ABSTRACT
In order to study the effects of phosphorothioated antisense oligodeoxynucleotides (ASODN) on the expression of VEGF in human lymphoma cell line Namalwa cells, human lymphoma cell line Namalwa cells were incubated with VEGF ASODN (the final concentrations of VEGF ASODN were 5, 10, 20 micromol/L respectively), or scrambled sequence for 24 or 48 hours. The expressions of VEGF mRNA and VEGF protein were detected by reverse transcriptase-polymerase chain reaction and streptavidin peroxidase (SP) immunohistochemistry respectively. The results showed that the expression levels of VEGF mRNA in Namalwa cells treated with three concentration levels (5, 10, 20 micromol/L of ASODN) were 1.38, 0.96 and 0.57 respectively. Those in PBS-treated cells and scrambled sequence treated cells were 1.79 and 1.84. When treated with 20 micromol/L VEGF ASODN for 48 hours, VEGF protein of Namalwa cells decreased greatly. Meanwhile, there was no obvious change in the scrambled sequence treated group. It is concluded that VEGF ASODN can suppress the VEGF expression in Namalwa cells in vitro.
Subject(s)
Humans , Cell Line, Tumor , Lymphoma , Metabolism , Pathology , Oligonucleotides, Antisense , Pharmacology , RNA, Messenger , Metabolism , Transfection , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
To study the effect of VEGF fully phosporothioated antisense oligodeoxynucleotide (VEGF-ASODN) on VEGF expression in acute monocyte leukemic cell line U937 in vitro, U937 cells were incubated with VEGF-ASODN at concentrations of 10, 20 and 30 micro mol/L or scrambled sequence as compared with negative control. The expression of VEGF mRNA was measured by semi-quantitative RT-PCR. The expression of VEGF protein was measured by Western blot. The result showed that VEGF-ASODN had obviously inhibitive effect on expression of VEGF in U937 cell, as compared with scrambled sequence and negative control (P < 0.05). Scrambled sequence group had no significant difference compared with negative control group (P > 0.05). It is concluded that the expressions of VEGF mRNA and protein in leukemic cell line U937 are down-regulated by VEGF-ASODN.
Subject(s)
Humans , Oligodeoxyribonucleotides, Antisense , Pharmacology , RNA, Messenger , U937 Cells , Vascular Endothelial Growth Factor A , GeneticsABSTRACT
<p><b>OBJECTIVE</b>Graft versus host disease (GVHD), a major cause of graft failure in allo-hematopoietic cell transplantation, was associated with the presence of major histocompatibility complex class II (MHCII), also called human leukocyte antigen (HLAII), on the tissues and organs of host. MHCII played a critical role in the induction of immune responses by presenting fragments of allo-antigenic peptides to CD(4)(+) T lymphocytes, then by resulting in CD(8)(+) T lymphocytes activation. Therefore, it was very important for compatibility of MHCII in allo-transplantation. But it was impossible to down-modulate MHCIIexpression directly. There were codominance and multiple allele for MHCII molecules which was owing to their complicated polymorphism, therefore it was difficult to repress every MHCII molecule expression. MHC class II transactivator (CIITA) was the major rate-limiting factor for both constitutive and inducible MHCIIexpression., and with rare exceptions, its expression paralleled that of MHCII transcripts. This study investigated the effect of anti-CIITA hammerhead ribozyme (Rz) on interferon (IFN)-gamma induced MHCII expression in Jukart cell line.</p><p><b>METHODS</b>Three hammerhead Rz specific to 134, 218, 464 sites of CIITA gene were synthesized and named as Rz134, Rz218, Rz464, respectively. Then they were cloned into the EcoRI/BamHI of vector pGEM-7zf(+). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted also into the pGEM-7zf(+) plasmid. The above recombinant plasmids were screened out by sequence analysis. Hammerhead Rz and their target RNA were transcribed and then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was selected as the one with the highest activity, and then inserted into the plasmid with internal ribosome entry site-enhanced green fluorescent protein (pIRES2-EGFP), pRz464. Stable transfectants of Jukart cell line with pRz464 (pRz464-J) were tested for classic MHCII (HLA-DR, DP, DQ) induction by recombinant human IFN-gamma. mRNA abundance of CIITA was measured by RT-PCR.</p><p><b>RESULTS</b>When induced with IFN-gamma, the expression of HLA-DR, DP, DQ in pRz464 positive (pRz464+) Jukart cells was 2.7%, 6.4% and 2.1%, respectively, and that in Jukart cells transfected by non-related ribozyme was 10.1%, 57.8% and 5.1%, respectively. Therefore, Rz464 suppressed IFN-gamma-induced up-regulation of HLA-DR, DP and DQ by 73.27%, 88.93% and 58.82%, respectively. Meanwhile, the mRNA content of CIITA was reduced significantly (P < 0.01).</p><p><b>CONCLUSION</b>CIITA hammerhead ribozyme transfer inhibited MHC-II expression in Jukart cells. The above result provided insight into the future application of anti-CIITA hammerhead ribozyme for the antigen-specific tolerance induction and anti-GVHD treatment in the hematopoietic stem cell transplantation.</p>