| 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... |
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