Function Study On CML28, A New Leukemia-associated Gene

[Objective]1.The aim of chapter one was designed to study mRNA expression of CML28 gene in leukemia cell lines and primary leukemia cells by reverse-transcriptase polymerase chain reaction.2 . The objective of chapter two was to induce CML28-specific cytotoxic T lymphocytes response by constructing dendritic cell vaccine loaded CML28 cDNA by electropotation. To enhance the anti-tumor efficacy of CML28 nucleic acid vaccination, siRNA silencing mediated by vector-based technology was performed to down regulate mRNA expression of SOCS1 gene.3.The objective of chapter three was to explore the function of CML28/hRrp46p gene involved in the apoptosis and proliferation of K562 cell line by siRNA silencing the mRNA expression of CML28/hRrp46p in K562 cell line.[Methods]1.The mRNA expression of CML28 in leukemia cells were detected by RT-PCR. Total RNAs were extracted from PBMCs obtained from patients'bone marrow samples using TRIzol reagent. RT-PCR was performed with the primers CML28-5P (5'-GGTAAACGCTAAGCCACG-3') and CML28-3P (5'-GAAGGAA- GCAGAGCCATCT-3') for CML28 and primers GAPDH-5P (5'-GCTGGCGC- TGAGTACGTCGT-3') and GAPDH-3P (5'-TGGGTGTCGCTGTTGAAGTC-3') for GAPDH as internal control.2.The full length CML28 cDNA was amplified from K562 by reverse-transcriptase polymerase chain reaction with the following primers, CML28-5P1 (5'-CGGAATTCTGCGGACGCCCAGGAGGATG-3') CML28-3P1 (5'-GCTCTAG- AGAGTGGGTGGAGGCAATG-3'). The fragment of RT-PCR products was ligated into the multi-clone sites (MCS) of EcoRⅠand XbaⅠsite of pcDNA3.1 HisA to generate recombinant plasmid pcDNA3.1 His-CML28. To facilitate the detection of CML28 protein, the open read frame (ORF) of His-CML28 fusion protein was obtained from recombinant pcDNA3.1His-CML28 by PCR with the primers CML28-5P2 (5'-CGCTCGAGCGGAGCTTACCATGG-3') and CML28-3P2 (5'-TCCCCGCGG- GGAGAGTGGGTGGAGGCAATC-3'). Digest the PCR fragment with XhoⅠand SacⅡand cloned into a bicistronic vector pIRES2-EGFP to generate CML28 nucleic acid vaccination pHis-CML28-IRES2-EGFP, verified by DNA sequencing. Dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMCs) of healthy donor. To enhance the CML28 nucleic acid DC vaccine, we construct the recombinant vector psiRNA-SOCS1 encoding siRNA targeting suppressors of cytokine signaling 1 (SOCS1) sequences using a vector-based RNA interference technology. Co-transfect CML28 nucleic acid vaccination and recombinant siRNA expression vector psiRNA-SOCS1 into DCs by electroporation. Validate SOCS1 gene silencing efficacy by RT-PCR. The expression products of CML28 nucleic acid vaccination, His-CML28 fusion protein and GFP protein, were measured by Western Blotting and fluorescence microscope respectively. Labeling nonadherent fraction of autologous PBMCs as responder cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) and then used for mixed lymphocyte reaction (MLR) culture with co-transfected DCs. Proliferation in response cells was assessed by CFSE proliferation assay. The standard LDH–release assay/51Cr release assay was performed to measure cytotoxicity of stimulated CTL.3.The selection of siRNA sequences targeting CML28 was based upon web-based program siRNA Wizard and optimized according to Brummelkamp et al, analyzed by BLAST.siRNA1:Oligo 1: TCC C AA CAC GTC TTC CGT TTC TA T CAA GAG TAG AAA CGG AAG ACG TGT T TTOligo 2: CAA AAA AAC AGG TCT TCC GTT TCT A CT CTT GA T AGA AAC GGA AGA CGT GTT;siRNA2:Oligo1: TCCC AACA CGTC TTCC GTTT CTACC TTCAAGAGA GGTAG AAAC GGAA GA CC TGTT TTOligo2: CAAAA AACA CGTC TTCC GTTT CTACC TCTCTTGAA GGTAG AAAC GGAA GA CC TGTT;siRNA 3:Oligo 1: TCCC AATC GCTG CAGA GGCGTTACT TTCAAGAGA AGT AACGCC TCTGCAGCGATT TTOligo 2: CAAAA AATC GCTG CAGA GGCGTTACT TCTCTTGAA AGT AACGCC TCTGCAGCGATT.The siRNA sequences of sense and antisense oligonucleotides (25μM each) was annealed in 0.15 M NaCl at 80°C for 2 min and then maintained until the temperature reached 35°C in a thermal unit. Annealed siRNA was ligated into the BbsI sites of the psiRNA-hH1neo vector, downstream of the H1 promoter, and the plasmids were sequenced and amplified. The efficacy of gene silencing on CML28 was measured by RT-PCR with RT primer, 5'-AGCC CAAT CTTC G-3', PCR Primer: Sense: 5'-GCTG CCAG GCGG AAGT GA-3'Anti-sense: 5'-CTGT TCGC AGGC AAAG TG-3'. The siRNA expression vector targeting CML28 was delivered to K562 cell line and FACS analysis of CFSE staining of K562 was performed to detect the influence of CML28 silencing on proliferation of K562. FACS analysis of annexin-V/PI double staining of K562 was performed to detect apoptosis of K562 post electropotation.[Results]1. CML28 mRNA is expressed at high level in AML, CML-AP and CML-BP, but low level in ALL and CML-CP.2. Full length sequence of CML28 cDNA was amplified from K562 cell successfully by RT-PCR and the recombinant plasmids of pcDNA3.1 His-CML28 and pHis-CML28-IRES2-EGFP contains the right sequence of CML28 fragment verified by restriction digestion and DNA sequencing. CML28 nucleic acid vaccination could encode CML28 mRNA, His-CML28 and GFP protein in DCs successfully, verified by RT-PCR, western blotting and fluorescence microscope, respectively. SiRNA targeting SOCS1 significantly suppress the expression of SOCS1 in DCs. Down regulation of SOCS1 resulted in higher expression level of CD80, CD86 and CD83 in co-tranfected DCs and more rounds of cell division of responder cells in CFSE-MLR. CTLs induced by co-transfected DCs exhibit stronger CML28-specific cytotoxicity against HLA-matched target cells comparing to CTLs induced by CML28 nucleic acid vaccination transfected DCs only.3. The recombinant plasmids psiRNA-CML28 obtained from blue/white selection and kanamycin selection contain the right siRNA sequence targeting CML28 mRNA, verified by DNA sequencing. It's verified by RT-PCR that the expressions of CML28 mRNA were down-regulated by siRNA targeting CML28 with different contents. FACS analysis of CFSE staining of K562 revealed that the proliferation of K562 was suppressed after CML28 silencing. Apoptosis assay of annexin-V/PI staining revealed K562 cell of CML28 silencing underwent apoptosis with different contents.[Conclusions]1. In our study, CML28 is expressed at high level in both primary leukemia cell and cell lines of AML, CML-AP and CML-BP, but low level in ALL and CML-CP, which is the first report of CML28 expression in ALL. In this term, successful induction of CML28-specific CTL response will provide a promising immunotherapy sword for a very broad population. 2. In this study, the transfected DCs effectively induced the autologous CML28-specific CTL responses. DC generated from PBMCs of healthy donor obtained typical phenotype of immature DC,Down regulation of SOCS1 by siRNA resulted in higher expression levels of costimulative moleculars and stronger capability to stimulate proliferation of responder cell in DCs. The CTLs induced by SOCS1-silenced DCs exhibited stronger CML28-specific cytotoxicity against target cells compared to the ones induced by SOCS1-expressing DCs. The gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of the CML28 DNA Vaccine in DCs.3. Gene-silencing of CML28 mediated by siRNA suppressed the proliferation of K562 and contributed to the apoptosis of K562 cell. Given the broad expression of CML28 in leukemia cells, we suppose that the high expression of CML28/hRrp46p in leukemia is a hallmark of hyperfunction of exosome, and furthermore, exosome might be deeply involved in proliferation and apoptosis in leukemia cells and participated in the signal transduction by CML28-hRrp46p-exosome-ZAP-HOX interaction.