【摘要】：目的探討瘦素(Leptin)促人乳腺癌細(xì)胞MCF-7、SK-BR-3的M2型丙酮酸激酶(M2-pyruvate kinase,PKM2)表達(dá)及其誘導(dǎo)上皮間質(zhì)化(epithelial-mesenchymal transition,EMT)的作用及機(jī)制,初步闡明Leptin通過(guò)上調(diào)糖代謝關(guān)鍵酶PKM2而影響乳腺癌EMT及其作用機(jī)制。方法1.采用蛋白質(zhì)印記法(western blotting,WB)和免疫熒光法(immunofluorescence,IF)檢測(cè)人乳腺癌細(xì)胞MCF-7、SK-BR-3、MDA-MB-468中Leptin長(zhǎng)受體OB-Rb、短受體OB-Rt的表達(dá);采用人源Leptin處理三株細(xì)胞,觀察細(xì)胞形態(tài)變化;WB檢測(cè)細(xì)胞EMT相關(guān)標(biāo)志蛋白的表達(dá)。2.Leptin處理人乳腺癌細(xì)胞MCF-7、SK-BR-3后,采用QRT-PCR和WB,分析PKM2 m RNA及蛋白水平的變化。3.采用化學(xué)合成的PKM2-si RNA干擾乳腺癌細(xì)胞MCF-7、SK-BR-3的PKM2,劃痕及Transwell侵襲實(shí)驗(yàn)分別檢測(cè)Leptin對(duì)兩株細(xì)胞遷移及侵襲能力的影響;WB檢測(cè)Leptin對(duì)兩株細(xì)胞EMT相關(guān)標(biāo)志蛋白表達(dá)的影響。4.采用Leptin聯(lián)合PI3K/AKT、JAK/STAT、MAPK/ERK1/2信號(hào)通路抑制劑處理乳腺癌細(xì)胞MCF-7和SK-BR-3,WB檢測(cè)PKM2的表達(dá)。采用Leptin受體中和抗體處理乳腺癌細(xì)胞MCF-7和SK-BR-3后,WB檢測(cè)Leptin對(duì)PKM2表達(dá)及對(duì)PI3K/AKT信號(hào)通路激活的影響;采用WB檢測(cè)PI3K/AKT通路抑制劑處理后,乳腺癌細(xì)胞MCF-7和SK-BR-3中PKM2及EMT相關(guān)蛋白的表達(dá)。5.構(gòu)建穩(wěn)定干擾PKM2的乳腺癌SK-BR-3細(xì)胞株,利用該細(xì)胞株建立裸鼠移植瘤模型,測(cè)量腫瘤大小,觀察裸鼠生存期。免疫組織化學(xué)法(immunohistochemistry,IHC)檢測(cè)腫瘤組織EMT相關(guān)蛋白的表達(dá),HE染色檢測(cè)腫瘤肝肺轉(zhuǎn)移。結(jié)果1.WB及IF結(jié)果表明,乳腺癌細(xì)胞MCF-7、SK-BR-3和MDA-MB-468均表達(dá)OB-Rb和OB-Rt;Leptin能引起三株細(xì)胞形態(tài)發(fā)生EMT樣改變,且下調(diào)上皮性標(biāo)志物E-cadherin,上調(diào)間質(zhì)性標(biāo)志物Vimentin和Fibronectin的表達(dá)。2.QRT-PCR及WB結(jié)果顯示,Leptin能顯著上調(diào)MCF-7、SK-BR-3細(xì)胞PKM2的m RNA及蛋白水平。3.為進(jìn)一步探討PKM2在Leptin促乳腺癌細(xì)胞EMT過(guò)程中的作用,采用PKM2-si RNA干擾細(xì)胞表達(dá)PKM2,劃痕及Transwell實(shí)驗(yàn)結(jié)果表明,MCF-7、SK-BR-3細(xì)胞的遷移和侵襲能力被抑制;WB檢測(cè)提示,細(xì)胞的E-cadherin水平升高,Vimentin、Fibronectin水平降低,即干擾細(xì)胞PKM2表達(dá)后Leptin促EMT作用被抑制。4.WB結(jié)果表明,抑制PI3K/AKT信號(hào)通路能減弱Leptin促PKM2表達(dá)的作用。采用中和性抗體封閉Leptin受體OBR,PI3K/AKT信號(hào)通路的激活被抑制,Leptin促PKM2表達(dá)的作用也被抑制。采用WB檢測(cè)發(fā)現(xiàn)PI3K/AKT信號(hào)通路抑制后,Leptin促乳腺癌細(xì)胞PKM2表達(dá)及EMT的作用被抑制。5.裸鼠乳腺癌移植瘤模型分為SKBR-3、SKBR-3-LV和SKBR-3-sh PKM2組,其中SKBR-3、SKBR-3-LV為對(duì)照組,SKBR-3-sh PKM2為實(shí)驗(yàn)組。實(shí)驗(yàn)結(jié)果表明SKBR-3-sh PKM2組腫瘤體積、重量均明顯小于對(duì)照組。HE染色SKBR-3、SKBR-3-LV組肝肺轉(zhuǎn)移灶較SKBR-3-sh PKM2組明顯增多。瘤組織IHC結(jié)果顯示,與對(duì)照組相比,SKBR-3-sh PKM2組E-cadherin表達(dá)明顯升高,Vimentin、Fibronectin表達(dá)明顯降低,SKBR-3-sh PKM2組小鼠生存期明顯延長(zhǎng)。結(jié)論1.Leptin促進(jìn)乳腺癌細(xì)胞MCF-7、SK-BR-3、MDA-MB-468發(fā)生EMT。2.Leptin激活PI3K/AKT信號(hào)通路上調(diào)乳腺癌細(xì)胞MCF-7、SK-BR-3的PKM2表達(dá),進(jìn)而促進(jìn)乳腺癌細(xì)胞發(fā)生EMT。3.抑制PKM2能夠減弱乳腺癌細(xì)胞移植瘤的生長(zhǎng),延長(zhǎng)荷瘤小鼠生存期,減少乳腺癌肝肺轉(zhuǎn)移,PKM2可能是影響乳腺癌侵襲轉(zhuǎn)移的重要靶點(diǎn)。
[Abstract]:Objective To investigate the effect and mechanism of leptin on the expression of M2-pyruvate kinase (PKM2) and epithelial-mesenchymal transition (EMT) in human breast cancer cells MCF-7, SK-BR-3. Methods 1. Western blotting (WB) and immunofluorescence (IF) were used to detect the expression of Leptin long receptor OB-Rb and short receptor OB-Rt in MCF-7, SK-BR-3 and MDA-MB-468 breast cancer cells. The morphological changes of three cells were observed by human Leptin treatment. The expression of EMT-related marker protein was detected by WB. After treatment of human breast cancer cells MCF-7, SK-BR-3, the changes of PKM2 m RNA and protein levels were analyzed by QRT-PCR and WB. 3. The effects of Leptin on the migration and invasion of breast cancer cells MCF-7, SK-BR-3 were detected by interfering with PKM2, scratch and Transwell invasion assays with chemically synthesized PKM2-si RNA, and WB was used to detect the effects of Leptin on the migration and invasion of the cells. Leptin combined with PI3K/AKT, JAK/STAT, MAPK/ERK1/2 signal pathway inhibitors were used to treat breast cancer cells MCF-7 and SK-BR-3. WB was used to detect the expression of PKM2. Leptin receptor neutralizing antibodies were used to treat breast cancer cells MCF-7 and SK-BR-3. WB was used to detect the expression of PKM2 and activation of PI3K/AKT signaling pathway. The expression of PKM2 and EMT-related proteins in breast cancer cells MCF-7 and SK-BR-3 after treatment with PI3K/AKT pathway inhibitors was detected by WB. 5. The SK-BR-3 cell line stably interfering with PKM2 was constructed. The nude mice transplanted tumor model was established by using this cell line, the tumor size was measured and the survival time was observed. Results WB and IF results showed that breast cancer cells MCF-7, SK-BR-3 and MDA-MB-468 all expressed OB-Rb and OB-Rt; Leptin could induce EMcadT-like changes in three cell lines, and down-regulate the epithelial marker E-cadherin and up-regulate the interstitial marker Vimenti Vimenti. QRT-PCR and WB results showed that Leptin could significantly up-regulate the expression of M RNA and protein of PKM2 in MCF-7, SK-BR-3 cells. 3. To further explore the role of PKM2 in the process of Leptin promoting EMT in breast cancer cells, PKM2-si RNA was used to interfere with the expression of PKM2 in cells. Scratch and Transwell experiments showed that MCF-7, SK-BR-3 cells migrated. WB assay showed that E-cadherin level increased, Vimentin and Fibronectin levels decreased, that is, Leptin-induced EMT was inhibited after interfering with PKM2 expression. 4. WB assay showed that inhibition of PI3K/AKT signaling pathway could attenuate Leptin-induced PKM2 expression. The activation of the pathway was inhibited and the effect of Leptin on PKM2 expression was inhibited. WB assay showed that the effect of Leptin on PKM2 expression and EMT in breast cancer cells was inhibited after PI3K/AKT signaling pathway was inhibited. 5. Nude mice breast cancer xenograft models were divided into SKBR-3, SKBR-3-LV and SKBR-3-sh PKM2 groups, SKBR-3-LV as control group, SKBR-3-sh PKM2 as control group. The results showed that the tumor volume and weight of SKBR-3-sh PKM2 group were significantly smaller than those of control group. The liver and lung metastases of SKBR-3, SKBR-3-LV group were significantly more than those of SKBR-3-sh PKM2 group. The survival time of R-3-sh PKM2 group mice was significantly prolonged. Conclusion 1. Leptin can promote the occurrence of EMT in breast cancer cells MCF-7, SK-BR-3, MDA-MB-468. Leptin can activate PI3K/AKT signaling pathway and up-regulate the expression of PKM2 in breast cancer cells MCF-7, SK-BR-3, thereby promoting the occurrence of EMT.3 in breast cancer cells. PKM2 may be an important target for breast cancer invasion and metastasis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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