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PGE2经EP4受体激活AKT/NF-κB信号转导通路上调Snail促进肝癌侵袭转移的机制研究

The Mechanism Underlying PGE2/EP4R Promoting the Invasiveness in Hepatocellular Carcinoma by Upregulating Snail Expression via AKT/NF-κB Signaling Transduction Pathway

【作者】 张敏

【导师】 冷静;

【作者基本信息】 南京医科大学 , 病理学与病理生理学, 2014, 博士

【摘要】 研究背景:前列腺素E2(Prostaglandin E2, PGE2)是花生四烯酸环氧合酶代谢产物,为二十碳不饱和脂肪酸,PGE2主要通过细胞膜上的四种G蛋白偶联受体(GProtein-Coupled Receptors, GPCRs),即EP1、EP2、EP3、EP4发挥跨膜信号转导作用。多项研究证实,PGE2能够显著促进多种肿瘤的增殖、侵袭和转移,本实验室前期研究结果也表明PGE2能够显著增加肝细胞癌和胆管细胞癌的增殖侵袭能力。有报导,肿瘤的侵袭转移与Snail蛋白水平的增高密切相关,但PGE2通过调控Snail蛋白水平促肝癌侵袭的具体机制尚不清楚。本课题在实验室前期已有工作基础上,进一步探讨PGE2调控Snail促进肝癌侵袭转移的分子机制。研究目的:阐明PGE2通过EP4受体激活AKT/NF-κB信号转导通路上调Snail,从而促进肝癌侵袭转移的机制。研究内容:1. Snail蛋白与人肝癌侵袭关系的研究2.PGE2通过激活EP4受体对Snail的调控研究3.EP4受体激动上调肝癌细胞Snail表达的信号转导通路和分子机制研究方法:1.免疫组织化学实验检测临床肝癌标本中Snail蛋白及信号通路中相关靶点蛋白的表达情况。2.常规方法培养肝细胞癌Huh7细胞。3.WST实验检测PGE2和EP4受体激动剂处理对肝细胞癌细胞生长的影响。4. Transwell实验检测PGE2和EP4受体激动剂处理对肝细胞癌细胞侵袭能力的影响。5. Real-Time PCR实验检测肝细胞癌细胞中PGE2和EP4受体激动剂处理对Snail mRNA水平的影响。6.RNA干扰处理,抑制肝细胞癌细胞EP4受体的表达后,检钡Snail蛋白及信号通路中信号靶点的改变。7. Western blot实验检测PGE2经EP4受体上调Snail蛋白表达的信号转导通路中相关靶点的蛋白表达改变。8.荧光素酶报告基因实验检测NF-κB-P65蛋白是否结合在Snail蛋白的起始部位,并促进了Snail蛋白的转录。9.激光共聚焦显微实验检测EP4受体激动剂处理对肝癌细胞中NF-κB-P65亚基入核的影响。10. Western blot实验检测EP4受体激动剂引起GSK-3β的活化后,在泛素化水平对Snail蛋白表达的影响。研究结果:1.免疫组织化学EliVisionTM super二步法实验结果表明,与癌旁正常肝组织相比,肝癌组织中Snail蛋白表达显著上调。低分化组与对照组相比,Snail蛋白表达上调了1.64倍,高分化组与对照组相比,Snail蛋白表达上调了0.47倍。Transwell实验结果表明当Huh7细胞转染了Snail蛋白的干扰质粒P-Super-Snail shRNA之后,PGE2和EP4受体激动剂处理促肝癌细胞侵袭能力较空载对照组明显下降。2.在肝癌细胞Huh7细胞系中,外源性PGE2处理肝癌细胞Huh7细胞24h,细胞内Snail蛋白的表达水平显著上升,E-cadherin表达明显下降,其中10MPGE2处理细胞导致Snail蛋白上升及E-cadherin下降作用最显著,与对照组相比,Snail上调了1.78倍,E-cadherine下降了2.5倍;EP4受体激动剂Prostaglanding E1Alchoal处理Huh7细胞可以显著上调细胞内Snail的蛋白表达,其中10μM时上调作用最显著,达到对照组的3.67倍(图4A和B),同时E-cadherin下调作用显著(图4C和D),达到对照组的4.08倍。3.在Huh7细胞中,通过Transwell实验观察,当EP4受体被干扰之后,PGE2诱导细胞内Snail蛋白水平较单纯PGE2刺激组相比下降了2.01倍。4.EP4受体激动剂Prostaglanding E1Alchoal10μM处理Huh7细胞后,Realtime PCR实验结果显示细胞内Snail mRNA水平发生显著变化,24小时时mRNA水平较对照组0小时增加了2.02倍。EP4受体激动剂处理引起CREB的磷酸化水平上升,30分钟时作用最明显,为对照组的4.01倍。将抑制性DN-CREB质粒转染进Huh7细胞,以空载pRc/CMV质粒作为对照,再用EP4受体激动剂Prostaglanding E1Alchoal处理细胞,结果显示与对照比较,CREB功能被抑制后,并未阻断EP4受体激动剂对Snail蛋白表达的上调作用。5.EP4受体激动剂Prostaglanding El Alchoal处理Huh7细胞45分钟时,EGFR磷酸化水平明显上调,为对照组的2.5倍,0-60分钟时间段内均可见AKT Thr308的磷酸化水平增高,以30分钟时作用最明显,较对照组上调3.33倍。给予肝癌细胞EP4受体激动剂和EGFR抑制剂AG1478后,AKT的磷酸化水平明显下降,较单一受体激动剂处理组下调了1.69倍。6.10μM EP4受体激动剂Prostaglanding E1Alchoal处理Huh7细胞后,IκB磷酸化水平明显增高,以60分钟时磷酸化水平上调最为显著。上述处理细胞在0-3小时内未观察到NF-κB-P65蛋白的磷酸化水平明显增高,而在4小时时的时间点观察到NF-κB-P65的磷酸化水平明显增高。用PI3K特异性抑制剂LY294002预处理Huh7细胞1小时后,用EP4受体激动剂Prostaglanding E1Alchoal作用Huh7细胞4小时,结果显示EP4受体激动诱导的NF-κB-P65磷酸化水平上调作用明显被PI3K抑制剂阻断。7.10μM EP4受体激动剂Prostaglanding E1Alchoal处理Huh7细胞25分钟后胞核中NF-κB-P65染色水平明显增高(粉色显色,根据三原色原理,粉色为红色和蓝色融合后的颜色)。证明NF-κB-P65磷酸化后入核现象明显,且4小时时入核作用最明显。8.免疫组织化学EliVisionTM super实验结果显示,与正常对照组织相比,NF-κB在肝癌组织中表达明显增加,呈强阳性并且阳性表达定位于细胞核,正常肝细胞中胞膜、胞浆及胞核未见棕黄色颗粒沉着。免疫组织化学双抗体标记(Dou SPTM法)实验显示,NF-κB与Snail共同表达于肝癌中,NF-κB呈红色,Snail呈棕黄色。二者同时显色的细胞即为NF-κB/Snail阳性细胞(棕黑色)。9.双荧光素酶报告基因实验显示,用pRL-SV40为载体的NF-κB-P65过表达质粒和Snail的启动子质粒共转染Huh-7细胞,以pGL3-Control作为阴性对照,共孵育72小时,通过荧光强度的绝对值分析,结果显示NF-κB-P65对Snail发挥了正向转录调节作用,72小时时的转录水平达到峰值。10.10μM EP4受体激动剂Prostaglanding E1Alchoal处理Huh7细胞后,GSK-3β磷酸化水平明显增高,以30分钟时磷酸化水平上调最为显著,为对照组的1.67倍。结论:PGE2经EP4受体以非G蛋白偶联的方式,即EGFR反式活化后激活PI3K/AKT信号,活化转录因子NF-κB,上调了Snail在肝癌中的转录活性;此外,EP4受体激活后使GSK-3β磷酸化失活,降低了对Snail的泛素化降解作用,使Snail稳定性增加;这两方面的协同作用共同上调了Snail在肝癌中的表达。

【Abstract】 Background:Prostaglandin E2(Prostaglandin E2, PGE2),20carbon unsaturated fatty acid,derives from arachidonic acid liberated from phospholipids in the cell membrane.It exerts its biological actions through seven-transmembrane-domain,G-protein-coupled receptors,classtified as EP1、EP2、EP3、EP4. Several studies have confirmed that PGE2can significantly promote a wide variety of tumor proliferation, invasion and metastasis, our laboratory previous data showed that PGE2can significantly increases the proliferation of liver cancer and bile duct carcinoma invasion ability, but the exact mechanism of how PGE2involves in the pecific regulatory to Snail is not clear.Research Object:T o clarify Prostaglanding E2upregulates Snail expression via the E prostanoid4receptors of activated AKT/NF-KB,which involved in the metastasis of hepatocellular carcinoma.Research Content:1.To examine the role of Snail in the metastasis of hepatocellular carcinoma. 2.To clarify the mechanism which Snail was regulated by PGE2/EP4.3.To investigate the specific signal transduction pathways and molecular mechanisms which Snail involved in,and activated by the E prostanoid4receptors.Research Method:1. The expression of Snail and relavent protein in clinical specimens of liver cancer by IHC(immunohistochemical) methods.2. Conventional method to cultivate liver cell cancer HUH-7.3. The effects on liver cell cancer cell growth activated by PGE2and EP4agonist using the WST method.4. The effects on liver cell cancer cell migration activated by PGE2and EP4agonist using the transwell method.5. The influence of Snail mRNA level in liver cancer cell activated by PGE2and EP4agonist using the Real-Time PCR.6. Inhibition of EP4in liver cancer cell using RNA interference,affected Snail expression in signaling pathway.7. Snail expression in signaling pathway activated by PGE2/EP4using Western Blot test.8. P65gene is incorporated in the initial parts of the Snail protein and promoted the Snail protein transcription by the Luciferase reporter gene assay.9. The influence of EP4receptor agonist on NF-κB-P65subunits into the nucleus by Confocal assay. lO.The influence of the level of ubiquitination of the Snail protein expression induced by activation of GSK-3β.Research Results:1. Snail protein expression was significantly upregulated in primary hepatocellular carcinoma when compared with adjacent normal liver tissue by Immunohistochemical EliVisionTM Two-step test. Poorly differentiated group compared with the control group, Snail protein expression increased1.64times, well-differentiated group compared with the control group, Snail protein expression increased0.47-fold when Huh7cells transfected with a plasmid interference Snail protein P-Super-Snail shRNA,the ability of PGE2and EP4receptor agonist induced proliferation of hepatoma cell invasion decreased than the control group after Transwell method.2. In human hepatoma cell lines Huh7cells, a significant increase of Snail protein expression and decrease of E-cadherin in intracellular levels treated by exogenous PGE224h,and raised to2.68and decreased to1.68times respectively,compared to the control group.when treated by EP4receptor agonist Prostaglanding E1Alchoal,the effect of upregulation can reach3.67fold and downregulation reaches4.83times respectively compared to the control group.3. Snail protein level downregulated from110%to101%induced by PGE2In Huh7cells, and invasion of hepatoma cells was significantly decreased (down40%compared with the control group), By Transwell test,when EP4receptor interferenced.4. Snail mRNA levels in24hours increased2.02-fold treated by EP4receptor agonist,compared with the control group0hours. EP4agonist induced phosphorylation of CREB reaches the most significant effect after treated30 minutes.when the CREB function is inhibited, Snail protein expression induced by EP4receptor agonist treatment is not blocked compared with the control.5. EGFR phosphorylation reached the most significant,2.5fold increase compared of the control when treated by EP4receptor agonist,and it can cause the phosphorylation of AKT Thr308,3.33-fold increase compared of the control group. The phosphorylation levels of AKT was significantly inhibited,compared with a1.47-fold lower compared of the group while adding EP4receptor agonists and EGFR inhibitor AG1478in human hepatoma cell lines Huh7cells6. In Huh7cells.,when treaten by EP4receptor agonist,IκB can be phosphorylaed significantly and reaches the most significant in60minutes,and the phosphorylation of P65can be induced significantly in8hours,reaching to the high point after4hours.The phosphorylation of P65can be inhibited while adding AKT specific inhibitor LY294002and EP4receptor agonist.7. In Huh7cells.,P65appears in the nucleus (according to the principle of the three primary colors) after EP4agonist treatment and reached a significant increase after4hours.8. In clinical patient specimens, NF-κB expression was significantly increased compared with normal control tissueby Immunohistochemical EliVisionTM Two-step detection, and the expression was strongly positive in the nucleus of tumor tissues,and normal liver cells membrane, cytoplasm and cell nuclear have not brown granules. Immunohistochemical double staining (Dou SPTM method) was used to detect the co-expressionof NF-κB and Snail in hepatocellular carcinoma, NF-κB was red, Snail brownish yellow. NF-κB/Snail co-positive cells brown.9. LuciferaseReportor Aaaay showed that, the pRL-SV40vector with P65overexpression plasmid and Snail promoter plasmid cotransfection Huh7cell, pGL3-Control as a negative control,72hours of incubation, NF-Kb-P65upregulated Snail transcription by the analysis of the absolute intensity value,72hours reached a peak level of transcription.10. In Huh7cells.,after10μM EP4agonist Prostaglanding E1Alchoal treatment, GSK-3β phosphorylation levels were significantly increased on30minutes,3.33-fold increase compared of the control group.Conclusion:PGE2via EP4receptor in non-G protein-coupling way mediated PI3K/AKT signaling,stimulated by transactivation of the EGFR,and activated transcription factor NF-κB,increaing the Snail upregulation.GSK-3βwas phosphorylated by EP4receptor and reduced the ubiquitination degradation of Snail,so the stability of Snail increased. The synergistic effect of these two aspects co-upregulated expression of Snail in HCC

【关键词】 前列腺素E2肝癌SnailNF-κB肿瘤侵袭
【Key words】 PGE2Hepatocellular carcinomaSnailNF-κBMigrationInvasiveness
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