Study On The Mechanism Of Peripheral T-cell Lymphoma With ITK-SYK Fusion Gene

Part 1 ITK-SYK fusion gene expression vector construction and cell transfectionObjective: ITK-SYK is a newly discovered fusion gene in non-specific peripheral T lymphoma,which plays an important role in the development of peripheral T lymphoma.We transfected Jurkat cells to express SYK protein by constructing an overexpression lentiviral vector of the ITK-SYK fusion gene.Meanwhile,the biological behavior of Jurkat cells after infection entiviral vector of the ITK-SYK fusion gene was observed.Methods: The ITK and SYK gene fragments were amplified by PCR,and then fused by Fusion PCR to form an ITK-SYK fusion gene.The fusion gene fragment was inserted into a lentiviral overexpression vector.The inserted gene fragment was analyzed by enzyme digestion sequencing,and the packaged virus infected Jurkat cells after successful sequencing.The expression of the fusion gene ITK-SYK in Jurkat cells was analyzed by fluorescence microscopy,flow analysis of cell infection rate and western blotting experiments.The effects of ITK-SYK fusion gene on the biological behavior of Jurkat cells were analyzed by CCK-8,soft agar cloning assay and ELISA assay.Results: The lentiviral vector of ITK-SYK fusion gene was successfully constructed by restriction endonuclease digestion.The expression of EGFP was detected by immunofluorescence protein under fluorescence microscope.The transfection rate was 85.23% by flow cytometry.The expression of SYK protein was detected by western blotting.CCK-8 assay was performed for the growth of transfected cells.Soft agar cloning assay was performed to examine changes in colony forming ability.ELISA was used to detect changes of cytokine.Conclusion: The ITK-SYK fusion gene was successfully constructed.Compared with ITK-SYK-Jurkat cells,the growth rate of cells was increased,the cloning ability was enhanced,IL-2 secretion was increased in ITK-SYK+ Jurkat cells.Part 2 Gene chip detection of Jurkat cell genetic changes caused by ITK-SYK fusion geneObjective: To analyze differentially expressed genes(DEG)in peripheral T lymphoma of control and ITK-SYK by gene chip,and perform cluster analysis,annotation and classification,significance analysis and network analysis to screen for tumorigenesis.The signaling pathway explores the pathogenesis of the ITK-SYK fusion gene at an overall level.Methods: Jurkat cells transfected with control and ITK-SYK lentiviral vector were sequenced for whole gene expression,and the differential genes were clustered.Based on the GO(Gene Ontology)database,gene functions are annotated on differential genes,and the significant functions exhibited by differential genes are screened.Based on the KEGG database,differential pathways for differential gene participation were analyzed to screen for differential pathways.Using the interaction between genes and gene products in the KEGG database,we found the relationship between differential genes,and located upstream and downstream proteins to construct a network of interactions.Results: Compared with the control group,there were 722 differentially expressed genes in ITK-SYK+ Jurkat cells,of which 278 genes were significantly up-regulated and 444 genes were significantly down-regulated.These differential genes are annotated and are mainly involved in biological functions such as cell cycle,apoptosis,DNA repair,signal transduction,and cell proliferation,involving tumors,PI3K/Akt,MAPK,and JAK/STAT signaling pathways.Gene interactions suggest a high correlation between JAK3 and STATs,suggesting activation of the JAK/STAT pathway.Conclusion: The role of fusion gene in peripheral T lymphoma is not the biological behavior of a single molecule.The above biological functions and signal pathways are intermingled and interacted to form the molecular mechanism of PTCL with ITK-SYK fusion gene.Part 3 ITK-SYK causes activation of JAK/STAT pathway in Jurkat cellsObjective: Based on gene chip results,the JAK/STAT pathway plays an important role in the peripheral T lymphoma of the ITK-SYK fusion gene.This study first verified the JAK/STAT pathway protein expression,and further observed the effect of the JAK3-specific inhibitor tofacitinib on the biological behavior of tumor cells and the tumor burden of xenograft mouse model.Methods: Jurkat cells were transfected with control and ITK-SYK lentivirus.The total protein and phosphorylation levels of the JAK family in the control ITK-SYK-Jurkat and ITK-SYK+ Jurkat were compared by western blotting.According to the results,the total protein and phosphorylation levels of STAT3 and STAT5 in the downstream cells were detected.Meanwhile,the ratio of phosphorylated protein to total protein was calculated.CCK-8 assay was performed to observe the cell viability in ITK-SYK-Jurkat and ITK-SYK+ Jurkat The Jurkat cells induced by specific JAK3 inhibitors.Western blotting was used to detect the effects of different concentrations of specific JAK3 inhibitors on the levels of STAT5 total protein and phosphorylated protein in the cells.Flow cytometry detects apoptosis and cycle changes caused by JAK3 inhibitors.Western blotting was used to detect the expression level of apoptosis and cyclin protein.The xenograft mouse model was established.The tumor volume changes between the drug-treated group and the control group were compared.The volume change of the mouse was monitored with electronic calipers.Fluorescence imaging was performed at the end of the experiment.SYK protein was detected by immunohistochemistry and measured by semi-quantitative analysis.Results: Compared with ITK-SYK-Jurkat cells,the expression of JAK1,JAK2 and TYK2 proteins was not influenced in ITK-SYK+ Jurkat cells.The JAK3 protein level increased slightly,the phosphorylated JAK3 protein level increased significantly.The downstream STAT3 and STAT5 protein level were not influenced,while p-STAT5 protein level was increased in ITK-SYK+ Jurkat cells rather than p-STAT3.these inhibitory effects increased with tofacitinib concentration(0.5,2.5 and 5 μM)and treatment time(6,18,24 and 48 h),indicating dose and time dependency of the inhibitory effects on cell viability on ITK-SYK+ Jurkat cells(50% reduction by 2.5 μM at 48 h),but there was no significant inhibition on ITK-SYK-Jurkat cells.Similarly,ITK-SYK+ Jurkat cells were treated with increasing concentrations of tofacitinib(0.5,2.5 and 5 μM)for 24 h,which caused STAT5 phosphorylation to markedly decreased.ITK-SYK+ Jurkat cells treated with 2.5 μM tofacitinib exhibited a significantly higher apoptosis rate compared with those treated with DMSO(tofacitinib 17.11% vs.control 4.12%;P<0.001).The apoptosis rate of cells treated with 0.5 μM or 5 μM tofacitinib increased compared with those treated with DMSO(0.5 μM tofacitinib 11.83% vs.control 4.12%;5 μM tofacitinib 15.24% vs.control 4.12%;P<0.05).Consistent with the results obtained by flow cytometry,increased levels of cleaved caspase-3,together with decreased levels of full-length caspase-3,were observed in the tofacitinib group.Cell cycle analysis demonstrated a significant increase in the number of tofacitinib-treated cells in the G1/S phase compared with the control group(control 71.16% vs.tofacitinib 93.77%;P<0.05).Consistent with G1/S phase arrest,increased levels of p27 and decreased levels of CDK2 were observed in the tofacitinib group.5 x 106 ITK-SYK+ CEM cells were inoculated subcutaneously into mouse.Tofacitinib(20 mg/kg/day)or equivalent PBS was administered orally for 28 days.At the end of the experiment,mouse treated with tofacitinib showed a delay in tumor growth compared to control mouse.On day 13,the anti-tumor effect of tofacitinib on tumor growth was evident.In vivo imaging in mouse showed that tumor growth was significantly inhibited in the tofacitinib-treated group compared with the control group.Meanwhile,Immunohistochemical analysis showed that the immunostaining intensity of SYK was significantly stronger in the control group than in the tofacitinib group.Conclusion: The ITK-SYK fusion gene can increase the phosphorylation level of JAK3 and STAT5 in Jurkat cells.Using the specific JAK3 inhibitor tofacitinib,the proliferation of ITK-SYK+ Jurkat cells in vitro was decreased,the level of apoptosis was increased,and the cycle arrest was in the G1/S phase,and tumor growth in mouse was inhibited.Part 4 ITK-SYK causes JAK/STAT pathway activation dependent on IL2 RG receptorObjective: JAK3 is a tyrosine kinase involved in the process of cell proliferation,differentiation and apoptosis through a common gamma chain(IL2RG)receptor involved in the cytokine pathway.Effects in the biological behavior of the cells were observed by silencing IL2 RG.Methods: Three different anti-IL2 RG si RNAs and non-sense si RNAs(control group)were transfected into ITK-SYK+ Jurkat cells by electroporation.The ITK-SYK+ Jurkat cells were used as observation objects,and the experiment was divided into two groups,an experimental group(anti-IL2 RG si RNA)and a control group(non-sense si RNA).Additional IL-2 or IL-21 was added to the experimental group.Western blotting analysis showed that JAK3,STAT5 total protein and phosphorylated protein expression levels were influenced in the experimental group and the control group as well as in the four groups of IL-2 and IL-21,and the phosphorylated protein/total protein ratio was calculated.Cell Counting were performed to observe Growth curves of cell proliferation in both groups.The apoptosis cycle of the two groups was detected by flow cytometry,and the expression of apoptosis and cyclin was analyzed by Western blotting.Four groups of cells(IYK-SYK-Jurkat group,ITK-SYK+ Jurkat group,anti-IL2 RG si RNA group,non-sense si RNA group)were tested for cytokine levels by ELISA.and IL receptor protein levels of ITK-SYK+ and ITK-SYK-Jurkat cells were analyzed by Western blotting.Results: We analyzed the inhibitory effects of three IL2RG-specific si RNAs on IL2 RG in ITK-SYK+ Jurkat cells.Si IL2RG-2 potently inhibits IL2 RG levels compared to si IL2RG-1 and si IL2RG-3.The growth curve showed that IL2 RG knockdown significantly inhibited the survival of ITK-SYK+ Jurkat cells,which increased with treatment duration(24,48,72,96 hours).Similarly,IL2 RG knockdown induced an increase in apoptosis in ITK-SYK+ Jurkat cells compared to si RNA treated controls.Consistent with the results obtained by flow cytometry,an increase in the level of cleaved caspase-3 and a decrease in the level of full-length caspase-3 were observed in the si IL2 RG treated group.Cell cycle analysis showed a significant increase in the number of G1/S phase cells in si IL2RG-treated ITK-SYK+ Jurkat cells(control 76.23% vs si IL2RG-2 92.31%,p < 0.01).Consistent with G1/S phase arrest,Western blotting detected increased levels of P27 protein and decreased CDK2 protein levels in ITK-SYK+ Jurkat cells after treatment with si IL2RG-2.We then studied the secretion of IL2RG-associated cytokines in ITK-SYK+ Jurkat cells.Compared with ITK-SYK-Jurkat cells,the secretion of IL-2 and IL-21 was increased in ITK-SYK+ Jurkat cells,while the secretion of IL-4,IL-7,IL-9 and IL-15 was not significantly changed.There was no significant change in IL-2 and IL-21 production in IL-2RG knockdown ITK-SYK+ Jurkat cells compared to the nonsense si RNA control.Western blotting analysis the silencing of IL2 RG by si IL2RG-2 inhibited the activity of JAK3 and STAT5 in ITK-SYK+ Jurkat cells,while the empty vector did not affect JAK3 or STAT5 phosphorylation.At the same time,exogenous IL-2(25 ng/ml)and IL-21(25 ng/ml)could not restore the changes caused by si IL2RG-2.Consistent with cytokines,we found that the expression levels of IL2 R and IL21 R were elevated in ITK-SYK+ Jurkat cells,while the expression levels of IL7 R were decreased.Conclusion: JAK3/STA5 phosphorylation is dependent on the presence of IL2 RG in ITK-SYK+ Jurkat cells and is associated with the secretion of IL-2 and IL-21.These findings indicate that all of these data strongly suggest that IL2RG/JAK3/STAT5 activation is involved in peripheral T-cell lymphoma with the ITK-SYK fusion gene.