抑制PIK3C3和PI3K通路对肝癌干细胞自我更新的影响及机制研究
发布时间：2021-10-26 22:36
我们前期研究发现血清和糖皮质激素调节激酶3（Serum and glucocorticoid kinase 3,SGK3）能够促进肝癌细胞发生上皮间质转化（epithelial-mesenchymal transition,EMT）进而促进肝癌细胞的侵袭和迁移,但其是否参与调控肝癌干细胞干性还不清楚。PI3K信号通路（通常指I型PI3K）异常调节在各种恶性肿瘤中非常普遍,Akt一直被认为是PI3K信号通路中至关重要的中转信号分子,近年来,有研究证实SGK3是I型PI3K通路下游不依赖Akt的独立调控分子,而SGK3在I型PI3K信号通路中的作用及机制仍未阐明。Ⅲ型PI3K（PIK3C3）又被称作Vps34,在细胞自噬和胞吞过程中发挥重要作用。PIK3C3还是生成PtdIns（3）P的重要场所,PtdIns（3）P是细胞内的重要第二信使,能够激活含PX和FYVE结构域的蛋白质。有报道称,PIK3C3在结肠癌、乳腺癌等多种肿瘤的发生发展过程中发挥重要作用,但是其是否参与了肿瘤干细胞的调控还未见相关报道。第一部分抑制PI3K对肝癌干细胞自我更新的影响及机制研究目的:探究SGK3在肝癌干细胞自...
【文章来源】：重庆医科大学重庆市
【文章页数】：103 页
【学位级别】：博士
【部分图文】：
磁珠分选细胞分选架组装示意
图 1. SGK3 在肝癌干细胞中磷酸化程度明显增高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
重庆医科大学博士研究生学位论文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 促进肝癌干细胞自我更新为了深入探究 SGK3 对肝癌干细胞自我更新的调控作用，我们使用慢病毒载体转染 MHCC97H 和 Huh7 细胞并构建了 SGK3 稳定过表达细胞株。如图 2.A 和 B所示，慢病毒转染成功后 SGK3 在 MHCC97H 和 Huh7 细胞中明显过表达，并且SGK3 过表达明显地促进了肝癌干细胞相关基因 CD133、CD90、Oct4、Nanog、Bmi-1 和 Sox2 的表达。我们在蛋白水平验证了 SGK3 对干细胞相关基因表达的促进作用（图 2.C）。为了进一步证实 SGK3 对肝癌干细胞自我更新的影响，我们检测了两组细胞的成球能力，发现 SGK3 过表达明显促进肝癌细胞的成球能力（图2.D）。
【参考文献】：
期刊论文
[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)
本文编号：3460321
【文章来源】：重庆医科大学重庆市
【文章页数】：103 页
【学位级别】：博士
【部分图文】：
磁珠分选细胞分选架组装示意
图 1. SGK3 在肝癌干细胞中磷酸化程度明显增高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
重庆医科大学博士研究生学位论文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 促进肝癌干细胞自我更新为了深入探究 SGK3 对肝癌干细胞自我更新的调控作用，我们使用慢病毒载体转染 MHCC97H 和 Huh7 细胞并构建了 SGK3 稳定过表达细胞株。如图 2.A 和 B所示，慢病毒转染成功后 SGK3 在 MHCC97H 和 Huh7 细胞中明显过表达，并且SGK3 过表达明显地促进了肝癌干细胞相关基因 CD133、CD90、Oct4、Nanog、Bmi-1 和 Sox2 的表达。我们在蛋白水平验证了 SGK3 对干细胞相关基因表达的促进作用（图 2.C）。为了进一步证实 SGK3 对肝癌干细胞自我更新的影响，我们检测了两组细胞的成球能力，发现 SGK3 过表达明显促进肝癌细胞的成球能力（图2.D）。
【参考文献】：
期刊论文
[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)
本文编号：3460321
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