本文選題：MDM2 + 乳腺癌��； 參考：《南京醫(yī)科大學(xué)》2016年博士論文

【摘要】：研究背景：乳腺癌是世界上女性最常見的惡性腫瘤,其死亡率在女性惡性腫瘤中占第二位。上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transitions,EMT)是啟動(dòng)腫瘤侵襲和轉(zhuǎn)移的一個(gè)公認(rèn)的機(jī)制,上皮間質(zhì)轉(zhuǎn)化發(fā)生時(shí),乳腺癌上皮細(xì)胞獲得一個(gè)能動(dòng)的間質(zhì)表型。MDM2基因在人類多種惡性腫瘤,如軟組織肉瘤、乳腺癌、卵巢癌、宮頸癌、腦癌、肺癌、前列腺癌、結(jié)腸癌和腎癌中均高表達(dá)。在我們以前的研究中,我們發(fā)現(xiàn)MDM2能夠通過上調(diào)MMP9的表達(dá)促進(jìn)乳腺癌的侵襲和轉(zhuǎn)移。MDM2是否還通過其他途徑促進(jìn)乳腺癌的侵襲和轉(zhuǎn)移,值得我們進(jìn)一步深入研究。研究目的：研究MDM2在人乳腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化中的作用及其可能的分子機(jī)制。研究方法：我們首先通過定量PCR和western blotting法檢測(cè)了三種乳腺癌細(xì)胞系MCF-7、MDA-MB-231、MDA-MB-435和一種正常乳腺細(xì)胞系HBL-100中MDM2的mRNA和蛋白水平的表達(dá)情況。然后,我們以慢病毒為載體,構(gòu)建了過表達(dá)MDM2的乳腺癌穩(wěn)轉(zhuǎn)細(xì)胞株MCF-7-MDM2-a,MCF-7-MDM2-d及對(duì)照細(xì)胞株MCF-7-pCMV,通過光鏡觀察三種細(xì)胞株的形態(tài),定量PCR及western blot檢測(cè)上皮標(biāo)記物E-鈣粘蛋白的表達(dá),轉(zhuǎn)錄因子Snail的表達(dá)以及間質(zhì)標(biāo)記物N-鈣粘蛋白和波形蛋白的表達(dá)。然后,我們通過干擾RNA在MDA-MB-231細(xì)胞中敲低MDM2的表達(dá),通過光鏡觀察兩種細(xì)胞株的形態(tài),定量PCR及western blotting檢測(cè)上皮標(biāo)記物E-鈣粘蛋白的表達(dá),間質(zhì)標(biāo)記物N-鈣粘蛋白和波形蛋白的表達(dá)以及轉(zhuǎn)錄因子Snail的表達(dá)。接著我們通過干擾RNA在MCF-7-MDM2-a細(xì)胞中敲低Snail的表達(dá),通過光鏡觀察兩種細(xì)胞株的形態(tài),定量PCR及western blotting檢測(cè)上皮標(biāo)記物E-鈣粘蛋白的表達(dá)及間質(zhì)標(biāo)記物N-鈣粘蛋白和波形蛋白的表達(dá)。然后,我們?cè)贛CF-7細(xì)胞中敲低Snail后再瞬時(shí)轉(zhuǎn)染入MDM2質(zhì)粒,定量PCR及western blotting檢測(cè)EMT標(biāo)記物E-cadherin,N-cadherin及Vimentin的表達(dá)情況。接著,我們進(jìn)一步在MCF-7細(xì)胞中敲低NF-kappaB/P65亞基后再瞬時(shí)轉(zhuǎn)染入MDM2質(zhì)粒,定量PCR及western blotting檢測(cè)轉(zhuǎn)錄因子Snail的表達(dá)。然后,我們用構(gòu)建的乳腺癌穩(wěn)轉(zhuǎn)細(xì)胞株MCF-7-MDM2-a及對(duì)照細(xì)胞株MCF-7-pCMV接種雌性裸鼠腋下成瘤,六周后處死裸鼠,測(cè)量腫瘤的大小及重量,并通過定量PCR、western blotting及免疫組化法檢測(cè)瘤體中上皮標(biāo)記物E-鈣粘蛋白的表達(dá),轉(zhuǎn)錄因子Snail的表達(dá)以及間質(zhì)標(biāo)記物N-鈣粘蛋白和波形蛋白的表達(dá)。最后,我們通過人乳腺癌芯片研究上皮標(biāo)記物E-鈣粘蛋白,間質(zhì)標(biāo)記物N-鈣粘蛋白和波形蛋白以及轉(zhuǎn)錄因子Snail在人乳腺癌組織中的表達(dá),并研究其相關(guān)性。研究結(jié)果：MDM2在侵襲性乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-435中均高表達(dá),在MCF-7細(xì)胞中,MDM2過表達(dá)通過NF-kappa/B P65亞基上調(diào)Snail的表達(dá),上調(diào)的Snail可以促進(jìn)細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化。而在MDA-MB-231細(xì)胞中,敲低MDM2促進(jìn)間質(zhì)上皮轉(zhuǎn)化。在MCF-7-MDM2-a細(xì)胞中,通過干擾RNA敲低Snail可導(dǎo)致MCF-7-MDM2-a細(xì)胞發(fā)生間質(zhì)上皮轉(zhuǎn)化,細(xì)胞恢復(fù)上皮性狀。在動(dòng)物水平,我們發(fā)現(xiàn)MDM2可以促進(jìn)腫瘤生長,誘導(dǎo)乳腺癌細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化及賦予人乳腺癌細(xì)胞轉(zhuǎn)移潛能。最后,在乳腺癌芯片中,我們發(fā)現(xiàn)MDM2的表達(dá)與上皮標(biāo)記物E-cadherin的表達(dá)呈負(fù)相關(guān),而與間質(zhì)標(biāo)記物N-cadherin、Vimentin以及轉(zhuǎn)錄因子Snail的表達(dá)呈正相關(guān)。Snail的表達(dá)與上皮標(biāo)記物E-cadherin的表達(dá)呈負(fù)相關(guān)。研究結(jié)論：MDM2通過NF-kappa/B P65亞基上調(diào)Snail的表達(dá),上調(diào)的Snail促使人乳腺癌細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化,MDM2有可能成為乳腺癌轉(zhuǎn)移治療和預(yù)防的一個(gè)潛在靶點(diǎn)。
[Abstract]:Background: breast cancer is the most common malignant tumor in women in the world. The mortality rate is second in female malignant tumors. Epithelial-mesenchymal transitions (EMT) is a recognized mechanism for the initiation of tumor invasion and metastasis. Interstitial phenotypic.MDM2 gene is highly expressed in a variety of human malignant tumors, such as soft tissue sarcoma, breast cancer, ovarian, cervical, brain, lung, prostate, colon and renal cancer. In our previous study, we found that MDM2 can promote the invasion of breast cancer and transfer.MDM2 through other routes through the up regulation of MMP9. The path to promote the invasion and metastasis of breast cancer is worthy of further study. Objective: To study the role of MDM2 in the epithelial mesenchymal transition of human breast cancer cells and its possible molecular mechanism. Research methods: we first detected three kinds of breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-435 by quantitative PCR and Western blotting method. And the expression of mRNA and protein levels of MDM2 in a normal mammary cell line HBL-100. Then, we use lentivirus as the carrier to construct a breast cancer cell line that overexpresses MDM2, MCF-7-MDM2-a, MCF-7-MDM2-d and the control cell line MCF-7-pCMV, and observe the morphology of three cell lines by light microscopy, quantitative PCR and Western blot detection of epithelium. Expression of marker E- cadherin, expression of transcription factor Snail and expression of stromal marker N- cadherin and vimentin. Then, we observe the morphology of two cell lines by interfering with RNA in MDA-MB-231 cells and observe the morphology of the cell lines by light microscopy, quantitative PCR and Western blotting detection of the epithelial markers E- cadherin. Expression, expression of stromal marker N- and vimentin and expression of transcription factor Snail. Then we observe the morphology of two cell lines by interfering with RNA in MCF-7-MDM2-a cells and observe the morphology of the two cell lines by light microscopy, quantitative PCR and Western blotting to detect the expression of the epithelial markers E- cadherin and the interstitial marker N. The expression of cadherin and vimentin. Then, we transfected into MDM2 plasmids after knocking down Snail in MCF-7 cells, and quantified PCR and Western blotting to detect the EMT marker E-cadherin, N-cadherin and Vimentin expression. Then, we further transfected into the substance after knocking down the NF-kappaB/P65 subunit in the MCF-7 cell. The expression of transcription factor Snail was detected by grain, quantitative PCR and Western blotting. Then, we used the constructed breast cancer cell line MCF-7-MDM2-a and the control cell strain MCF-7-pCMV to be inoculated into the axillary tumor of female nude mice. After six weeks, the nude mice were killed and the size and weight of the tumor were measured, and the quantitative PCR, Western blotting and immunohistochemistry were detected. Expression of E- cadherin, expression of transcription factor Snail and expression of stromal marker N- cadherin and vimentin in the tumor. Finally, we study the epithelial markers E- cadherin by human breast cancer microarray, interstitial marker N- cadherin, wave white egg white and transcription factor Snail in human breast cancer tissue The expression of MDM2 is highly expressed in invasive breast cancer cell lines MDA-MB-231 and MDA-MB-435. In MCF-7 cells, MDM2 overexpression increases the expression of Snail through the NF-kappa/B P65 subunit, and up-regulated Snail can promote epithelial mesenchymal transition. In MDA-MB-231 cells, low MDM2 promotes low MDM2. Mesenchymal epithelial transformation. In MCF-7-MDM2-a cells, RNA knockdown Snail can lead to interstitial epithelial transformation in MCF-7-MDM2-a cells and cells restore epithelial traits. At animal levels, we found that MDM2 can promote tumor growth, induce epithelial stroma transformation of breast cancer cells and give human breast cancer cell metastasis potential. Finally, In the breast cancer chip, we found that the expression of MDM2 was negatively correlated with the expression of the epithelial marker E-cadherin, but the expression of the interstitial marker N-cadherin, Vimentin and the transcription factor Snail was positively correlated with the expression of.Snail and the expression of the epithelial marker E-cadherin. The conclusion: MDM2 is up Snail by the NF-kappa/B P65 subunit. The expression of upregulation of Snail promotes epithelial mesenchymal transition in human breast cancer cells. MDM2 may be a potential target for breast cancer metastasis therapy and prevention.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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