Na+,K+-ATP酶表达对人结肠癌干细胞生物学特性的影响

Na+, K+-ATPases, as a signal transducer, mediates cell proliferation and death related signal transmissions, and participates in the prosess of metastasis, multidrug resistances and epithelial-mesenchyme transition(EMT) in tumor, which could be a target for tumor therapy . However, whether the expression of Na+, K+-ATPases was related to the biological characteristics of colon cancer stem cells, whether to be a treament target for colon cancer stem cells(CSCs), as well as the molecular machanism that effects on biological characteristics of stem cells when it interacts with PI3K/Akt/mTOR pathway were still undetermined. Colon cancer cell line SW480 was used to be subject in this study. Stably resistant to oxaliplatin were developed by exposing SW480 cells to oxaliplatin in Leibovitz's L-15 Medium with 10% FBS, stem cells were obtained by magnetic bead sorting(MACS).We first compare the actibity of Na+, K+-ATPases and the expression differences of key molecules such as Na+, K+-ATPases subunits、PI3K、Akt、mTOR and EMT in colon parental cell SW480, oxaliplatin-resistance cells SW480/OXR and CSCs.Taking those subunits which closely relate to the biological characteristics of SW480/OXR and CSCs in colon cancer as treament targets. We then test the specificity of these targets by gene overexpression or lower expression and clarify the mechanism of transformation among different subunits as well as the effects of biological characteristics in stem cells when they work with PI3K/Akt/mTOR pathway.

Na+, K+-ATP酶作为信号转导蛋白，介导细胞增殖与死亡相关信号传递，参与肿瘤的转移、多药耐药及上皮间质化（EMT）发生，并成为肿瘤治疗的靶点。Na+, K+-ATP酶表达是否与结肠癌干细胞生物学特性有关，能否成为干细胞治疗靶点，及其与PI3K/Akt/mTOR通路相互作用对干细胞"干性"影响的机制尚未阐明。本项目拟以人结肠癌细胞株（SW480）为研究对象，通过建立耐药细胞株及免疫磁珠分选干细胞，比较结肠癌亲本细胞、耐药细胞及干细胞Na+, K+-ATP酶活性、Na+, K+-ATP酶亚单位、PI3K、Akt、mTOR及EMT关键分子基因表达的差异，从Na+, K+-ATP酶亚单位中筛选出与耐药细胞及干细胞生物学特性密切相关的亚单位为治疗靶点。采用基因技术验证靶点的特异性，并阐明不同亚单位间相互转换及其与PI3K/Akt/mTOR通路对干细胞生物学特性影响的机制。

Na+, K+-ATP 酶参与肿瘤的转移、多药耐药及上皮间质化(EMT)发生等病理生理过程，而Na+, K+-ATP酶表达是否与结肠癌干细胞生物学特性有关及可能机制尚未明确。因此，本项目探讨Na+, K+-ATP 酶亚单位表达与PI3K/Akt/mTOR 通路相互作用对干细胞"干性"的调控机制；以人结肠癌SW480细胞为研究对象,建立耐药细胞株及流式细胞技术分选干细胞, 比较结肠癌SW480、耐药细胞SW480/OxR及干细胞（CSCs）Na+, K+-ATP酶活性、Na+, K+-ATP 酶亚单位、PI3K、Akt、mTOR 及 EMT 关键分子基因表达差异, 从 Na+, K+-ATP酶亚单位中筛选出与SW480/OxR及CSC生物学特性密切相关的亚单位为治疗靶点。本项目部分内容增加了结肠癌SW1116和SW620细胞及2个CSCs标志物以增加研究结果可靠性，另增加了人转录激活因子（ATF5）、钠通道1.5（Nav1.5）和细胞自噬与结肠癌多药耐药、EMT和CSC生物特性相关研究内容。结果发现：CSCs、SW480/OxR CD44/EpCAM 阳性细胞显著高于亲本细胞，CD44/ EpCAM阳性细胞具有高致瘤能力；在CSCs “富集”过程中，Na+, K+-ATP酶α3亚单位表达显著上调、ATP酶活性下降、细胞内“钙超载”及凋亡减少；CSCs 和SW480/OxR具有EMT表型，结肠癌CD44+细胞表达与α3亚单位表达水平正相关；Na+, K+-ATP酶抑制剂哇巴因能抑制SW480/OxR增殖及下调α3亚单位表达水平；沉默α3亚单位能显著抑制CSCs生长，促进CSCs凋亡及降低CSCs致瘤能力；ATF5参与CSCs “富集”及分化过程；PI3K /AKt /mTOR通路及信号分子Raptor、Rictor及GβL表达显著增加，可能参与CSCs“干性”维持；Nav1.5在结肠癌耐药细胞膜离子通道密度显著增加，而在CSCs显著减小，参与结直肠癌多药耐药及EMT过程；自噬在耐药细胞显著增加，而在CSCs却消失。综上，Na+, K+-ATP 酶α3亚单位与 PI3K/AKt/mTOR通路相互作用参与结肠癌CSCs“干性”维持，α3亚单位可能是 结肠癌CSCs潜在的治疗靶点；ATF5和Nav1.5 可能参与结肠癌多药耐药、EMT、CSCs “富集”及分化过程。