Study On The Mechanism Of SAMHD1 Inhibiting Gastric Cancer Proliferation And Its Expression Regulation

Background and Objective Gastric cancer is one of the most common malignant tumors in digestive system.Currently,the improvement of overall survival of patients with gastric cancer is still dissatisfactory after comprehensive treatment including surgery,thus energetically exploring the molecular mechanism of development of gastric cancer and targeted drugs will be helpful to break through the bottleneck of treatment.Trastuzumab,as HER2 inhibitor,is the most common targeted drug in gastric cancer,which combining with first-line chemotherapy significantly improves overall survival in HER2-positive patients.However,in clinical practice,the positive rate of HER2 is less than 20%,which leads to the limited benefits,suggesting that the discovery of new moleculars regulating gastric cancer proliferation will provide more options for future targeted therapies.SAMHD1 is an important antiviral gene,and also associated with the development of a congenital inflammatory disease of the nervous system—Aicardi-Goutières syndrome.Recent studies have shown that SAMHD1 involves in regulating the development of lung cancer and cutaneous T-cell lymphoma.But,it was not clear whether SAMHD1 had an effect on the proliferation of gastric cancer.Our preliminary experimental results show that the decreased expression of SAMHD1 in gastric cancer tissues is related to tumor size,depth of invasion and TNM stage,suggesting that lower expression of SAMHD1 might be an important factor in the development of gastric cancer.In this study,we aim to clarify the mechanism of SAMHD1 on regulating proliferation of gastric cancer,to explore the transcriptional expression of SAMHD1 regulated by KLF4 and to discover the effect and mechanism of phosphorylation at Thr592 on the role of SAMHD1 in suppressing gastric cancer proliferation.Methods1.Immunohistochemistry was used to detect the expression levels of SAMHD1 protein in 58 cases of gastric cancer and adjacent normal mucosa tissues,and we also analyzed the relationship between its expression levels and clinicopathological factors of the patients.Western blot and q RT-PCR were used to detect the expression of SAMHD1 proteins and m RNAs in gastric cancer(MKN-45,MGC-803,HGC-27 and AGS)and normal gastric mucosa epithelial cell lines,respectively.2.After knockdown of SAMHD1 expression in HGC-27 and MGC-803 cells,the proliferation was detected by CCK-8 assay,and the ability of clone formation was also assessed.After overexpression of SAMHD1 in MKN-45 cells,the proliferation and ability of clone formation were detected,and the cell cycle and DNA synthesis were detected using flow cytometry.In vivo,subcutaneous tumor model of nude mice was used to assess the effect of SAMHD1 expression on the growth of gastric cancer.3.RNA-seq was performed in HGC-27 cells with stable knockdown of SAMHD1 and AGS cells with stable overexpression of SAMHD1,and then bioinformatic analysis was applied to screen out the potential signaling pathway and differentially expressed genes related with expression levels of SAMHD1.Moreover,q RT-PCR and Western blot were used to detect the expression of MAP2K6 m RNAs and proteins,and activation of MAPK p38,respectively.HGC-27 cells were treated with MAPK p38 inhibitor(SB203580),and the proliferation and activation of MAPK p38 were detected using CCK-8 and Western blot assay,respectively.4.The core promoter of SAMHD1 gene was identified using double luciferase reporting assay.We analyzed the differentially expressed transcription factors in gastric cancer tissues from the TCGA and GSE54129 databases.UCSC and JASPAR websites were used to predict the potential transcription factors binding to the core promoter of SAMHD1.Additionally,we detected and analyzed the relationship between the expression levels of SAMHD1 and KLF4 using q RT-PCR and Western blot.KLF4 protein binding to core promoter of SAMHD1 was verified through double luciferase reporting and chromosomal immunoprecipitation assay.Using the q RT-PCR and Western blot assay,we detected the expression of SAMHD1 m RNA and protein after overexpression of KLF4 in gastric cancer cells.The sh KLF4(or sh NC)and p LVXSAMHD1(or p LVX-vector)plasmids were co-transfected into AGS cells,and then CCK-8 assay was applied to detect the proliferation.5.Using the q PTM database,we analyzed the type of post-translational modifications and phosphorylated sites of SAMHD1 proteins in tumors.In 58 cases of paired gastric cancer and adjacent normal tissues,the expression levels of phosphorylated SAMHD1 at Thr592 were detected using immunohistochemistry assay.The p LVX-SAMHD1-WT,-T592 A,-T592 E or-HD/AA plasmids were transfected into gastric cancer cells,and then CCK-8 assay was used to detect the proliferation.6.Gastric cancer cells were treated with CDK4/6 inhibitor(Palbociclib),and then the cell proliferation was detected by CCK-8 assay and Western blot was used to detect the phosphorylation levels of CDK2,RB and SAMHD1 protein.7.Co-immunoprecipitation and mass spectrometry were used to screen out the SAMHD1 interacting proteins in HGC-27 cells.Meanwhile,SAMHD1 interacting proteins in Hit Predict、Gene MANIA and Bio GRID databases were also determined.Next,the common SAMHD1 interacting proteins from four datasets were identified.Coimmunoprecipitation and Western blot assay were used to verify the interaction between NRK and SAMHD1 proteins.Western blot was used to detect the phosphorylation levels of SAMHD1 at Thr592 after overexpression of NRK.Results1.SAMHD1 was down-regulated in gastric cancer tissues and cell lines,which was correlated with tumor size,depth of invasion and TNM stage,suggesting that SAMHD1,as a tumor suppressor,regulated the proliferation of gastric cancer.2.Knockdown of SAMHD1 promoted proliferation and clone formation of HGC-27 and MGC-803 cells,while overexpression of SAMHD1 inhibited proliferation,clone formation and DNA synthesis,and arrested cell cycle in G1/G0 phase of MKN-45 cells.In vivo,downregulation of SAMHD1 promoted the growth of subcutaneous grafts in nude mice.3.In HGC-27 cells with stable downregulation of SAMHD1,differentially expressed genes were mainly enriched in MAPK signaling pathway using KEGG analysis;in AGS cells with stable overexpression of SAMHD1,differentially expressed genes were mainly enriched in MAPK and PI3K/AKT signaling pathways using KEGG analysis;among the above enriched signaling pathways,MAPK was the common signaling pathway related with expression levels of SAMHD1.MAP2K6,as one of upstream kinases of MAPK p38 signaling pathway,was the most significant differentially expressed genes.Overexpression of SAMHD1 inhibited the expression of MAP2K6 m RNA and protein and the phosphorylation of MAPK p38 in AGS cells,while knockdown of SAMHD1 promoted the expression of MAP2K6 m RNA and protein and the activization of MAPK p38 in HGC-27 cells.SB203580 could reversed the activization of MAPK p38 and cell proliferation induced by knockdown of SAMHD1.4.The core promoter region was within the first 285 bp before translation start site of SAMHD1 gene.In TCGA and GSE54129 databases,there were 43 differentially expressed transcription factors in gastric cancer tissues.Among them,KLF4 was the only transcription factor that could bind to the core promoter region of SAMHD1.The expression levels of SAMHD1 m RNA and protein were positively correlated with the expression levels of KLF4 protein in gastric cancer cells.Chromatin immunoprecipitation and dual luciferase reporting assay showed that KLF4 protein was combined with the specific sequence in the core promoter region of SAMHD1,promoting its transcriptional activity.Overexpression of KLF4 increased the expression levels of SAMHD1 m RNA and protein in gastric cancer cells.Down-regulation of KLF4 promoted the proliferation of AGS cells,which could be reversed partially by overexpression of SAMHD1.5.In q PTM database,phosphorylation was the most common post-translational modification of SAMHD1 protein,and Thr592 was the most common phosphorylated site in tumors.The phosphorylation levels of SAMHD1 at Thr592 in 58 gastric cancer tissues were higher than that in normal adjacent mucosa tissues.The non-phosphorylated SAMHD1 at Thr592 inhibited the proliferation of gastric cancer cells,while the phosphomimetic SAMHD1 at Thr592 had no effect on the proliferation,suggesting that phosphorylation of SAMHD1 at Thr592 abrogated its activity to suppress gastric cancer proliferation.6.Palbociclib inhibited phosphorylation levels of SAMHD1 at Thr592 in a dosedependent manner,enhancing its ability to suppress gastric cancer proliferation.7.NRK was the common SAMHD1 interacting protein in four databases,and the interaction of SAMHD1 and NRK was verified through co-immunoprecipitation and Western blot.Overexpression of NRK elevated phosphorylation levels of SAMHD1 at Thr592 in gastric cancer cells.Conclusion1.SAMHD1 suppressed the proliferation of gastric cancer cells via negatively regulating the activation of MAPK p38 signaling pathway.2.Transcription factor KLF4 binded to the core promoter region of SAMHD1,promoting its transcription in gastric cancer cells.And overexpression of SAMHD1 suppressed the the proliferation of gastric cancer cells induced by knockdown of KLF4.3.Phosphorylation of SAMHD1 at Thr592 compromised its activity to inhibit the proliferation of gastric cancer cells.Palbociclib enhanced the role of SAMHD1 against gastric cancer proliferation through suppressing phosphorylation of SAMHD1 at Thr592.4.NRK interacted with SAMHD1,and overexpression of NRK increased the phosphorylation levels of SAMHD1 at Thr592.