Regulation Of Secreted Type Ⅱ CGMP-dependent Protein Kinase On Gene Expression Of Human Gastric Cancer Cell Line

Protein phosphorylation is one of the most common post-translational modifications of proteins.Phosphorylation of most proteins occurs in cells and is catalyzed by intracellular protein kinases.Phosphorylation of secreted protein casein was detected in 1883,but the protein kinase that phosphorylated casein was not reported until 130 years later.Since then,a variety of secreted protein kinases such as VLK and c-Src tyrosine kinase have been discovered.We found that PKG Ⅱ(Type Ⅱ c GMP dependent protein kinase)is also a secreted protein kinase.Previous studies showed that PKG Ⅱ can inhibit proliferation and migration of gastric cancer by blocking the signal transduction initiated by various cell receptor tyrosine kinase family members such as vascular endothelial cell growth factor receptor(VEGFR)in gastric cancer cells.However,the effect of PKG Ⅱ in extracellular and its target is unclear.In this paper,the recombinant protein GST-PKG Ⅱ is applied to gastric cancer cells directly to simulate the action mode of secreted PKG Ⅱ.The effect of secreted PKG Ⅱ on gene expression of AGS and HGC-27 gastric cancer cell lines is detected and verified by q RT-PCR and Western blotting on the basic of transcriptome sequencing analysis(RNA-Seq)to preliminary exploring the function of PKG Ⅱ outside cells and its potential targets.The experimental contents and methods of this study:(1)RNA-Seq was used to detect the effect of GST-PKG Ⅱ on the expression of AGS gastric cancer cell line.(2)q RT-PCR was used to detect the effect of GST-PKG Ⅱ on the transcription level of genes related to cell proliferation in gastric cancer cell line.(3)Western blotting was used to detect the effect of GST-PKG Ⅱ on the protein level of target genes in gastric cancer cell line.(4)Western blotting was used to detect the effect of growth factor EGF and VEGF-A on EGF expression in AGS and HGC-27 cells.(5)Western blotting was used to detect the effect of GST-PKG Ⅱ on VEGF-A mediated ERK1/2 phosphorylation.(6)Nuclear protein was extracted and the effect of GST-PKG Ⅱ on nuclear translocation of HIF-1α was detected by Western blotting.The experimental results of this study(1)A total of 579 differentially expressed genes were selected by RNA-Seq: 267 genes were significantly up-regulated and 312 genes were significantly down-regulated under the treatment of GST-PKG Ⅱ.q RT-PCR was performed on 9 genes related to tumor cell proliferation and cell apoptosis for the further verification.(2)Under the treatment of GST-PKG Ⅱ and 8-p CPT-c GMP,the m RNA levels of EGF,DFNA5,DACT1,and PSCA showed a consistent trend with RNA-Seq both in AGS and HGC-27 gastric cell line.The trend of GLI1 in AGS is also consistent with the result of RNA-Seq.(3)Western blotting results showed that the protein level of Pro-EGF was downregulated under the treatment of GST-PKG Ⅱ and 8-p CPT-c GMP,which was consistent with the results of RNA-Seq and q RT-PCR.(4)Western blotting results showed that EGF and VEGF-A can upregulate the protein level of Pro-EGF in both AGS and HGC-27 cells.(5)GST-PKG Ⅱ can inhibit the phosphorylation of ERK1/2 in AGS and HGC-27,and the inhibition was more obvious under the stimulation of 8-p CPT-c GMP.(6)Nuclear proteins were extracted and the nuclear translocation of HIF-1α was detected by Western blotting.The results showed that nuclear translocation of HIF-1α was suppressed effectively with or without the stimulation of 8-p CPT-c GMP both in AGS and HGC gastric cancer cell line.The conclusion of this studySecreted PKG Ⅱ affects the m RNA levels of many genes in AGS human gastric cancer cells.The inhibition of secreted PKG Ⅱ on EGF expression in human gastric cancer cells may be caused by inhibiting the activation of VEGF-A mediated MEK/ERK signaling pathway,and then inhibiting HIF-1α nuclear translocation to reduce its transcriptional activity.