本文選題：LPS + Hippo信號通路　； 參考：《揚州大學》2017年碩士論文

【摘要】：目的:炎癥已經(jīng)被認為是癌癥的第七種生物學特性。很多肺部慢性炎癥也能夠增加肺癌發(fā)生的風險,并在一定程度上促進肺癌的進展。脂多糖(Lipopolysaccharides,LPS)是革蘭氏陰性桿菌釋放的重要致炎因子,具有引起炎性免疫應答及內(nèi)毒素休克等效應。一些既往研究顯示,Hippo信號通路參與了肺癌細胞的增殖。本研究擬探究Hippo信號通路在LPS誘導的肺腺癌A549細胞增殖過程中的作用及其機制。方法:1、培養(yǎng)A549細胞株,應用CCK-8檢測LPS對A549增殖能力的影響;2、應用Western blot(WB)分別檢測不同濃度LPS處理A549后Hippo信號通路相關(guān)蛋白(Mst1、SAV1、LATS1、MOB1、P-MOB1、YAP 和 P-YAP(ser127))的表達變化;應用WB分別檢測LPS處理不同時間點A549中Hippo信號通路相關(guān)蛋白(Mst1、YAP、P-YAP(ser127))的表達變化;應用免疫熒光檢測LPS處理后A549中YAP蛋白的定位變化;3、A549轉(zhuǎn)染Cy3標記的siRNA,應用免疫熒光顯微鏡觀察轉(zhuǎn)染效率;應用WB、逆轉(zhuǎn)錄-多聚酶鏈反應(Reverse transcription polymerase chain reaction,RT-PCR)檢測目的蛋白和mRNA的抑制率;4、A549轉(zhuǎn)染 Mst1 siRNA、YAPsiRNA 后,抑制 Mst1、YAP 表達,并應用 CCK-8檢測LPS對轉(zhuǎn)染Mst1 siRNA、YAP siRNA后A549增殖能力的變化;5、A549轉(zhuǎn)染Mst1siRNA、YAPsiRNA后,抑制 Mst1、YAP表達,應用 WB檢測Mst1、YAP的表達變化;應用免疫熒光技術(shù)檢測YAP蛋白定位變化。結(jié)果:1、0.1 μg/ml的LPS可顯著促進A549細胞增殖;2、LPS能夠下調(diào)A549細胞中Mst1、LATS1和p-mob1總蛋白的表達,上調(diào)SAV1、Mob1、YAP和p-YAP總蛋白的表達以及促進YAP的核易位;LPS對肺癌A549細胞中YAP蛋白的定位無明顯影響;3、Mst1 siRNA和YAP siRNA能夠成功轉(zhuǎn)染至肺癌A549細胞中,并且顯著抑制了A549細胞中Mst1和YAP的表達;4、轉(zhuǎn)染Mst1 siRNA后LPS對A549細胞的增殖作用無明顯變化,而轉(zhuǎn)染YAP siRNA后LPS對A549細胞的促增殖作用明顯抑制;5、轉(zhuǎn)染Mst1 siRNA后,LPS能夠進一步減少A549中Mst1的表達,并且顯著增加YAP的表達;轉(zhuǎn)染YAP siRNA后,LPS對A549中Mst1的表達無明顯影響,而對YAP表達的促進作用明顯減弱。6、轉(zhuǎn)染Mst1 siRNA和YAP siRNA后,LPS對于A549細胞中YAP的表達定位無明顯影響。結(jié)論:1、低濃度LPS能夠通過上調(diào)YAP表達促進A549細胞的增殖;2、YAP并不完全通過Hippo信號通路參與LPS誘導的A549細胞的增殖。
[Abstract]:Objective: inflammation has been recognized as the seventh biological characteristic of cancer. Many chronic lung inflammation also increases the risk of lung cancer and, to some extent, promotes its progression. Lipopolysaccharide lipopolysaccharide (LPSs) is an important inflammatory factor released by gram-negative bacilli and has the effects of inflammatory immune response and endotoxic shock. Some previous studies have shown that Hippo signaling pathway is involved in the proliferation of lung cancer cells. The purpose of this study was to investigate the role of Hippo signaling pathway in the proliferation of A549 cells induced by LPS and its mechanism. Methods: A549 cell line was cultured with CCK-8. The expression of Hippo signaling pathway related protein (Mst1SAV1, MOB1, P-MOB1OYAP and P-YAPser127A) was detected by Western blotWB. The expression of Hippo signal pathway related protein (Mst1SAV1) and P-YAPser127were detected by Western blotWB. The expression of Hippo signal pathway related protein (Mst1) and P-YAPP ser127A in A549 treated with LPS were detected by WB. The localization of YAP protein in A549 treated with LPS was detected by immunofluorescence. The Cy3 labeled siRNAs were transfected with A549 and the transfection efficiency was observed by immunofluorescence microscope. Reverse transcription-polymerase chain reaction (reverse transcription polymerase chain reverse PCR) was used to detect the inhibition rate of target protein and mRNA. Mst1 siRNA-YAPsiRNA was transfected into A549 to inhibit the expression of Mst1 siRNA-YAPsiRNA, and CCK-8 was used to detect the change of proliferation ability of A549 transfected with Mst1 siRNA-YAP siRNA, and 5A1-siRNA-5A549 was transfected into Mst1siRNA-YAPsiRNA. The expression of YAP protein was detected by WB, and the localization of YAP protein was detected by immunofluorescence technique. Results LPS with 0.1 渭 g/ml of 1 渭 g/ml could significantly promote the proliferation of A549 cells. LPs could down-regulate the expression of Mst1, LATS1 and p-mob1 total proteins in A549 cells. Upregulating the expression of total protein of SAV1 and p-YAP and promoting the nuclear translocation of YAP had no significant effect on the localization of YAP protein in lung cancer A549 cells. The expression of Mst1 siRNA and YAP siRNA in lung cancer A549 cells could be successfully transfected into A549 cells. The expression of Mst1 and YAP in A549 cells was significantly inhibited, and the proliferation of A549 cells was not changed after transfection of Mst1 siRNA with LPS. The proliferation of A549 cells was inhibited by LPS after transfection of YAP siRNA, the expression of Mst1 in A549 was further decreased and the expression of YAP was significantly increased after transfection of Mst1 siRNA, and the expression of Mst1 in A549 was not significantly affected by LPS after transfection of YAP siRNA. However, the promotion of YAP expression was significantly weakened. The expression of YAP in A549 cells was not significantly affected by Mst1 siRNA and YAP siRNA transfection. Conclusion low concentration of LPS can promote the proliferation of A549 cells by up-regulating the expression of YAP. (2) Yap does not participate in the proliferation of A549 cells induced by LPS through Hippo signaling pathway.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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