SHP1 Decreases The Level Of P-STAT3 (Ser727) And Inhibits Proliferation And Migration Of Pancreatic Cancer Cells

Background: The incidence of pancreatic cancer has been increasing rapidly worldwide.Given this gloomy situation,any attempt to improve the survival rate of patients with pancreatic cancer is worthwhile.SHP-1 has attracted wide attention as a therapeutic target for many cancers,and several drugs have been developed.SHP1 regulates the activity of oncogenic STAT3,thereby inhibits tumor growth,promots apoptosis,and even prevents chemoradiotherapy resistance.PTPN6 is the gene that encodes SHP1 and is generally considered to be a tumor suppressor gene.The role of SHP1 in tumors may vary with different biological backgrounds.The direct role and possible mechanisms of SHP1 in pancreatic cancer tissue itself,rather than lymphocytes,have not been investigated.Previous studies focused on the negative regulation of STAT3 activation by SHP1 dephosphorylation of Tyr705,and the effect of SHP1 on Ser727 has not been reported.Objectives:In this situation,the main purpose of this study is to explore the function and mechanism of endogenous SHP1 expression in pancreatic cancer.To explore the regulatory relationship between SHP1 and p-STAT3 in pancreatic cancer tissue and the possible mode of action.Methods: The expression of SHP1 in human pancreatic cancer tissues was detected by immunohistochemical staining,and Spearman correlation analysis was performed by SPSS software.Western blot was used to detect the expression of SHP1 in pancreatic cancer cells.PANC-1 cells were overexpressed with lentivirus and SHP1 was knocked down for cell function test.The experimental grouping information is as follows: NC(KD)group: normal target cells,plus negative control virus infected cells group;KD group:normal target cells and cells infected with sh RNA virus of SHP1 gene;NC(OE)group:normal target cells,plus negative control virus infected cells;OE group: normal target cells and cells infected with SHP1 gene overexpression virus.Cell viability,colony formation,apoptosis,migration and invasion were used to detect the function of SHP1 in pancreatic cancer cells.Quantitative proteomics analysis of PRM phosphorylation modification verified the effect of SHP1 overexpression on p-STAT3(Ser727)changes.Non-standard quantitative proteomics analysis was conducted to determine the effect of SHP1 overexpression and reduction of p-STAT3(Ser727)levels on the protein expression levels of related signaling pathways in pancreatic cancer cells.Immunoprecipitation combined with mass spectrometry analysis and verification of the relationship between STAT3 and JAK1 and p-STAT3(Ser727).Results:(1)In 55 cases of human pancreatic ductal adenocarcinoma,the expression of SHP1 was absent in 44 cases,decreased in 11 cases,and was moderately expressed in all 55 adjacent non-malignant pancreatic acinus or ducts.There was no significant correlation between SHP1 expression and baseline information such as age,gender,smoking,alcohol consumption,diabetes,hypertension,allergy history(P>0.05);There was no significant correlation with CA199,tumor location,degree of differentiation,lymph node metastasis and the 8th AJCC stage.The positive expression cases were concentrated in pancreatic cancer patients with no history of alcohol consumption,diabetes mellitus,hypertension or allergy.At the same time,it was found that the patients with SHP1 positive expression were mostly of well differentiation,no lymph node metastasis,and AJCC stage I and II.SHP1 can be detected in pancreatic cancer cells PANC-1,As PC-1,Bx PC-3 and SW1990.The expression of target protein SHP1 was not detected in CFPAC-1,MIA PACA-2 and PATU-8988 cells.(2)MTT assay showed that SHP1 inhibited PANC-1 cell proliferation;The results of Celigo scratch assay showed that SHP1 inhibited PANC-1 cell migration.Annexin V-APC staining flow cytometry showed that SHP1 inhibited PANC-1 cell apoptosis.(3)Phosphoropeptide enrichment techniques and targeted proteomic quantification techniques based on mass spectrometry identified the target peptide FICVTPTTCSNTIDLPMS(ph)PR,reduced serine phosphorylated peptide level at 727 of STAT3,indicating that overexpression of SHP1 reduced p-STAT3(Ser727)level.(4)Non-standard quantitative proteomic analysis indicated that SHP1 was overexpressed(nearly 15 fold upregulated),and T-test p-value<0.05.1.5 fold(or 0.667fold)was used as the threshold of differential expression change.When 0.05 was the significance threshold,45 proteins were up-regulated and 18 proteins were down-regulated in PANC-1 cells.The biological functions of up-regulated and down-regulated proteins were mainly be involved in signal transduction mechanism by KOG classification analysis.Among of these,RPRD1 A,EFEMP1 and OTUD6 B were up-regulated and MADD was down-regulated.By searching the Uniprot protein database,it was found that their functions may involve negative regulation of cell cycle and regulation of cell growth and proliferation.This could theoretically explain the inhibition of PANC-1 cell proliferation by SHP1 in the above cell function experiments.At the same time,MADD subtype 1 May induce cell apoptosis,which may explain the inhibition of Panc-1 apoptosis by SHP1 to some extent.C3ORF38 may also be involved in apoptosis.GSEA software analysis showed that SHP1 overexpression decreased p-STAT3(Ser727)levels and changed the expression levels of 25 proteins in the JAK/STAT pathway,10 proteins were up-regulated and 15 proteins were down-regulated.The main products are IL6 ST,PTPN2,PTPN11,STAT1,STAT2,STAT3,STAT5 A,STAT5B,STAT6,PIK3 CA,PIK3CB,PIK3R1 and so on.Among them,the expression levels of IL6 ST,PTPN2 and PIK3 CB were significantly changed,while those of other proteins were less changed.L6 ST activates STAT,PTPN2 dephosphorylates JAK/STAT,PIK3 CB is the catalytic subunit of PIK3,and PIK3 phosphorylates PIP3,thereby regulating cell growth,differentiation,and survival.These results suggest that changes in SHP1 in Panc-1 cells affect protein levels in the Jak/Stat pathway and thus signal transduction in the JAK/STAT pathway.(5)Qualitative analysis of proteins by Co-IP Shotgun method showed that 128 proteins were detected in NC group;377 proteins were detected in the OE group.274 specific proteins of the OE group were detected,including STAT3 and JAK1 proteins concerned in this study.In the preliminary experiment,STAT3 protein expression was not detected in PANC-1 cells,but it could be detected in positive control cells.However,p-STAT3(S727)protein expression could be detected in Panc-1 cells and in He La cells(S727),the positive control cell.The interaction between p-STAT3(S727)and SHP1 was verified in the Co-IP validation experiment.FLAG was detected in the OE group.Flag was detected in the OE group of Input and IP.Compared with NC(OE)group,the expression level of p-STAT3 in IP group was approximately the same,suggesting that there may be no direct structural interaction between p-STAT3 and SHP1 protein.In Input and IP,the expression levels of JAK1 in OE group and NC(OE)group were roughly the same,suggesting that there may be no direct structural interaction between JAK1 and SHP1 protein.Conclusion: This study first investigated the expression of SHP1 in pancreatic cancer tissues and cells and its function in pancreatic cancer cells,and found that:(1)The expression of SHP1 in human pancreatic cancer cells was different,and it was expressed in Panc-1,Aspc-1,BXPC-3 and SW1990 cells.No expression was found in CFPAC-1,MIA PACA-2 and PATU-8988.The expression of SHP1 was absent or weakened in human pancreatic cancer tissues,while it was moderately expressed in adjacent normal pancreatic acinus and ductal epithelial tissues.(2)SHP1 inhibits proliferation and migration in human pancreatic cancer cells;(3)SHP1 promotes apoptosis in human pancreatic cancer cells;According to the above results,the expression of SHP1 in pancreatic cancer was mainly absent,and a few were reduced.Patients with weakly expressed pancreatic cancer are characterized by early staging.The main function of SHP1 in pancreatic cancer cells is to inhibit proliferation and migration,and it is generally a tumor suppressor gene.However,SHP1 inhibits cell apoptosis in pancreatic cancer cells,showing different biological effects,reflecting the complex mechanism of SHP1 in solid tumors.This study was the first to investigate the effect of SHP1 on p-STAT3(Ser727)levels,and to explore the possible ways of action of both,and found that:(4)SHP1 reduced the level of p-STAT3(Ser727)in PANC-1 cells;(5)SHP1 did not interact directly with JAK1 and p-STAT3(Ser727)in the form of protein complex.Our results reveal a new mechanism by which SHP1 regulates STAT3 and provide a new target for the study of its functions and effects.