Survivin通过上调IKK-β转录促使食管鳞状上皮永生化细胞恶变的机制研究

The expression of Survivin is elevated in esophageal cancer tissue, but the mechanism that it causes the carcinomatous change of esophageal squamous epithelial cells is unclear. We have clarified in our previous work that Survivin can up-regulate the transcription of IKK-β, then activate the NF-κB signaling pathway in esophageal cancer ECA109 cells. And there has much established data that confirmed NF-κB can up-regulate the expression of Survivin. Therefore our scientific hypothesis is that there must have the positive feedback loop circuit of Survivin→IKK-β→NF-κB→Survivin, to activate the expression of Survivin continually. And persistently elevatory expression of Survivin causes the canceration of esophageal squamous epithelial cells. In order to verify this idea, we use molecular biologic methods to raise or decrease Survivin levels, and apply different pharmacological inhibitors to block different nodes of Survivin→IKK-β→NF-κB→Survivin loop in esophageal squamous epithelial immortalized cell lines.Then we will test variation tendency of Survivin, IKK-β, NF-κB mRNA and proteins levels to testify existence of this loop in esophageal squamous epithelial immortalized cell lines. To confirm whether our hypothetical loop plays crucial role in the carcinomatous change of esophageal squamous epithelial cell lines, we elevating Survivin levels and blocking functions of different proteins of the loop by inhibitors, then test changes of esophageal squamous epithelial cells’ biological behavior and tumorigenic ability by clone formation assay, soft agar culture clone forming experiments, transwell assay, and nude mouse transplantation tumor experiments. We believe our work should deepen the understanding about the roles of Survivin in pathogenesis of esophageal squamous cell cancer.

Survivin在多数食管癌中高表达，但其参与食管癌发生早期的机制仍不明确。因我们的研究表明：在食管癌细胞中，Survivin可正调控IKK-β的表达，而激活NF-κB信号通路；且文献报道NF-κB可正调控Survivin表达。故我们的科学问题是：可能存在Survivin→IKK-β→NF-κB→Survivin的正反馈环路，循环激活Survivin的表达，起始了食管鳞状上皮细胞恶性转化。为了验证此设想，我们以食管鳞状上皮永生化细胞为研究对象，通过上调及下调Survivin水平，并使用抑制剂阻断上述设想环路的不同环节，来验证此环路在所研究细胞中是否存在；后通过上调Survivin水平，并结合抑制剂阻断环路中不同蛋白作用，观察所研究细胞生物学行为及裸鼠成瘤能力的变化，来确定上述调控环路是否可使食管鳞状上皮细胞恶性转化。本研究有望加深对Survivin在食管癌发生过程中所起作用。

为证实是否存在Survivin→IKK-β→NF-κB→Survivin的正反馈环路，循环激活Survivin的表达，起始了食管鳞状上皮细胞恶性转化这一设想，首先通过将SHEE和Het-1A细胞转染GV219-3Flag-Survivin质粒后，检测到Survivin mRNA水平明显上调，相应的IKK-βmRNA水平随之增高，在蛋白水平检测到带有Flag标签的外源性Survivin 蛋白水平明显增加，IKK-β、磷酸化p-NF-κB (p65)蛋白水平随之增加，最终检测到内源性Survivin水平增高。然后用NF-κB抑制剂CAPE处理SHEE和Het-1A细胞后，随着CAPE浓度的增加，NF-κB (p65) 蛋白活化水平明显降低，IKK-β、Survivin mRNA及蛋白表达水平均随之降低；当用 IKK-β抑制剂TPCA-1处理后，随着TPCA-1浓度的增加，IKK-βmRNA及蛋白表达水平受抑制后逐渐降低，NF-κB (p65) 蛋白活化、Survivin mRNA及蛋白表达水平也逐渐下降；当用Survivin抑制剂YM155处理后，随着YM155浓度的增加，Survivin mRNA及蛋白表达水平受抑制后逐渐降低，IKK-βmRNA及蛋白表达水平也相应下降，最终NF-κB (p65) 蛋白活化随之下降。说明在Survivin→IKK-β→NF-κB→Survivin这个设想的环路的不同环节下调靶基因表达水平，下游的基因均会发生相应的表达下调。从而从正负两个调控方向说明Survivin→IKK-β→NF-κB→Survivin调控环路的存在，并可使Survivin的表达起到自我增强的作用。最后用GV142-Survivin重组质粒转染SHEE和Het-1A细胞后，平板克隆形成实验、transwell实验和裸鼠成瘤能力显示，上调Survivin蛋白水平可使SHEE和Het-1A细胞克隆形成能力、侵袭能力、迁移能力及成瘤能力明显增强，说明Survivin表达上调促进了食管癌永生化细胞恶性转化。证实Survivin→IKK-β→NF-κB→Survivin调控环路使Survivin的表达自我增强，促进了食管上皮永生化细胞恶性转化。