抑制PIK3C3和PI3K通路對肝癌干細胞自我更新的影響及機制研究
發(fā)布時間：2021-10-26 22:36
我們前期研究發(fā)現血清和糖皮質激素調節(jié)激酶3（Serum and glucocorticoid kinase 3,SGK3）能夠促進肝癌細胞發(fā)生上皮間質轉化（epithelial-mesenchymal transition,EMT）進而促進肝癌細胞的侵襲和遷移,但其是否參與調控肝癌干細胞干性還不清楚。PI3K信號通路（通常指I型PI3K）異常調節(jié)在各種惡性腫瘤中非常普遍,Akt一直被認為是PI3K信號通路中至關重要的中轉信號分子,近年來,有研究證實SGK3是I型PI3K通路下游不依賴Akt的獨立調控分子,而SGK3在I型PI3K信號通路中的作用及機制仍未闡明。Ⅲ型PI3K（PIK3C3）又被稱作Vps34,在細胞自噬和胞吞過程中發(fā)揮重要作用。PIK3C3還是生成PtdIns（3）P的重要場所,PtdIns（3）P是細胞內的重要第二信使,能夠激活含PX和FYVE結構域的蛋白質。有報道稱,PIK3C3在結腸癌、乳腺癌等多種腫瘤的發(fā)生發(fā)展過程中發(fā)揮重要作用,但是其是否參與了腫瘤干細胞的調控還未見相關報道。第一部分抑制PI3K對肝癌干細胞自我更新的影響及機制研究目的:探究SGK3在肝癌干細胞自...
【文章來源】：重慶醫(yī)科大學重慶市
【文章頁數】：103 頁
【學位級別】：博士
【部分圖文】：
磁珠分選細胞分選架組裝示意
圖 1. SGK3 在肝癌干細胞中磷酸化程度明顯增高Fig 1. SGK3 is preferentially activated in liver CSCs(A) MHCC-97H cells were cultured in monolayer or ultra-low attachment conditions. ThemRNA expression of liver CSC-related genes and SGK3 in spheroids and attached cells wascompared by RT-PCR. (B) Western blot analysis for levels of CD133, active Akt (phosphorylated atSer473)/total Akt and active SGK3 (phosphorylated at Thr320)/total SGK3 between spheroids andattached cells. (C) Flow cytometry analysis of CD133+ cell distribution in CD133- and CD133+cells isolated using CD133 MicroBead Kit. (D) Representative images of CD133+ and CD133- cellssorted from MHCC97H HCC cells cultured in serum-free culture medium after 7 days. Scale bars,100 μm. (E) CD133+/CD133- cells were subcutaneously injected into 6-week-old female athymicnude mice, and tumourigenicity was evaluated 5 weeks post-inoculation. (F) Levels of CD133,active Akt (phosphorylated at Ser473)/total Akt and active SGK3 (phosphorylated at Thr320)/total
重慶醫(yī)科大學博士研究生學位論文SGK3 were compared by western blot analysis between CD133+ and CD133- cells. β-actin was usedas a loading control. All experiments were performed in triplicate. *P < 0.05.2.2 SGK3 促進肝癌干細胞自我更新為了深入探究 SGK3 對肝癌干細胞自我更新的調控作用，我們使用慢病毒載體轉染 MHCC97H 和 Huh7 細胞并構建了 SGK3 穩(wěn)定過表達細胞株。如圖 2.A 和 B所示，慢病毒轉染成功后 SGK3 在 MHCC97H 和 Huh7 細胞中明顯過表達，并且SGK3 過表達明顯地促進了肝癌干細胞相關基因 CD133、CD90、Oct4、Nanog、Bmi-1 和 Sox2 的表達。我們在蛋白水平驗證了 SGK3 對干細胞相關基因表達的促進作用（圖 2.C）。為了進一步證實 SGK3 對肝癌干細胞自我更新的影響，我們檢測了兩組細胞的成球能力，發(fā)現 SGK3 過表達明顯促進肝癌細胞的成球能力（圖2.D）。
【參考文獻】：
期刊論文
[1]Cancer stem cell plasticity and tumor hierarchy[J]. Marina Carla Cabrera,Robert E Hollingsworth,Elaine M Hurt. World Journal of Stem Cells. 2015(01)
本文編號：3460321
【文章來源】：重慶醫(yī)科大學重慶市
【文章頁數】：103 頁
【學位級別】：博士
【部分圖文】：
磁珠分選細胞分選架組裝示意
圖 1. SGK3 在肝癌干細胞中磷酸化程度明顯增高Fig 1. SGK3 is preferentially activated in liver CSCs(A) MHCC-97H cells were cultured in monolayer or ultra-low attachment conditions. ThemRNA expression of liver CSC-related genes and SGK3 in spheroids and attached cells wascompared by RT-PCR. (B) Western blot analysis for levels of CD133, active Akt (phosphorylated atSer473)/total Akt and active SGK3 (phosphorylated at Thr320)/total SGK3 between spheroids andattached cells. (C) Flow cytometry analysis of CD133+ cell distribution in CD133- and CD133+cells isolated using CD133 MicroBead Kit. (D) Representative images of CD133+ and CD133- cellssorted from MHCC97H HCC cells cultured in serum-free culture medium after 7 days. Scale bars,100 μm. (E) CD133+/CD133- cells were subcutaneously injected into 6-week-old female athymicnude mice, and tumourigenicity was evaluated 5 weeks post-inoculation. (F) Levels of CD133,active Akt (phosphorylated at Ser473)/total Akt and active SGK3 (phosphorylated at Thr320)/total
重慶醫(yī)科大學博士研究生學位論文SGK3 were compared by western blot analysis between CD133+ and CD133- cells. β-actin was usedas a loading control. All experiments were performed in triplicate. *P < 0.05.2.2 SGK3 促進肝癌干細胞自我更新為了深入探究 SGK3 對肝癌干細胞自我更新的調控作用，我們使用慢病毒載體轉染 MHCC97H 和 Huh7 細胞并構建了 SGK3 穩(wěn)定過表達細胞株。如圖 2.A 和 B所示，慢病毒轉染成功后 SGK3 在 MHCC97H 和 Huh7 細胞中明顯過表達，并且SGK3 過表達明顯地促進了肝癌干細胞相關基因 CD133、CD90、Oct4、Nanog、Bmi-1 和 Sox2 的表達。我們在蛋白水平驗證了 SGK3 對干細胞相關基因表達的促進作用（圖 2.C）。為了進一步證實 SGK3 對肝癌干細胞自我更新的影響，我們檢測了兩組細胞的成球能力，發(fā)現 SGK3 過表達明顯促進肝癌細胞的成球能力（圖2.D）。
【參考文獻】：
期刊論文
[1]Cancer stem cell plasticity and tumor hierarchy[J]. Marina Carla Cabrera,Robert E Hollingsworth,Elaine M Hurt. World Journal of Stem Cells. 2015(01)
本文編號：3460321
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