Par-4 Gene Expression In Acute Leukemia And Its Pro-apoptotic Role

ObjectiveProstate apoptosis response-4 (Par-4) is a proapoptotic gene originally isolated from apoptotic prostate cancer cells. It was firstly identified as a molecular chaperones and inhibitive factor of Wilms'tumor suppressor-1(WT1). WT1 gene is abnormally overexpressed in different kinds of leukemias and is related to malignant transformation of normal cells. In this study, we will chiefly observe expressions of mRNA and protein for Par-4 and WT1 in bone marrow cells from acute leukemia patients and non-leukemia patients by means of Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction (RQ-RT-PCR) and Western blotting, and furtherly approach their expression changes in apoptotic leukemia cells. Then we will design and construct the eukaryotic expression vector of human Par-4 gene, transfect it into human leukemia cells K562, investigate the effect of Par-4 to WT1mRNA and protein expression level in K562, and finally observe the influence of Par-4 gene up regulation on K562 biological behaviour such as cell proliferation, apoptosis and cell cycle. This study will provide further theoretical evidences to leukemia pathogenesis and therapy target to leukemia gene therapy .Methods1. Detection of Par-4 and WT1 mRNA expression level by means of Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction (FQ-RT-PCR) in bone marrow cells from acute leukemia patients.Prepared Par-4,WT1gene andβ-actin positive standard substances, constructed Real-time Fluorescent Quantitation RT-PCR amplification system, to detect Par-4 and WT1 mRNA expression level in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients by means of Real-time Fluorescent Quantitation RT-PCR. Meanwhile, to approach the correlation between CR rate and Par-4,WT1 expression level.2. Exploration of changes of Par-4 and WT1 genes expression during the apoptosis of human leukemic K562 cells induced arsenic trioxide (As₂O₃).After K562 cells had been treated with arsenic trioxide (2-10umol/L) for 24-72 hours, cell smear was stained by Wreigt-Gmisa, cell morphous observed by microscope; cell survival rate evaluated by MTT assay and cell apoptosis analyzed using flow cytometry. Real-time Fluorescent Quantitation RT-PCR was used to test the expression of Par-4 and WT1 mRNA, and Western blotting was used to test the expression of protein for Par-4 and WT1.3. Construction of human Par-4 gene eukaryotic expression vector . Using pDNR/Par-4 plasmid as a template, the full length Par-4 cDNA was amplified by PCR and subsequently cloned into T-A vector, then subcloned into pIRES2-EGFP vector. Finally,restriction enzyme digestion and sequencing analysis was conducted to the recombinant plasmids which was named pIRES2-EGFP/Par-4.4. Influence of transfection of Par-4 gene on WT1 gene expression level and the biological behaviour of K562 cells.K562 cells were transfected with transfection complex comprising optimal proportion of pIRES2-EGFP/Par-4 plasmid and Superfect reagents, before and 48h after the transfection, obeservation for EGFP was made by fluorescent microscope, and transfection efficiency was detected by flow cytometery (FCM). Total RNA and protein was extracted from EGFP positive K562 cells ( sorted by FCM). Par-4,WT1mRNA and protein expression level was evaluated by Real-time Fluorescent Quantitation RT-PCR and Western-blotting. Then cytostasis rates was measured by MTT assay; cell apoptosis was detected by FCM. Total RNA and protein of pCon group (K562 cells transfected with pIRES2-EGFP) and non transfected group were also evaluated as control.5. Study on the auxo-apoptosis action of Par-4 gene in K562 cells.48h after K562 cells being transfected with pIRES2-EGFP/Par-4, 2umol/L As₂O₃ was added, then cytostasis rates was measured by MTT assay,cell apoptosis and distribution of cell cycle was detected and analysed by FCM. Meanwhile, non- transfected group,non- transfected group with As₂O₃ and Par-4 transfected non- As₂O₃ group were also evaluated as control.Results1. Expressions of Par-4 and WT1 mRNA by Real-time Fluorescent Quantitation Reverse Transcription Polymerase Chain Reaction (FQ-RT-PCR) in bone marrow cells from acute leukemia patients.FQ-RT-PCR result showed that Par-4 mRNA was expressed in bone marrow cells from 78 acute leukemia patients and 23 non-leukemia patients.Compared with control groups, the expression levels of Par-4 mRNA were significantly suppressed (p<0.05). Compared with initial treatment groups and relapse groups, the expression levels of Par-4 mRNA in remission groups were significantly up-regulated but were still significantly lower than that in control groups. There was no significance difference between initial treatment groups and relapse groups. No apparent association was found between Par-4 expression level and CR rate(p>0.05).WT1 gene was overexpressed in bone marrow cells from acute leukemia patients, but the expression levels of WT1 mRNA were significantly lower in bone marrow cells from control groups(p<0.05). Compared with initial treatment groups and relapse groups, the expression levels of WT1 mRNA in remission groups were significantly down-regulated but were still significantly higher than that in control groups. There was no significance difference between initial treatment groups and relapse groups . There was significance difference between different WT1 expression levels and CR rates (p<0.05).2. Changes of Par-4 and WT1 genes expression lever during human leukemic K562 cells apoptosis induced by arsenic trioxide (As₂O₃).After the K562 cells were treated with As₂O₃ of different concentrations, arsenic trioxide could efficiently inhibit the growth of K562 cells and induce apoptosis with the increase of As₂O₃ concentration. At the same time, the mRNA and protein expressions of Par-4 were up-regulated, while the mRNA and protein expressions of WT1 were down-regulated.3. Construction of human Par-4 gene eukaryotic expression vector.Restriction enzyme digestion and sequencing analysis showed that human Par-4 gene eukaryotic expression vector had been constructed successfully.4. Influence of transfection of Par-4 gene on WT1 gene expression level and the biological behaviour of K562 cells.4.1 Transfection of Par-4 plasmidAfter K562 cells in well growth conditions(2×106/ml)was transfected with the optimal transfection proportion of pIRES2-EGFP/Par-4 to Superfect reagent as 1.5μg: 6μl, transfection efficiency was evaluated by fluorescence microscope and FCM, it showed that the transfection efficiency was increased with the prolongation of time and reached its peak(75.34±5.84)% at 48h. There were significance difference compared with control groups(p<0.05).4.2 Influence of transfection of Par-4 gene on WT1 gene expression level48h after K562 cells was transfected, EGFP positive cells were sorted by FCM, purity rates reached 98.0%±1.2%. Total RNA and protein were extracted, then Par-4,WT1mRNA and protein were evaluated by FQ-RT-PCR and Western blotting. The result showed: compared with control groups and the pCon group, the expression levels of Par-4 mRNA and protein in Par-4 plasmid group were significantly increased (p<0.05); but there were no significance difference in expression levels of WT1 mRNA and protein(p>0.05).4.3 Influence of the expression of Par-4 gene on the proliferation and apoptosis of K562 cellsAfter K562 cells'being transfected with pIRES2-EGFP/Par-4, cytostasis rates was measured by MTT. The result showed: compared with non-transfected group and pCon group, proliferation inhibition ratio had no significance difference (p>0.05). Apoptosis rates of EGFP positive cells were analyzed applying AnnexinV/PI double parameter method by FCM, the result showed: compared with non-transfected group and pCon group, apoptosis rates had no significance difference (p>0.05).5. Study on the auxo-apoptosis action of human Par-4 gene in K562 cells.5.1 Cell proliferative inhibition rates assayed by MTTThe result showed:compared with non-transfected group, cell proliferative inhibition rates of different transfection groups had no significance difference(p>0.05); When As₂O₃ was added, cell proliferative inhibition rates of each group were increased gradually; significance difference emerged gradually with the prolongation of time (P<0.05); cell proliferative inhibition rates of Par-4 plasmid +As₂O₃ group was higer than other groups at each time point (P<0.05).5.2 Influence of Par-4 gene on K562 cells cycle distribution analyzed by FCMThe result showed:cells rates of S phase were higher in non-transfected group. Compared with non-transfected group, cell rates of G1 phase were a little decreased, cell rates of G2 phase were a little increased in Par-4 plasmid group and non-transfected+As₂O₃ group, while cells rates of S phase showed no obvious change (p>0.05); But cell rates of G2 phase were increased significantly (P<0.05), while cells rates of S and G1 phase were decreased significantly(P<0.05) in Par-4 plasmid +As₂O₃ group. Inhibition of G2/M phase emerged.5.3 K562 Cell apoptosis rate analyzed by FCM AnnexinV/PIThe result showed:compared with non-transfected group, cell apoptosis rates of different transfected groups had no significance difference(p>0.05); when 2μmol/L As₂O₃ was added , cell apoptosis rates of each group were increased gradually; significance difference emerged gradually with the prolongation of time (P<0.05); cell apoptosis rates of Par-4 plasmid +As₂O₃ group was higer than other groups at each time point (P<0.05).Conclusion1. The result of FQ-RT-PCR testing confirmed that Par-4 mRNA expression is lower, while WT1 is higer in acute leukemia. Par-4 and WT1 gene presentates mutually exclusive expression patterns. There was no apparent association between Par-4 expression level and CR rate.2. Higher concentration As₂O₃ could efficiently inhibit the growth of K562 cells and induce apoptosis. The induction of K562 cells apoptosis was accompanied by up-regulation of Par-4 mRNA and protein expression but down-regulation of WT1.3. Human Par-4 gene eukryotic expression vector was constructed successfully.4. Giant molecule balsam tranfection reagent Superfect can transfect pIRES2-EGFP/Par-4 into K562 cells efficiently and mediate Par-4 gene expression in K562 cells. Par-4 gene expression in K562 cell could not efficienly inhibit the expression of WT1mRNA and protein. There was no straight inhibition action to cell proliferation and apoptosis.5. Up-regulation of Par-4 gene in K562 cells can promote inhibition action to cell proliferation and apoptosis induced by As2O3. Inhibition of G2/M phase emerged.