The Expression And Function Of Ubiquitin Ligase SIAH2 In Acute T Lymphocyte Leukemia

PART ONE THE LOCALIZATION AND EXPRESSION OF UBIQUITIN LIGASE SIAH2 IN ACUTE T LYMPHOCYTE LEUKEMIA BMMNCSObjective: To Study the localization and expression level of ubiquitin ligase Seven-in-Absentia Homolog 2(SIAH2) in acute T lymphocyte leukemia(T-ALL) bone marrow mononuclear cells(BMMNCs) and analyze the relationship between the expression level of SIAH2 and T- ALL clinical indicators, to explore the expression of SIAH2 relations with T-ALL clinical manifestations.Methods: Collected 35 cases of new diagnosed T-ALL and 10 cases new diagnosed idiopathic thrombocytopenic purpura(ITP, control group)BMMNCs from November 2011 to June 2015. Immunofluorescence(IF)evaluation of SIAH2 protein expression and location were conducted in Jurkat cell(a T-ALL cell line) as well as in BMMNCs from T-ALL and ITP patients. The expression of SIAH2 m RNA and protein were also examined by quantitative real-time PCR(q RT-PCR) and cell immunohistochemical(CIH) in these cells.Results:1) Cell immunofluorescence staining analysis showed that highlighted granular dyed red fluorescence were mainly detected in the cytoplasm of T- ALL BMMNC and Jurkat cell, which was almost absence in ITP BMMNC;Cell immunohistochemical results showed that the positive expression rate of SIAH2 proteinin in the new diagnosed in children with T- ALL was71.4%(25/35), which was significantly higher than the control group(P <0.05);q RT-PCR analysis showed that the m RNA expression of SIAH2 in BMMNCs from new diagnosed T-ALL patients was significantly higher than cells from ITP patients(P < 0.05);There were significant positive associations between SIAH2 m RNA expression and the extramedullary infiltration(EMI)(P < 0.001), especially with the mediastinal lymph node metastasis(P < 0.05) and the pleural effusion(P < 0.05). However, SIAH2 expression in T-ALL BMMNCs was not correlated with age, gender, white cell count,chromosome abnormalities,gene abnormalities,prednisone induced remission or the clinical risk classification.Conclusion:The expression level of SIAH2 in BMMNCs of children with T-ALL was higher,and significant positive associated with theextramedullary infiltration,especially with the mediastinal lymph node metastasis and the pleural effusion, prompting SIAH2 may play an important role in the development of T-ALL.PART TWO THE ROLE OF LV-SHSIAH2 IN THE BIOLOGICAL BEHAVIOR OF JURKAT CELL LINES ANDITS POTENTIAL MECHANISMSObjective:Lentivirus-packed sh RNA targeting on SIAH2(Lv-sh SIAH2) was used to knock down SIAH2 expression in Jurkat cells to investigate the effect of down-regulation of SIAH2 expression on proliferation, apoptosis, invasion biological behavior of Jurkat cell lines and its potential mechanisms.Methods: Group of Experiments: 1)Experiment group: Jurkat cell lines transfected Lv-sh SIAH2; 2)Blank control group: Jurkat cell lines;3)Negative control group: Jurkat cell lines transfected negative lentivirus-packed sh RNA; Cell proliferation, cell cycle, apoptosis, invasion were then determined by CCK-8 assay, flow cytometry, annexin V-PI assay,transwell assay, respectively; ELISA assay was used to determin the protein expression level in the supernatant of the three groups of Jurkat cell lines;q RT-PCR 、 Western-blot assays were used to determine the m RNA andprotein expression level of SIAH2、P27、Cyclin E1、CDK2、c-myc、BCL2、VEGF、VEGFR1、VEGFR2、MMP-9、MMP-13,to observe the interference effect of Lv-sh SIAH2 on SIAH2 gene in Jurkat cell lines and to investigate the impact of Lv-sh SIAH2 on cell cycle, apoptosis, invasion related molecular in the three groups of Jurkat cell lines.Results:1)q RT-PCR and Western blot assays showed that the m RNA and protein expression level of SIAH2 were down regulated significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared to the control group and negative control group(P<0.05);2)CCK-8 assay showed that the proliferation was inhibited in Jurkat cell lines after infected by Lv-sh SIAH2 in day four compared to the control group and negative control group(P<0.05), and inhibited significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight compared to the control group and negative control group(P<0.001);3)Flow cytometry assay showed that the proportion in G0 / G1 phase increased and in S phase decreased in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared to the control group and negative control group(P<0.05);4)Annexin V-PE/7-AAD double staining combined with flow cytometry assay showed that the early apoptosis rate increased significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared tothe control group and negative control group(P<0.05);5)Transwell assay showed that the cell number of invasioned through the transwell chamber decreased significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared to the control group and negative control group(P<0.05);6)EIASA assay showed that the protein expression of VEGF in cell supernatant decreased significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared to the control group and negative control group(P<0.05)7)In the eight day of Jurkat cell lines infected by Lv-sh SIAH2, the m RNA and protein expression level of P27 increased significantly,and c-myc、Cyclin E1 decreased significantly compared to the control group and negative control group(P<0.05); the m RNA and protein expression level of CDK2 did not change significantly, compared to the control group and negative control group(P>0.05);8)In the eight day of Jurkat cell lines infected by Lv-sh SIAH2, the m RNA and protein expression level of Caspase3 increased significantly,and Bcl2 decreased significantly, compared to the control group and negative control group(P<0.05);9)In the eight day of Jurkat cell lines infected by Lv-sh SIAH2, the m RNA and protein expression level of VEGF 、 VEGFR2 、 MMP-13 decreased significantly compared to the control group and negative controlgroup(P<0.05); the m RNA and protein expression level of VEGF 、VEGFR2、MMP-13 did not change significantly, compared to the control group and negative control group(P>0.05);Conclussion:1)Interference the gene expression of SIAH2 could inhibit the proliferation and block the DNA in G0 / G1 phase of Jurkat cell lines, the possible mechanism was due to up regulat the gene and protein expression level of P27, at the same time down regulat c-myc and cyclin E1 by interference the gene expression of SIAH2, which inhibited the the combination of cyclin E-CDK2 complex to arrest the cell cycle in G0 / G1phase;2)Interference the gene expression of SIAH2 could promote the early apoptosis of Jurkat cell lines,the possible mechanism was due to up regulat the gene and protein expression level of Caspase3, at the same time down regulat Bcl2 by interference the gene expression of SIAH2, which promoted the apoptosis of Jurkat cell lines through the mitochondrial apoptosis pathway;3)Interference the gene expression of SIAH2 could decrease the invasion ability of Jurkat cell lines,the possible mechanism was due to up regulat the gene and protein expression level of VEGF 、 VEGFR2 、MMP-13,which blocked the important part of angiogenesis and the extracellular matrix degradation.PART THREE THE INFLUENCE OF LV-SHSIAH2 ON PHD/HIF-1 SIGNALING PATHWAY OF JURKAT CELL LINESObjective:Lentivirus-packed sh RNA targeting on SIAH2(Lv-sh SIAH2) was used to knock down SIAH2 expression in Jurkat cells to investigate the effect of down-regulation of SIAH2 expression on PHD/HIF-1 signaling pathway in Jurkat cell lines.Methods: Group of Experiments: 1)Experiment group: Jurkat cell lines transfected Lv-sh SIAH2; 2)Blank control group: Jurkat cell lines;3)Negative control group: Jurkat cell lines transfected negative lentivirus-packed sh RNA; q RT-PCR 、 Western-blot assays were used to determine the m RNA and protein expression level of SIAH2 to observe the interference effect of Lv-sh SIAH2 on SIAH2 gene in Jurkat cell lines;Western-blot assays was used to determine the protein expression level of SIAH2, PHD1/2/3, HIF-1αin total protein and HIF-1αin nuclear protein in the three groups of Jurkat cell lines under normal oxygen concentration and1% oxygen concentration respectively.1)q RT-PCR and Western blot assays showed that the m RNA and protein expression level of SIAH2 were down regulated significantly in Jurkat cell lines after infected by Lv-sh SIAH2 in day eight, compared to the control group and negative control group(P<0.05);2)Under the condition of 1% oxygen concentration training after 48 hours, the gene expression level of SIAH2 、 HIF-1 α had no obvious change,compared to the blank control and the negative control group in corresponding condition(P>0.05). There was no significant difference between the blank control and the negative control group(P>0.05);3)Under the condition of normal and 1% oxygen concentration training after 48 hours respectively, the gene expression level of SIAH2 decreased significantly while HIF-1αhad no significantly change of Jurkat cell lines infected by Lv-sh SIAH2 after 7 days, compared to the blank control and the negative control group in corresponding condition(P<0.05);There was no significant difference between the corresponding groups under corresponding condition(P>0.05);4)Under the condition of 1% oxygen concentration training after 48 hours, the protein expression level of SIAH2 increased significantly while PHD1/2/3 decreased significantly, the protein expression level of HIF-1αincreased significantly on total and nuclear protein of the blank control and the negative control group of Jurkat cell lines, compared to the corresponding groups of Jurkat cell lines under the normal oxygen Results:concentration(P<0.05);There was no significant difference between the blank control and the negative control group(P>0.05);3)Under the condition of normal and 1% oxygen concentration training after 48 hours respectively, the protein expression level of SIAH2 decreased significantly while PHD1/2/3 increased significantly, the protein expression level of HIF-1 α decreased significantly on total and nuclear protein of Jurkat cell lines infected by Lv-sh SIAH2 after 6 days, compared to the blank control and the negative control group under corresponding condition(P<0.05);There was no significant difference between the corresponding groups under corresponding condition(P>0.05);.Conclussion:1)Under the low oxygen concentration condition, SIAH2 was up regulated, PHD was downregulated, HIF-1αwas up regulated, the possible mechanism was due to the low oxygen concentration could induce the expression of SIAH2, promoting the degradation of PHD through ubiquitin-proteasome by combined with PHD specifically, enhancing the stability of HIF-1αto accumulate in the cells, prompting HIF-1α transfer into the nuclear, promoting the transcription of its downstream gene.2)Under the low oxygen concentration and normal oxygen concentration conditions, interference the gene expression of SIAH2 could up regulate the protein expression level of PHD and down regulate the protein expression level of HIF-1 α, the possible mechanism was due tointerference the gene expression of SIAH2 reduced the degradation of PHD through ubiquitin-proteasome, destroyed the stability of HIF-1 α,accelerated the degradation HIF-1 α by ubiquitin-proteasome, reduced HIF-1 α transposition into nuclear, inhibited transcription its downstream gene transcription..3)SIAH2 could regulate the cell proliferation, apoptosis and invasion biology behavior in Jurkat cell lines by promoting PHD/HIF- 1 signaling pathways.