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@#[Abstract] Objective: This study aimed at investigating the epigenetic regulation mechanism of abnormally low expression of SHP-1 gene in esophageal squamous cell carcinoma (ESCC). Methods:Atotal of 71 cases of ESCC tissues and corresponding para-cancer tissues(2 cm from the edge of the cancer) resected during surgery at the Department of thoracic surgery of Hebei Province, the Fourth Hospital of Hebei Medical University from 2008 to 2011 were collected for this study. The expression level of SHP-1 mRNA and protein was detected in esophageal cancer cell lines (Eca109, Kyse170, Yes-2) before and after 5-Aza-dC or TSA treatment by RT-qPCR and Western blotting methods respectively. The methylation status of CpG sites in promoter region of SHP-1 was analyzed by bisulfite genome sequencing (BGS) in three esophageal cancer cell lines before and after 5-Aza-dC treatment. The methylation status of SHP-1 was studied by methylation-specific polymerase chain reaction (MSP) method in esophageal cancer cell lines, ESCC tissues and para-cancer tissues. The association between the SHP-1 promoter methylation status and clinic pathological parameters were analyzed in ESCC patients. Dual-luciferase reporter assay systems method was applied to detect the impacts of methylation status of CpG island in SHP-1 promoter region on gene transcription activity. For prognostic analysis of SHP-1 methylation, survival curves were constructed using the Kaplan-Meier method and the log-rank. Results: After treated with 5-Aza-dC, the expression level of SHP-1 mRNA and protein was significantly up-regulated in Eca109, Kyse170 and Yes-2 cells, meanwhile the methylation status of SHP-1 was decreased (P<0.05). The expression level of SHP-1 had no obviously change after treated with trichostatin A(TSA). The methylation frequency of promoter in ESCC tumor tissues was significantly higher than that in corresponding para-cancer tissues (P<0.05). When stratified for clinic pathologic characteristics, methylation frequency of SHP-1 was associated with TNM stage, pathological differentiation, and LN metastasis (P<0.05). The mRNAexpression level of SHP-1 in the ESCC tissues with SHP-1 methylation was significantly decreased compared to the ESCC tissues with unmethylation of SHP-1 (P<0.05). It was associated with methylation of promoter (P<0.05). The activity of fluorescein reporter vector in methylase treatment group was significantly lower than that in untreated group (P<0.05), indicating that SHP-1expression can be silenced by methylation of SHP-1 promoter. The result of Kaplan-Meier shown that SHP-1 promoter methylation was correlated with ESCC patients’poor survival. Conclusion: The transcriptional activity of SHP-1 can be inhibited with hypermethylated SHP-1 promoter region. The hypermethylated SHP-1 promoter induced the silencing of SHP-1. Therefore, SHP-1 gene may serve as one of prognostic methylation biomarkers for ESCC patients.
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Objective To explore the effects of Qa-1 and PD-L1 loaded artificial liposomal treatment in allograft rejection and its outcomes .Methods The extracellular domains of Qa-1 and PD-L1 were loaded on liposome surface by streptavidin-biotin system . Mixed lymphocyte reaction (MLR) was performed for measuring Qa-1/PD-L1 liposome biological function .Then liposome was co-transplanted with allo-islets via portal vein .The levels of blood glucose and C-peptide were detected daily after transplantation .Also hepatic lymphocytes after transplantation were isolated for determining the proportion of activated cells and signaling pathway changes .Results Artificial liposome could be easily loaded with biotinylated peptide and its diameter was between 50 to 500 nm . Qa-1/PD-L1 liposome could significantly suppress lymphocyte proliferation , activation and secretion of IFN-γ in MLR by an activation of SHP1/2 and an inhibition of Syk pathway .Qa-1/ PD-L1 liposomes could suppress the activation of hepatic lymphocytes in vivo by activating SHP1/2 ,protecting islet allografts and maintaining a normal level of blood glucose in recipients .Conclusions Qa-1/PD-L1 loaded liposome can effectively suppress allograft rejection and improve the outcomes of islet transplantation .
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Objective To investigate the effect of high expression of protein tyrosine phosphatase SHP-1 on the tumor cell invasiveness and chemosensitivity of esophageal squamous cell carcinoma.Methods EC9706 ceils of esophageal squamous cell carcinoma were divided into three groups:the blank group was not given any treatment;the experimental group used SHP-1 mimic instantaneous vector to transfect cells for 24 h,and used 10 μmol/L cisplatin to treat EC9706 cells for 12 h;the control group selected 10 μmol/L cisplatin to treat EC9706 cells for 12 h.Cell proliferation was detected by using methyl thiazolyl tetrazolium (MTT) assay,and cell invasion was detected by using Transwell chamber.The expression of SHP-1 mRNA was detected by using fluorescence quantitative polymerase chain reaction.The expression level of SHP-1 protein was detected by using Western blot.Results The expression of SHP-1 mRNA in the experimental group (3.42t0.14) was higher than that in the control group and the blank group (1.09±0.13,0.42±0.24,F =9.143,P ＜ 0.05).Compared with the control group and the blank group,the cell growth inhibition rate and the apoptosis index in the experimental group were increased (both P ＜ 0.05),and the differences between any two groups were also statistically significant (all P ＜ 0.05).The cell invasion rate in the experimental group,the control group and the blank group was (6.5±1.3) ％,(18.5±2.5) ％ and (45.2±7.2) ％,respectively,and the difference was statistically significant (F =11.853,P ＜ 0.05).Conclusion High expression of SHP-1 can inhibit the invasion of esophageal squamous cell carcinoma,improve chemosensitivity,promote apoptosis and inhibit cell proliferation,which could lay a theoretical foundation for improving the efficacy of chemotherapy for esophageal squamous cell carcinoma.
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Objective To explore the relationship between SHP1 and CD99 expression in Hodgkin lymphoma (HL) .Methods RT‐PCR and Western blot to detect the expression of CD99 and SHP1 mRNA and protein expression in IM9、KM3、L428、L428‐CD99 cells ;fluorescence confocal to observe the expression and co‐localization of CD99 and SHP1 ;transiently interference CD99 in IM9 cells and then detect the expressing of SHP1 mRNA and protein levels .Results SHP1 mRNA was low expressed in L428、KM3 and high expressed in IM9;gene and protein expression were consistent trend;in L428‐CD99 cell SHP1 mRNA expression and protein levels were higher than L428 cell;CD99 expressed in membrane and SHP1 expressed in cell plasma;transient interference CD99 ,SHP1 decreased in mRNA expression and protein levels .Conclusion In HL cells ,SHP1 expression related to the missing expression of CD99 ,and its mechanism remains to be studied .
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Objective To investigate the effect of angiotensin-converting enzyme 2 (ACE2) over-expression on ventricular remodeling in rat model of acute myocardial infarction (AMI) and the related mechanisms. Methods Totally 75 male SPrague- Dawley ratswere randomlydivided into five groups (n = 15): Sham group, AMI group, AMI + normal saline (AMI+NS) group, AMI+adenovirus-EGFP (AMI + AdEGFP) group, and AMI+ adenovirus-ACE2 (AMI+AdACE2) group. AMI models were established by ligating the left anterior descending coronary artery of rats. Rats in the AMI+NS, AMI+AdEGFP and AMI+AdACE2 groups received intramyocardial injection of NS, AdEGFP and AdACE2 in five different infarction border zones, respectively. Rats in the Sham and AMI groups received no injection intervention. Four weeks later, left ventricular end-diastolic pressure (LVEDP) and heart weight/body weight (HW/BW) were examined. Myocardial structure changes and collagendeposition were evaluated histopathologically. The expression of angiotensin (Ang) n (Angn), Ang-(1-7) and crsmooth muscle actin (crSMA) proteins was assessed by immunohistochemical staining. The relative protein expression of ACE2, Srchomology2domain-containingprotein tyrosine phosphatase 1 (SHP-1), ERK1/2, p-ERK1/2, p38, p-p38, crSMA and transforming growth factor (TGF-fii) was measured by Western blotting analysis. Results (1) Compared with the other four groups, the protein expression levels of ACE2 and Ang-(1-7) were significantly increased in myocardial tissues in AMI+AdACE2 group (P<0. 05). (2) Compared with the Sham group, LVEDP, the values of HW/BW, collagen deposition, and the expression levels of AngH, Ang-(1-7), SHP-1, p-ERK1/2/ERK1/2, p-p38/p38, crSMA and TGF-(3i were all signficantly upgraded in AMI, AMI+NS and AMI+AdEGFP groups (P<0. 05). (3) Compared with AMI, AMI+NS and AMI +AdEGFP groups, the expression levels of Ang-(1-7) and SHP-1 were significantly increased in AMI+AdACE2 group (P<0. 05); while p-ERK1/2/ERK1/2, p-p38/p38, mSMA and TGF-(3i protein expression levelswere significantly decreased in AMI + AdACE2 group (P<0. 05). Conclusion This study suggests that over-expression of ACE2 can improve ventricular fibrosis and ameliorate ventricular remodeling after myocardial infarction in rats, which may be due to that ACE2 increases SHP-1 protein expression, and the latter negatively regulates renin-angiotensin system (RAS) and mitogen-activated protein kinases (MAKPs) pathway.
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Objective To detect the significance of the SHP-1,p15 and SOCS-1 genes methylation status in the genesis and development of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of SHP-1,p15 and SOCS-1 genes CpG islands were measured by methylation-specific polymerase chain (MSP) reaction.Results The positive rates of aberrant methylation for SHP-1,p15 and SOCS-1 genes in 50 specimens of DLBCL were 96 ％ (48/50),52 ％ (26/50) and 56 ％ (28/50) respectively.In control group,however,15 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1,p15 and SOCS-1 genes promoter.Conclusion Aberrant methylations of the SHP-1,p15 and SOCS-1 genes exist in the patients with DLBCL.The methylations of SHP-1,p15 and SOCS-1 genes may be associated with the occurrence and development of DLBCL.This study provides a theoretical basis of treatment methylation for DLBCL.
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Objective: To investigate the role of SHP1 gene in inducing apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562. Methods: The full length cDNA of SHP1 gene was cloned by RT-PCR and was subcloned into mammalian expression vector pcDNA3.0. The cDNA sequence of the cloned gene was validated by enzyme digestion and DNA sequencing. Then the recombinant plasmid was used to transfect K562 cells via lipofectin. The apoptosis of K562 cells was examined by Hoechst 33258 staining assay and Annexin V/PI double-labeled assay; the differentiation of K562 cells was observed by benzidine staining and expression of glycophorin A (GPA). Results: RT-PCR and Western blotting analysis showed expression of SHP1 in K562 cells after transfection with pcDAN3-SHP1 plasmid. Apoptotic cells were detected in the K562 cells 48 h after treatment with pcDNA3-SHP1, with the apoptosis rate being 16.84%, which was significantly higher than that in cells transfected with pcDNA3.0 (6.23% , P=0.000). The positive rate of benzidine staining was 14.67% and the positive rate of GPA expression was 19.38% in cells treated with pcNDA3-SHP1, both were significantly different from those in the cells transfected with pcDNA3.0 (P=0.005). Conclusion: Over-expression of SHP1 can effectively induce apoptosis and erythroid differentiation in K562 cells.
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Objective To investigate the expression of SHP-1 and JAK1 mRNA in acute leukemia patients and their impact on disease development,and outcome of the primary chemotherapy.Methods Semi-quantitative reverse transcription polymerase chain reaction was used to measure the expression of SHP-1 and Janus kinase 1(JAK1)mRNA in 93 patients with acute leukemia(AL)and 20 healthy adults as normal 、controls(NC).Results The expression of SHP-1 mRNA in de novo AL patients was significantly lower than that in NC group(P=0.000),which was elevated when complete remission(CR)was achieved(P=0.032)and decreased after the disease relapsed (P=0.015).The expression of JAK1 mRNA in NC group was a lower than that in de novo AL group, but with no statistical significance(P=o.051).While there was statistical significance between NC group and relapsed AL group(P=0.047).The complete remission(CR)rate of the primary chemotherapy in SHP-1 positive group Was 88.9%,but 60.38%in negative group,and there was a statistical significance between them(P=0.018).There Was a negative correlation between the expression level of SHP-1 and JAKI mRNA (P=0.048).Conclusion The expression of SHP-1 mRNA Was significantly decreased or absent in the specimens of acute leukemia patients,and the positive expression of SHP-1 mRNA may be proposed as a factor of preferable therapeutic efficacy in de novo AL and a marker for the progress of the disease.The abundance of JAK1 mRNA was possibly elevated in patients with acute leukemia.
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BACKGROUND: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. METHODS: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. RESULTS: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T cell lysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. CONCLUSION: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.
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Cell Proliferation , Cytoplasm , Killer Cells, Natural , Phosphotyrosine , Protein Tyrosine Phosphatases , T-Lymphocytes , TransfectionABSTRACT
Objective To investigate alteration of SHP-1 mRNA and protein of bone marrow cells of leukemia mice after ?-ray exposure.Methods A total of 318 BALB/c mice were exposed to ~(60)Co ?-ray once a week for 4 weeks,and the total dose of ?ray received by mice was 7.00 Gy.By pathological examination,39 mice developed thymic lymphoma,14 acute lymphocytic leukemia,21 T-lymphoblast leukemia/lymphoma,1 spiroma,4 malignant yolk sac tumor.Exposed to ?-ray,the mice that developed leukemia or were free of canceration were used in our study(n=10 in each group),and another 10 mice free from irradiation served as control.SHP-1 mRNA and protein in femoral bone marrow cells were detected by real-time fluorescence quantitative PCR(FQ-PCR) and Western blotting respectively.Results SHP-1 mRNA in leukemia mice was(5.26?2.14),significantly higher than that of mice free of canceration(3.68?1.27) and controls(2.95?1.09)(P