Study Of Cml28/hrrp46p Effecting On Regulation Of Proliferation And Apoptosis In Aml Cells

Objective:1. We detected the mRNA expression of CML28/hRrP46P & WT1 and their correlation in the K562 cells and patients of acute myeloid leukemia (AML) by reverse transcription polymerase chain reaction (RT-PCR) and real time quantitative polymerase chain reaction (RQ-PCR).2. It inhibits the expression of CML28/hRrP46P by transfecting specific small interfering RNA (siRNA) in K562 cells and assesses the effect of interference by RQ-PCR; 3. After down-regulation of CML28/hRrP46P using specific siRNA in K562 cells, we check out the proliferation and apoptosis of K562 cells and study the function of CML28/hRrP46 in K562 cells.Methods:1. Total mRNA was extracted from K562 cell and and patients of AML using Trizol reagent, the first cDNA was synthesized using Fermenats's reverse transcription kit, we have designed specific primers of CML28/hRrP46P and WT1 using primer 5.0 software and detected their mRNA expression in K562 cell and patients of AML by RT-PCR and RQ-PCR. And the mRNA relative expression levels of CML28/hRrP46P and WT1 were quantitated by relevant quantitative formula 2–ΔΔCт. The correlation coefficient was calculated using SPSS 15.0 statistical software to apply to evaluate their correlation.2. Three specific CML28-siRNAs were electransfected respectively in K562 cell by electroporation apparatus with different electric voltages and mRNA expression of CML28/hRrP46P and WT1 in K562 cell were checked out by RT-PCR and RQ-PCR before and after electransfection.3. Flow CytoMeter (FCM) stained Annexin V– PI was applied to detect the apoptosis of the K562 cell which was eletransfected CML28-siRNA. 4. The CFSE labled cell proliferation trial was served to check out the proliferation inhibition rate of the K562 cell after targeted silencing CML28.Results:1. There was a high expression level of CML28/hRrP46P and WT1 in the K562 cell and patients of AML at the same time. We have relatively quantified the mRNA expression of CML28/hRrP46P and WT1 by RQ-PCR in 29 cases bone marrow specimens of newly diagnosed AML, the results show a positive correlation between their mRNA expression.2. Specific CML28-siRNA can be efficiently electransfected into K562 cell and the mRNA expression level of CML28 were obviously down regulated in CML28-siRNA treatment group.3. The apoptotic ratio of K562 cell treated specific CML28-siRNA was significantly higher than group treated unspecific negative control siRNA and normal group, meanwhile,their proliferation were remarkably inhibited.Conclusions:1. CML28/hRrP46P and WT1 are highly expressed in K562 cell and a variety of acute leukemia, especially they were higher in the AML.their expression are higher during those malignant proliferative leukemic cells, these results suggest that they have played an important role in modulating cell proliferation.2. The specific CML28-siRNA can effectively down regulated the mRNA expression of CML28/hRrP46P and WT1 in K562 cell , in addition, it can significantly inhibit K562 cell proliferation and induce K562 cell apoptosis, these results enough indicate that they have taken on an indispensable part in regulating the K562 cell apoptosis and proliferation.3. Through observing the mRNA expression of CML28/hRrP46P and WT1 in 29 cases bone marrow specimens of newly diagnosed AML, they showed apparent positive correlation, the above reveals that WT1 is a pivotal molecule which produces a capital effect together with CML28/hRrP46P in patients of AML; Relevant experiments have confirmed that the targeted reduction of WT1 mRNA targeted reduction can also induce apoptosis and inhibit cell proliferation, comparisons with our test results reveal them identical. These consequences emerged that CML28 probably played a more important and complex part in accommodating cell apoptosis and proliferation, not just as a known tumor associated antigen (TAA), and WT1 is only one during many related elements; besides, another relevant experiment also has confirmed that WT1 have played an critical role in regulating cell proliferation and differentiation by recruiting hRrP46P, one of six critical components of the human EXOSME, which is a multiprotein complex involved in 3'processing of RNA. XiaoFeng-Yang and his colleagues also have verified CML28 to be identical to hRrP46P, comparisons with the published sequence of hRrP46P reveal that the 5'coding region of CML28 is 33 amino acids longer than hRrP46P, No other differences in nucleotide sequence compared with hRrP46P were found [3]. To some extent, WT1 complete probably play a critical role with CML28 theoretically, the concrete mechanism of action was seen in following study in our research group.