ABSTRACT
OBJECTIVE@#To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1).@*METHODS@#The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group.@*RESULTS@#The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05).@*CONCLUSION@#The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.
Subject(s)
Humans , Eukaryota , Genetic Vectors , Glycoproteins , Metabolism , Green Fluorescent Proteins , TransfectionABSTRACT
<p><b>OBJECTIVE</b>Our research was aim to investigate the effect of microRNA-328 (miR-328) on proliferation of chronic myeloid leukemia(CML) cell K562 and the mediated effect of C/EBPα.</p><p><b>METHODS</b>The eukaryotic expression vectors of miR-328 targeting gene and suppressor gene (hsa-miR-328 and hsa-miR-328-inhibitor) were constructed, and transfected into K562 cells respectively. The mRNA expression levels of miR-328 and C/EBP α were detected by real-time fluorescence quantitative RT-PCR; C/EBP α protein expression was detected by Western blot; CCK-8 was used to estimate the cell viability.</p><p><b>RESULTS</b>The recombinant genes of hsa-miR-328 and hsa-miR-328-inhibitor were successfully constructed and transfected into K562 cells. Fluorescent cells were observed after 24 h, and the visible fluorescence cells were gradually increased after 48 h or 72 h, the miR-328 showed no effect on the mRNA expression of C/EBPα detected by RT-PCR. Meanwhile, miR-328 showed recovering effect on C/EBPα translation and inhibition of K562 cells proliferation.</p><p><b>CONCLUSION</b>miR-328 has been successfully constructed and transfected into K562 cells, miR-328 inhibits the proliferation of K562 cells by up-regulation of C/EBPα.</p>