STAT-3信號(hào)通路在27-羥基膽固醇促進(jìn)乳腺癌細(xì)胞侵襲轉(zhuǎn)移中的作用
發(fā)布時(shí)間：2018-07-28 11:43
【摘要】：21世紀(jì)乳腺癌已經(jīng)成為危害女性健康的重要因素,在女性腫瘤中占有相當(dāng)重要的地位。然而腫瘤細(xì)胞的侵襲轉(zhuǎn)移是乳腺癌發(fā)展過(guò)程和致死過(guò)程中的重要機(jī)制之一。腫瘤細(xì)胞的侵襲轉(zhuǎn)移過(guò)程復(fù)雜多樣,例如癌細(xì)胞的上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)以及基質(zhì)金屬蛋白酶(matrix metalloproteases,MMPs)降解細(xì)胞外基質(zhì)等等。眾多文獻(xiàn)報(bào)道細(xì)胞上皮間質(zhì)轉(zhuǎn)化可以使腫瘤細(xì)胞由多邊形、方形變?yōu)殚L(zhǎng)梭形伴有上皮指標(biāo)E-鈣黏蛋白(E-cadherin)等下降、間質(zhì)指標(biāo)波形蛋白(Vimentin)、N-鈣黏蛋白(N-cadherin)和E盒結(jié)合鋅指蛋白1(ZEB1)等上升而具有更強(qiáng)的運(yùn)動(dòng)遷移能力,是癌癥進(jìn)展的一個(gè)標(biāo)志;已有研究表明 RECK(the reversion-inducing cysteine-rich protein with kazal motifs)蛋白可以通過(guò)下調(diào)MMP2和MMP9的表達(dá)進(jìn)而抑制腫瘤細(xì)胞的侵襲轉(zhuǎn)移,因此在腫瘤發(fā)展過(guò)程中RECK表達(dá)的下調(diào)成為腫瘤惡轉(zhuǎn)的一個(gè)標(biāo)志。轉(zhuǎn)錄激活因子-3(signal transducers and activators of transcription-3,STAT-3)的活化在腫瘤侵襲轉(zhuǎn)移過(guò)程中也發(fā)揮重要作用,可以激活下游侵襲轉(zhuǎn)移指標(biāo)的表達(dá)。27-羥基膽固醇(27-hydroxyl cholesterol,27HC)是人體內(nèi)膽固醇的代謝產(chǎn)物,由細(xì)胞色素P450酶CYP27A1代謝產(chǎn)生,并由CYP7B1分解。高膽固醇血癥患者體內(nèi)27HC水平明顯升高;婦女尤其是絕經(jīng)后婦女肥胖導(dǎo)致膽固醇水平升高進(jìn)而催生27HC引發(fā)各種包括腫瘤在內(nèi)的疾病,嚴(yán)重危害人類健康。2013年Science報(bào)道,27HC可與ER受體結(jié)合促進(jìn)腫瘤細(xì)胞增殖及與LXR受體結(jié)合促進(jìn)腫瘤細(xì)胞侵襲轉(zhuǎn)移,但是目前關(guān)于27HC能否通過(guò)下調(diào)腫瘤抑制基因RECK的表達(dá)從而影響STAT-3信號(hào)通路促進(jìn)腫瘤細(xì)胞的侵襲轉(zhuǎn)移尚未有報(bào)道。目的:本研究以人ER陽(yáng)性乳腺癌細(xì)胞系MCF7、T47D和人三陰乳腺癌細(xì)胞系MDA-MB-231為研究對(duì)象,探討27HC促進(jìn)乳腺癌細(xì)胞侵襲轉(zhuǎn)移的機(jī)制及STAT-3在其中的作用,從而為腫瘤的預(yù)防及治療提供更多的實(shí)驗(yàn)數(shù)據(jù),打開(kāi)新的思路。方法:檢測(cè)27HC對(duì)細(xì)胞活力的影響:CCK8實(shí)驗(yàn);檢測(cè)27HC對(duì)乳腺癌細(xì)胞的侵襲轉(zhuǎn)移能力的影響:細(xì)胞劃痕實(shí)驗(yàn),預(yù)鋪matrigel膠和未鋪matrigel的Transwell實(shí)驗(yàn);檢測(cè)MMP2和MMP9的活性:明膠酶譜實(shí)驗(yàn);檢測(cè)目的基因的mRNA表達(dá)水平:RT-PCR和實(shí)時(shí)定量PCR(qRT-PCR)實(shí)驗(yàn);檢測(cè)基因的蛋白水平:蛋白質(zhì)免疫印跡實(shí)驗(yàn);si-RNA敲除RECK和STAT-3:細(xì)胞轉(zhuǎn)染實(shí)驗(yàn);檢測(cè)ROS:活性氧檢測(cè)實(shí)驗(yàn)。結(jié)果:(1)27HC能夠促進(jìn)乳腺癌MCF7和T47D細(xì)胞的侵襲遷移能力:首先,CCK8實(shí)驗(yàn)和劃痕愈合實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)4.0μM是處理乳腺癌細(xì)胞72小時(shí)不影響細(xì)胞活力的最高濃度;采用預(yù)鋪膠和未鋪膠的Transwell實(shí)驗(yàn)證實(shí)27HC能夠在不影響細(xì)胞活力的前提下,上調(diào)乳腺癌細(xì)胞的侵襲遷移能力;RT-PCR和qRT-PCR實(shí)驗(yàn)結(jié)果顯示27HC可以呈劑量依賴性地上調(diào)乳腺癌細(xì)胞內(nèi)MMP9的mRNA,但并不影響MMP2的mRNA水平;明膠酶譜實(shí)驗(yàn)結(jié)果顯示27HC可以上調(diào)MMP9的活性,但并不影響MMP2的活性。另外,27HC處理后,Western Blot實(shí)驗(yàn)發(fā)現(xiàn),乳腺癌細(xì)胞內(nèi)EMT相關(guān)指標(biāo)E-cadherin下降,Vimentin和ZEB1上升。(2)27HC下調(diào)RECK的蛋白水平和mRNA水平并上調(diào)MMP9的表達(dá)和活性:在分子機(jī)制探索研究中,我們發(fā)現(xiàn)經(jīng)過(guò)27HC處理后乳腺癌細(xì)胞的REECK的蛋白水平和mRNA均呈現(xiàn)下調(diào)趨勢(shì),采用RECK siRNA干擾RECK蛋白表達(dá)后,乳腺癌細(xì)胞內(nèi)MMP9表達(dá)、活性及細(xì)胞侵襲能力上升,但是MMP2的表達(dá)和活性不變。(3)27HC可以通過(guò)ERα非依賴性途徑激活STAT-3信號(hào)通路進(jìn)而促進(jìn)乳腺癌細(xì)胞的侵襲遷移能力:另外,27HC處理ER陽(yáng)性乳腺癌細(xì)胞MCF7和T47D及ER陰性乳腺癌細(xì)胞MDA-MB-231后,p-STAT-3上升,STAT3信號(hào)通路被激活,GREB1的表達(dá)也上升,說(shuō)明27HC也激活了經(jīng)典的ER信號(hào)通路。應(yīng)用siSTAT-3敲除STAT-3之后,27HC所引起的乳腺癌侵襲轉(zhuǎn)移能力增強(qiáng)有所下降,其下游MMP9下降,E-cadherin上升,Vimentin和ZEB1下降。表明STAT-3信號(hào)通路在27HC誘導(dǎo)的乳腺癌侵襲轉(zhuǎn)移過(guò)程中發(fā)揮重要作用。(4)27HC通過(guò)激活細(xì)胞內(nèi)ROS水平并促進(jìn)乳腺癌細(xì)胞侵襲遷移能力:27HC和陽(yáng)性參照H2O2處理ER陽(yáng)性乳腺癌細(xì)胞MCF7和T47D后,加入碧云天ROS檢測(cè)試劑,并應(yīng)用熒光顯微鏡拍照,結(jié)果顯示27HC可以促進(jìn)ROS產(chǎn)生和蓄積,引起細(xì)胞氧化應(yīng)激。(5)27HC通過(guò)ROS引起甲基化導(dǎo)致RECK的表達(dá)下調(diào)進(jìn)而激活STAT-3:27HC,甲基轉(zhuǎn)移酶抑制劑(AZA),甲基供體(SAM)和陽(yáng)性參照H2O2處理ER陽(yáng)性乳腺癌細(xì)胞MCF7后,發(fā)現(xiàn)聯(lián)合AZA處理后,27HC所引起的RECK下調(diào)有所逆轉(zhuǎn),SAM處理后RECK的表達(dá)呈現(xiàn)下降趨勢(shì)。采用RECK siRNA干擾RECK蛋白表達(dá)后乳腺癌細(xì)胞內(nèi)p-STAT-3水平下降,STAT-3信號(hào)通路被阻斷。所以27HC通過(guò)ROS甲基化抑制RECK的表達(dá)進(jìn)而激活STAT-3最終正向調(diào)控乳腺癌細(xì)胞的侵襲轉(zhuǎn)移能力。另外采用STAT-3 siRNA干擾STAT-3后乳腺癌細(xì)胞內(nèi)RECK的mRNA水平上調(diào)。提示在這一過(guò)程中RECK與STAT-3信號(hào)通路存在相互作用關(guān)系。結(jié)論:在人乳腺癌細(xì)胞中,27HC通過(guò)激活ROS,甲基化下調(diào)RECK的mRNA和蛋白水平進(jìn)而激活STAT-3信號(hào)通路,升高M(jìn)MP9的轉(zhuǎn)錄水平和活性以及導(dǎo)致EMT相關(guān)分子指標(biāo)E-cadherin下降、Vimentin和ZEB1上升,使乳腺癌細(xì)胞降解細(xì)胞外基質(zhì)的能力和運(yùn)動(dòng)遷移的能力上升,最終促進(jìn)其侵襲轉(zhuǎn)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
本文編號(hào)：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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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3 林娜;姚曉光;李南方;;細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制因子3的研究進(jìn)展[J];中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào);2012年02期
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