Relationship Between Circadian Clock Protein CRY1 And CAMP-PKA Signaling Pathway In Gastric Cancer Cells And Its Effect On Tumor Cell Growth

OBJECTIVE: To detect the expression of clock protein CRY1 in gastric cancer cell lines BGC-823,HGC-27 and normal gastric mucosal epithelial cell lines GES-1,and the content of cAMP in each cell line.To investigate the changes of cell proliferation,migration and invasion,and the levels of cAMP and related signaling pathways by modifying the expression level of CRY1 in gastric cancer cell line and applying agonists of cAMP-PKA signaling pathway.The final purpose of this study is to explore whether the changes of CRY1 expression could affect the effects of cAMP agonists on gastric cancer cells,and to elucidate the relationship between CRY1 protein and the cAMP-PKA signaling pathway,and its effect on the growth of tumor cells.METHODS:(1)Normal gastric mucosal epithelial cells GES-1 and gastric cancer cells BGC-823 and HGC-27 were cultured,and total RNA and total protein were extracted,and the expression level of CRY1 was detected in different cell lines by Real time PCR and Western Blot.(2)A lentivirus vector for CRY1 overexpression was constructed.The HGC-27 cell line was infected to overexpress the CRY1 protein.(3)In the cell lines with different CRY1 expression levels,isoproterenol(ISO)was administrated at various concentrations and at different time.The proliferation of the cells was detected by CCK-8 method.The changes of cell cycles were analyzed by flow cytometry.The migration and invasion abilities of the cells were investigated by scratch test and Transwell method.(4)The cAMP levels in the cells were detected by ELISA kit before and after ISO adminstration.(5)Total proteins of the cells were extracted.And the levels of the proteins in the related signaling pathway were detected by Western Blotting.(6)The proteins in the cAMP-PKA pathway that intereacting with CRY1 were verified by pull-down and co-IP experiments.Result:(1)The mRNA and protein levels of CRY 1 in gastric cancer cell lines BGC-823 and HGC-27 were higher than those in gastric mucosal epithelial cells GES-1,and the difference was statistically significant(P < 0.05).And the expression level of CRY1 in gastric cancer BGC-823 cell was higher than that in gastric cancer HGC-27 cells,and the difference was statistically significant.(P <0.05).(2)The CRY1 overexpression cells constructed on the basis of HGC-27 cell line showed ~64 times higher levels of CRY1 mRNA and ~4 times higher levels of CRY1 protein than those of non-overexpression cells.The difference was statistically significant(P <0.05).(3)Compared with the non-overexpression HGC-27 cell lin,the CRY1 overexpression cells had less inhibitory effect on proliferation and migration after administration of high concentrations of ISO,and the difference was statistically significant.(P<0.05)(4)Prokaryotic expression plasmid was constructed,and GST-tagged CRY1 protein was expressed and purified.Pull-down experiment showed that CRY1 could interact with Gsα subunit in cells.(5)CRY1 protein and Gsα protein were overexpressed in 293 T cells.Co-IP showed that CRY1 could interact with Gsalpha subunit in 293 T cells.Conclusion:1.The expression of CRY1 in gastric cancer cell lines BGC-823 and HGC-27 was higher than that in gastric mucosal epithelial cells GES-1,and the expression of CRY1 in gastric cancer cell lines BGC-823 was higher than that in gastric cancer cell lines HGC-27.2.In the CRY1 overexpression HGC-27 cells,CRY1 can reduce the expression levels of cAMP,thereby attenuating the inhibition effects of ISO on the cell proliferation and migration by elevating the levels of cAMP.3.CRY1 can interact with Gsα protein,and prevent Gsα protein to active adenylate cyclase to produce cAMP.