A New Mechanism Improving The Survival And Metastasis Of Tumor Cell By Proton-Sensing Receptor Tdag8in The Acidic Condition

It’s well-known that outside of tumor cell is an acidic microenvironment, tumor cell survival and metastasis are closely related to the mechanism for a variety of complicated regulating process at the acidic microenvironment. Although several studies have shown that TDAG8(T-cell death-associated gene8encoded receptor, G protein-coupled receptor65, GPCR65) might involve in tumor cell survival on acidic condition, but its detailed molecular mechanism is still unclear. In this study, we found that acid stimulate tumor cell TDAG8expression and TDAG8induced MCT1(Monocarboxylate transporters1) MCT4(Monocarboxylate transporters4) and CD147expression in acidic condition, then enhance tumor cell survival and migration. So, the study was designed to demonstrate a new mechanism of proton sensing receptor TDAG8enhance tumor cell survival and metastasis in acidic condition resulting from Monocarboxylate transporters expression mediated by TDAG8and its signaling mechanism. Our findings will provide new evidence to understanding of tumor cell survival and metastasis in acidic environment. In addition, TDAG8and the signaling pathway involved in MCTs induction by TDAG8will be potential target for drug development of tumor treatment.The content of the dissertation is divided into four parts to describe as follows:Part1. Establishment of TDAG8gene stable over-expressing cell lineAims:To clone the humen TDAG8gene, and constract to the TDAG8gene expression vector and establish TDAG8gene over-expressing cell line. Methods: Reverse transcription PCR was used to cloned TDAG8gene from the total RNA of humen gastric cancer tissues, TDAG8gene was subcloned to the pEASY-Tl vector and through the sequencing, digestion, ligated into the eukaryotic expression vector pIRES2-EGFP. Eukaryotic expression vector pIRES2-EGFP-TDAG8was identified by sequencing, and transfected to A549cell through lipofectamineTM2000. TDAG8gene expression in transgenic cells was detected by Western Blot and Real time PCR technique. Result:Restriction enzyme digestion and sequencing results indicated that TDAG8gene had been cloned and pIRES2-EGFP-TDAG8expression vector was constructed. After screening by G418, positive cell clone was obtained and was confirmed by Real time and Western Blot assays. Summary:TDAG8gene was cloned and a eukaryotic expression vector pIRES2EGFP-TDAG8was constructed. TDAG8gene stable expressing cell line A549-TDAG8was obtainedPart2. TDAG8enhanced A549cell surviail and migration at acidic condition by inducing MCT1, MCT4and CD147expression.Aims:To demonstrate the effects of proton sensing receptor TDAG8on tumor cell survivals and migration in acidic condition. Methods:MTT assay and Wound closure cell migration assay were used to detect A549-TDAG8cell and A549-Vector cells survival and migration in acidic environment respectively. MCT1, MCT2, MCT4and CD147expression in A549-TDAG8cell were detected by Western Blot experiment. Result:A549-TDAG8cell survival and migration was significantly improved at acidic condition and which is inhibited by MCTs inhibitor. A549-TDAG8cell MCT1, MCT4and CD147expression induce by acid condition while MCT2not. Summary:TDAG8enhanced tumor cell survival and migration by inducing MCT1, MCT4and CD147expression.Part3Signaling pathways involved in TDAG8induced MCT1, MCT4and CD147expression.Aims:To study the signaling pathways involved in TDAG8inducing1MCT1, MCT4and CD147expression in A549-TDAG8cell. Methods:ELISAkit was used to determine cAMP accumulation in the A549-TDAG8cell. Effects of inhibitors specific to the pathway on cell survivals and migration in acidic condition were tested by MTT assay and Wound closure cell migration assay respectively. Western Blot experiment used to detect the TDAG8induced MCTl, MCT4and CD147expression and PKA, PKC, NF-кB and Spl activation in A549-TDAG8cell. Result:TDAG8induced cell survivals and migration were inhibited by H89, Staurosporine, BAY11-7082and Mithramycin A, whereas MCT1and MCT4expressions were inhibited by Staurosporine TDAG8induced CD147expression were also inhibited by H89, Staurosporine and Mithramycin A in A549-TDAG8cell. TDAG8induced NF-кBp65phosphorylation inhibited by H89and Staurosporine and Sp1phosphorylation inhibited by Staurosporine alone. Summary:TDAG8induced tumor cell survivals mediated by TDAG8-Gs-cAMP-PKA-NF-кBp65-MCT1/4signaling pathway; TDAG8induced tumor cells migration mediated by TDAG8-Gs-cAMP-PKA/PKC-Spl-CD147pathway in acidic condition.Part4Effect of TDAG8on tumor tissue formation.Aims:To detect the effect of TDAG8on in vivo tumor formation. Methods: pIRES2-EGFP-TDAG8and pIRES2-EGFP plasmid were transfected into the LLC cell with lipofectamine2000TM, and the positive cells were screened by G418. TDAG8induced cell survival was tested by MTT assay and MCT1, MCT4and CD147expression was detected by Western Blot. To observe the tumor formation after LLC-TDAG8cells and LLC-vector cells injection into BL/C57mice respectively, then Western Blot used to detect MCT1, MCT4and CD147expression in tumor tissues. Result:TDAG8significantly improved LLC cell survival and increased MCT1, MCT4and CD147expression in LLC-TDAG8cells. Both of tumor tissues formation and MCT1, MCT4, CD147expression increased in vivo experiment. Summary:TDAG8promote the growth of tumor in tumor model through the MCT1, MCT4and CD147expression.Conclusion1.TDAG8induced MCT1and MCT4expression mediated by H-/TDAG8-Gs-cAMP-PKA-NF-кBp65-MCT1/MCT4signaling pathway; 2. TDAG8induced CD147expression mediated by H+/TDAG8-Gs-cAMP-PKA/PKC-Spl-CD147signaling pathway;3. Acidic pH induced TDAG8expression; for this reason, TDAG8mediated MCT1, MCT4and CD147expression were related to acid resistance of tumor cell and Survivability of the cell.