The Research Of The Molecular Mechanism Underlying PGE₂Promoting The Invasiveness In Hepatocellular Carcinoma Through EP1-Snail Signal Transduction Pathway

Background:Prostaglandin E2(PGE2) is a kind of inflammatory mediator, which has significant promoting effects on the proliferation and invasiveness of cancer cells. In hepatocellular carcinoma cell lines, PGE2could bind with four kinds of E prostanoid receptors on the cell membrane, which is EP1, EP2, EP3and EP4respectively, and influence the biological properties of hepatocellular carcinoma cell lines from different aspects. According to our previous results, we showed that PGE2could greatly enhance the proliferation and invasiveness of hepatocellular carcinoma, and investigated the associated mechanisms on proliferation. But the mechanisms on how the PGE2promoting hepatocellular carcinoma invasiveness and metastasis is still unclear.On the basis of our previous research work, we further analyzed the molecular mechanisms underlying PGE2promoting invasiveness and metastasis in hepatocellualr carcinoma.Research Object:Clarifying the mechanisms and associated signal transduction pathways underlying PGE2promoting invasiveness and metastasis in hepatocellular carcinoma by upregulating the expression level of Snail protein throuth EP1receptor.Research Content:1Clarifying the role of the EP1receptor in PGE2enhancing the migration and invasion in hepatocellular carcinoma cell lines2Clarifying the role of the snail protein in PGE2enhancing the migration and invasion through EP1receptor in hepatocellular carcinoma cell lines3Investigating the signal transduction pathway and associated mechanisms underlying PGE2upregulating the expression level of Snail protein through EP1receptor.Research method:1. The hepatocellular carcinoma cell lines HepG2, Hep3B, Huh7were cultured in a conventional method.2. The effects of the PGE2and EP1receptor agonist (17-P-T-PGE2) on the migration and invasion in hepatocellular carcinoma celll line were investigated by using the transwell method.3. For some key points in the signal transduction pathway, chemical inhibitor, RNA interference and expression functional suppressive protein, combined with the Western Blot method, were used to analyzed the signal transduction pathway,.4. Realtime PCR method was used to analyzed the mRNA level of the associated proteins in the signal transduction pathway. 5. The effect of the PGE2on the translational efficiency of the snail through EP1receptor was analyzed by polysome profile method.Research Results:1. In Hep3B and Huh7cell lines, PGE2could increase the migration ability to223%and300%, and increase the invasion ability to228%and284%.17-P-T-PGE2could increase the migration ability to202%and267%, and increase the invasion ability to203%and235%. When knocking down the snail protein, PGE2could only increase the migration ability to126%and118%, and increase the invasion ability only to115%and127%.17-P-T-PGE2could increase the migration ability only to116%and104%, and increase the invasion ability to103%and102%.2. In HepG2, Hep3B, Huh7cell lines, PGE2could upregulate the expression level of snail protein from168%to216%,17-P-T-PGE2could upregulate the expression level of Snail protein from140%to223%. EP1receptor regulated the snail protein expression through Gai and Gβγ subunits. When PTX were used to block the effect of Gai subunits,17-P-T-PGE2could only regulate the expression level of snail protein from111%to139%. When the effect of Gβγ subunit was blocked by GRK2ct,17-P-T-PGE2could only regulate the expression level of snail protein to156%and134%, while in the mock plasmid transfected cell lines,17-P-T-PGE2could upregulate the expression level of snail to253%and272%.3. In our hepatocellular carcinoma cell lines, when treated with17-P-T-PGE2for24h, the results of the polysome profile analysis showed that EP1receptor could enhance the translational efficiency of snai mRNA. 4. In HepG2, Hep3B and Huh7cell lines, EP1receptor could activate the Erk kinase, then upregulate the phosphorylation level of mTOR from192%to461%; while PKC inhibitor BIS could greatly suppress the phosphorylation level of Erk kinase. EP1receptor could upregulate the expression level of Raptor from229%to581%, through activating the CREB transcription factor. GRK2ct could suppress the phosphorylation of CREB through blocking the Gβγ subunits. Where DN-CREB could significantly suppress the upregulation of Raptor through inhibiting the CREB function. The increasing level of mTOR phosphorylation, in cooperated with the upregulation of Raptor protein, could activate the mTORCl, thus increasing the phosphorylation level of Rps6, which could upregulate the expression level of YB-1from235%to299%, then enhance the translational efficiency of snail mRNA.Conclusion:PGE2activated the EPl receptor coupled with Gαi subunit, increasing the mTOR phosphorylation level through PKC, Erk kinase; upregulating the expression level of Raptor through GPy subunit, both of which activated the mTORC1in cooperation. mTORC1could increase the expression of snail protein through enhancing the translational efficiency, then promoting the migration and invasion in hepatocellular carcinoma.