FOG2在胰腺癌中的表达及其调控MAPK、PI3K信号通路串话的机制研究

发布时间：2018-08-27 19:09

【摘要】：第一部分：胰腺癌MAPK与PI3K信号通路存在串话目的：研究胰腺癌MAPK与P13K信号通路间是否存在串话及其作用方式。方法：分别向胰腺癌BxPC3、 PANC1细胞株中加入MAPK通路抑制剂PD98059或P13K通路抑制剂LY294002干预1 h,免疫印迹方法检测p-ERK、 ERK、 p-Akt、 AKt的蛋白表达水平。应用siRNA方法降低ERK表达水平,免疫印迹方法检测p-Akt、Akt的蛋白表达水平。结果：应用MAPK抑制剂抑制BxPC3、 PANC1细胞ERK活性后,p-Akt表达水平反而升高。使用siRNA转染BxPC3、 PANC1细胞沉默ERK的表达同样可以使p-Akt的蛋白表达水平升高。结论：胰腺癌MAPK, PI3K信号通路间存在串话,ERK的活化可以抑制PI3K/Akt信号通路的激活。第二部分：FoG2在胰腺癌中的表达及其在MAPK、 PI3K信号通路串话中的作用目的：研究FOG2在胰腺癌组织中的表达,及其调控MAPK、 PI3K信号通路串话的机制。方法：采用免疫组织化学方法检测108例临床胰腺癌样本中FOG2的蛋白含量。使用免疫印迹方法检测多种胰腺癌细胞株FOG2的蛋白表达水平,选择FOG2高表达细胞株a应用慢病毒颗粒转染胰腺癌细胞降低FOG2表达,对照组应用空白慢病毒载体。分别应用MAPK和P13K抑制剂抑制通路活性,免疫印迹方法检测p-ERK.ERK、p-Akt,Akt的蛋白表达水平。通过免疫共沉淀(Co-IP)方法,检测ERK与FOG2有无结合。结果：61.1%(66/108)患者的胰腺癌组织FOG2表达水平要强于对应的癌旁组织。FOG2在BxPC3、PANC1细胞中呈高表达。在对照组,抑制ERK的磷酸化可以显著增加Akt的磷酸化,活化的ERK可以抑制Akt的活化；应用慢病毒转染方法降低FOG2表达水平后,应用MAPK抑制剂抑制ERK活性,p-Akt的蛋白表达水平并没有明显变化。免疫共沉淀结果显示,ERK可以与FOG2特异性结合。结论：大多数胰腺癌肿组织中FOG2蛋白的表达水平要强于癌旁组织。ERK通过与FOG2特异性结合来调控Akt的活化。沉默FOG2可以干扰ERK对Akt信号通路的抑制作用。FOG2是调控胰腺癌MSPK、 PI3K信号通路串话的重要分子。第三部分：沉默FoG2对MAPK抑制剂作用效果的影响目的：研究沉默FOG2对MAPK抑制剂抑制胰腺癌细胞生物学行为的能力有无影响。方法：向慢病毒稳定转染的BxPC3、PANC1细胞中加入MAPK抑制剂,采用Transwell小室测定BxPC3、PANC1细胞的定向迁移能力,Flow Cytometry检测BxPC3、 PANC1的凋亡比例,采用平板克隆形成实验测定BxPC3、 PANC1细胞的成瘤能力,MTT检测BxPC3、 PANC1增殖能力。结果：Transwell assay结果显示,虽然沉默FOG2对BxPC3、 PANC1细胞的定向迁移能力没有明显影响,但沉默FOG2表达后,无论是单用MAPK通路抑制剂还是与P13K通路抑制剂联合应用,其抑制BxPC3、 PANC1细胞迁移的能力均得到增强。流式细胞术和平板克隆形成实验结果分别提示沉默FOG2可以增强PD98059对BxPC3、 PANC1细胞的促凋亡作用和抑制BxPC3、 PANC1细胞成瘤的能力。MTT结果显示,沉默FOG2后,PD98059抑制BxPC3、 PANC1细胞增殖的作用强度和持续时间均得到增强。结论：沉默FOG2可以增加胰腺癌细胞对ERK抑制剂的敏感性,可以增强MAPK抑制剂抑制BxPC3、 PANC1细胞增殖、存活、定向迁移和集落形成的能力。
[Abstract]:Part I: There is a crosstalk between MAPK and PI3K signaling pathway in pancreatic cancer. Objective: To study whether there is a crosstalk between MAPK and P13K signaling pathway in pancreatic cancer and its mechanism. The expression levels of ERK, p-Akt and AKt were decreased by siRNA, and the expression levels of p-Akt and Akt were detected by Western blotting. Results: The expression level of p-Akt was increased by using MAPK inhibitor to inhibit the ERK activity of BxPC3 and PANC1 cells. Conclusion: The activation of ERK can inhibit the activation of PI3K / Akt signaling pathway. Part II: FoG2 expression in pancreatic cancer and its role in MAPK, PI3K signaling pathway crosstalk. Objective: To study the expression of FOG2 in pancreatic cancer and its regulation of MAPK. Methods: Immunohistochemical method was used to detect FOG2 protein content in 108 clinical pancreatic cancer samples. Immunoblotting was used to detect FOG2 protein expression in various pancreatic cancer cell lines. Lentiviral particles were used to transfect pancreatic cancer cell line a with high expression of FOG2 to reduce FOG2 protein expression. The expression of p-ERK, p-Akt, and Akt was detected by Western blot. The binding of ERK to FOG2 was detected by Co-IP. Results: The expression of FOG2 in pancreatic cancer tissues of 61.1% (66/108) patients was higher than that of corresponding cancers. FOG2 was highly expressed in BxPC3 and PANC1 cells. In control group, inhibition of ERK phosphorylation significantly increased Akt phosphorylation, and activated ERK inhibited Akt activation. After lentivirus transfection decreased FOG2 expression, MAPK inhibitors inhibited ERK activity and p-Akt protein expression did not change significantly. ConclusionThe expression level of FOG2 protein in most pancreatic cancer tissues is higher than that in adjacent tissues. ERK regulates the activation of Akt by binding to FOG2 specifically. Silencing FOG2 can interfere with the inhibition of ERK on Akt signaling pathway. FOG2 regulates MSPK and PI3K signaling pathway strings in pancreatic cancer. Part 3: Effect of silencing FoG2 on the effect of MAPK inhibitors Objective: To study whether silencing FOG2 has any effect on the ability of MAPK inhibitors to inhibit the biological behavior of pancreatic cancer cells.Methods: BxPC3, PANC1 cells stably transfected with lentiviruses were added with MAPK inhibitors, and BxPC3 and PANC1 were measured by Transwell chamber. Flow Cytometry was used to detect the apoptotic ratio of BxPC3 and PANC1 cells, the tumorigenic ability of BxPC3 and PANC1 cells was determined by plate cloning formation assay, and the proliferation of BxPC3 and PANC1 cells was detected by MTT. Results: Transwell assay showed that although FOG2 silencing had no significant effect on the directional migration ability of BxPC3 and PANC1 cells, the sedimentation rate was not significant. The results of flow cytometry and plate cloning showed that silencing FOG2 could enhance the apoptosis-inducing effect of PD98059 on BxPC3, PANC1 cells and inhibit the apoptosis-inducing effect of BxPC3, PANC1 cells, respectively. MTT results showed that PD98059 inhibited the proliferation of BxPC3 and PANC1 cells after FOG2 silencing. CONCLUSION: Silencing FOG2 can increase the sensitivity of pancreatic cancer cells to ERK inhibitors and enhance the inhibition of proliferation, survival, directional migration and colony formation of BxPC3 and PANC1 cells by MAPK inhibitors. Ability.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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