MMPs) degradation of extracellular matrix and so on. Many literature reports that cell epithelial mesenchymal transformation can make tumor cells from polygon, square into long spindle with epithelial index E- calcium mucin (E-cadherin) and so on, interstitial index vimentin (Vimentin), N- calcium mucin (N-cadherin) and E box combined with zinc finger protein 1 (ZEB1) increase and have a stronger movement ability. It is a sign of cancer progression. The existing studies have shown that RECK (the reversion-inducing cysteine-rich protein with Kazal motifs) can inhibit the invasion of tumor cells by down regulation and expression. The downregulation of RECK expression in the process of tumor development has become a sign of tumor metastasis. The activation of the transcription activating factor -3 (signal transducers and activators of transcription-3, STAT-3) also plays an important role in the process of tumor invasion and metastasis, and can activate the expression of.27- hydroxyl cholesterol (27). -hydroxyl cholesterol, 27HC, a metabolite of cholesterol in the human body, is produced by the metabolism of cytochrome P450 enzyme CYP27A1, and is decomposed by CYP7B1. The level of 27HC in patients with hypercholesterolemia is significantly elevated; women, especially postmenopausal women, are obese and lead to higher levels of cholesterol, causing 27HC to cause a variety of diseases, including tumors. Science reports that serious harm to human health in.2013, 27HC can combine with ER receptor to promote tumor cell proliferation and LXR receptor binding to promote tumor cell invasion and metastasis. However, there has been no report on whether 27HC can affect the invasion and metastasis of tumor cells by decreasing the expression of the tumor suppressor gene RECK and thus affecting the invasion and metastasis of the tumor cells. In this study, ER positive breast cancer cell line MCF7, T47D and human three shade breast cancer cell line MDA-MB-231 were studied in this study. The mechanism of 27HC to promote invasion and metastasis of breast cancer cells and the role of STAT-3 in the breast cancer cells were discussed, so as to provide more experimental data for the prevention and treatment of cancer and to open new ideas. Methods: detection of 27HC to cell survival. Influence of force: CCK8 experiment; detection of the influence of 27HC on the invasion and metastasis of breast cancer cells: cell scratch test, prelaying Matrigel glue and unpaved Matrigel Transwell test; detection of MMP2 and MMP9 activity: gelatinase assay; detection of mRNA expression level of target genes: RT-PCR and real-time quantitative PCR (qRT-PCR) experiment; detection of gene eggs White level: protein immunoblotting test, si-RNA knockout RECK and STAT-3: cell transfection experiments, and detection of ROS: reactive oxygen test. Results: (1) 27HC can promote the invasion and migration of MCF7 and T47D cells in breast cancer: first, the results of CCK8 and scratch healing experiments found that 4 mu of breast cancer cells did not affect cell viability for 72 hours. The highest concentration of Transwell showed that 27HC could increase the invasion and migration ability of breast cancer cells without affecting cell viability, and the results of RT-PCR and qRT-PCR showed that 27HC could increase the mRNA of MMP9 in breast cancer cells in a dose-dependent manner, but did not affect the mRNA level of MMP2, gelatin. The results of enzyme spectrum test showed that 27HC could increase the activity of MMP9, but did not affect the activity of MMP2. In addition, after 27HC treatment, Western Blot experiment found that EMT related index E-cadherin decreased and Vimentin and ZEB1 increased in breast cancer cells. (2) 27HC down RECK protein levels and levels and up regulation of expression and activity: molecular mechanism exploration In the study, we found that the REECK protein level and mRNA of breast cancer cells were down downward after 27HC treatment. After RECK siRNA interference RECK protein expression, MMP9 expression, activity and cell invasiveness increased in breast cancer cells, but MMP2 expression and activity did not change. (3) 27HC can activate STAT through ER alpha non dependent pathway -3 signaling pathway further promotes the invasion and migration of breast cancer cells. In addition, after 27HC treatment of ER positive breast cancer cells MCF7 and T47D and ER negative breast cancer cells MDA-MB-231, p-STAT-3 rises, STAT3 signaling pathway is activated and GREB1 expression rises, indicating that 27HC also activates the classical ER signal pathway. The enhanced invasion and metastasis of breast cancer caused by 27HC decreased, its downstream MMP9 decreased, E-cadherin increased, and Vimentin and ZEB1 decreased. It showed that STAT-3 signaling pathway played an important role in the invasion and metastasis of breast cancer induced by 27HC. (4) 27HC can promote the invasion and migration of breast cancer cells by activating the ROS level in the cells and promoting the invasion and migration of breast cancer cells: 27HC and After positive reference to H2O2 to treat ER positive breast cancer cells MCF7 and T47D, ROS detection reagent was added to the blue cloud sky ROS, and the fluorescence microscope was photographed. The results showed that 27HC could promote the production and accumulation of ROS and induce cell oxidative stress. (5) 27HC through ROS caused methylation to reduce RECK and activate STAT-3:27HC, methyltransferase inhibitor ( AZA), after the treatment of ER positive breast cancer cells MCF7 by methyl donor (SAM) and positive reference H2O2, it was found that the RECK down regulation caused by 27HC was reversed after the combined AZA treatment. The expression of RECK decreased after SAM treatment. The level of mammary cancer cells decreased and the signaling pathway was blocked after the RECK siRNA interference protein was expressed. ROS methylation inhibits the expression of RECK and then activates STAT-3 eventually regulating the invasion and metastasis of breast cancer cells. In addition, the mRNA level of RECK in breast cancer cells is up regulated by STAT-3 siRNA interfering STAT-3. It is suggested that there is a interaction between RECK and STAT-3 signaling pathway in this process. Conclusion: in human breast cancer cells are fine. In the cell, 27HC activates ROS, reduces the mRNA and protein level of RECK and activates the STAT-3 signaling pathway, increases the transcriptional level and activity of MMP9, and leads to the decrease of the EMT related molecular index E-cadherin, Vimentin and ZEB1 increase, which makes the ability of the breast cancer cells to degrade the extracellular matrix and the ability to move the movement, and eventually promotes it. An increase in the ability to invasion and metastasis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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相关期刊论文 前3条
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2 韩春生;陈艳;伍学强;;STAT3在肿瘤形成中的作用[J];中国实验血液学杂志;2013年02期
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