Down-regulation of AnnexinⅡExpression in NB4 Cells Induced by all-trans Retinoic Acid and Its Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore effect of all-trans retinoic acid(ATRA) on annexin Ⅱ expression in NB4 cells and to analyze the luciferase activity of annexinⅡ promoter in condition of ATRA-induced treatment.</p><p><b>METHODS</b>NB4 cells were cultured in vitro, the transcriptional or translational expression levels of Annexin Ⅱ in NB4 cells treated with 1 µmol/L ATRA at different time points were detected by RT-PCR or Western blot respectively. Annexin Ⅱ-promoter was constructed, the recombinant plasmids pGL4.15 -Annexin Ⅱ -promoter were transfected into NB4 cells with electroporation, and after being treated with 1 µmol/L ATRA for 24 hours the luciferase acttivity of Annexin Ⅱ promoter was determined by luciferase activity assay.</p><p><b>RESULTS</b>The transcriptional expression of Annexin Ⅱ was down-regulated after 48 h. The translation expression of Annexin Ⅱ was slowly weakened after 24 h, and it was seriously reduced after 48 h. Further, Luciferase activity of AnnexinⅡ promoter in NB4 cells treated with 1 µmol/L ATRA was down-regulated, and showed a decreased tendency at indicated time points.</p><p><b>CONCLUSION</b>All-trans retinoic acid can induce the down-regulation of AnnexinⅡ expression on the membrane of NB4 cells, and the activity of Annexin Ⅱpromoter is down-regulated too. This study provide a basis for further study of molecular mechanism.</p>

Screening drugs for regulating tissue factor gene expression / 中国实验血液学杂志

This study was purposed to screen the drugs for regulating tissue factor (TF) gene expression through establishing stable cell line with luciferase gene having TF promoter transcription activity, so as to provide the basis for further studying the molecular mechanism of screened drugs. A series of luciferase reporter gene plasmids under control of 5'-truncated TF promoter (including -2174 bp - +128 bp, -684 bp - +128 bp, -247 bp - +128 bp and -201 bp - +128 bp) were constructed. The above plasmids were separately electroporated into U937 cells to establish stably transfected sublines. The function of stable cell line was testified by treatment with ATRA, the luciferase gene activity was analyzed by treating established cell line with bortezomib (BTZ) and CDA-II, and drugs for regulating TF gene expression were screened. The results indicated that the BTZ of 5 nmol/L could activate TF gene transcription activity, up-regulate the expression level of TF transcripts; CDA-II of 1 mg/ml could suppress TF gene transcription activity, down-regulate the expression level of TF transcripts. The functional analysis of TF promoter transcription revealed that the region of regulating TF promoter transcription activity by BTZ and CDA-II was between -201 to 0 bp. It is concluded that stable cell line U937 expressing luciferase activity of TF promoters is established, the novel drugs regulating TF gene expression are screened out by means of this established cell line. This study provides basis for screening the new drugs and further studying their molecular mechanisms.

Establishment of stable subline of K562 cells overexpressing high mobility group B1 protein / 中国实验血液学杂志

This study was aimed to establish a stable subline of K562 cells (K562-HMGB1) overexpressing HMGB1 protein and K562-HMGB1 sublines served as control, so as to provide a basis for exploring the role of hmgb1 gene in occurrence and development of leukemia and their mechanism. Protein-coding gene of hmgb1 was amplified by PCR with cDNA as template, which was synthesized by reverse transcription from total RNA extracted from U937 cells. The PCR-amplified hmgb1 gene was ligated into PMD18-T vector (PMD18-T-HMGB1 vector), and then transformed into E. coli strain DH5α. DH5α containing PMD18-T-HMGB1 vector were grown on LB agar plate supplemented with 100 µg/ml ampicillin overnight. The single ampicillin-selected DH5α clone was picked for culturing overnight and then harvested for plasmid extraction. The extracted plasmid was characterized to contain hmgb1 gene digested with the desired restriction enzymes of KpnI/XhoI. The correctness of hmgb1 sequence was confirmed with DNA sequencing. The insert of hmgb1 gene contained in PMD18-T-HMGB1 vector was cut out with restriction enzymes of KpnI/XhoI and then ligated into eukaryotic expression vector pcDNA3.1 to form pcDNA3.1-HMGB1 vector. 10µg of pcDNA3.1-HMGB1 or pcDNA3.1 plasmid was separately electroporated into K562 cells. At 48 hours after electroporation the cells were cultured with G418 at a final concentration of 800 µg/ml for over 2 weeks. Finally stably transfected sublines of K562 cells containing hmgb1 gene (K562-HMGB1), and of K562 containing pcDNA3.1 vector (K562-pcDNA3.1) served as a control, were obtained. The transcriptional or translational expression of hmgb1 gene was detected with RT-PCR or Western blot, respectively, to testify transfected efficiency and validity of stable subline of K562-HMGB1. The results indicated that the eukaryotic expression vector pcDNA3.1-HMGB1 plasmid was successfully constructed and was electroporated into K562 cells. The transcriptional or translational expression of hmgb1 gene in the stable subline of K562 cells containing hmgb1 gene was overexpressed. It indicated that stable subline of K562-HMGB1 cells was successfully established. It is concluded that the stable sublines of K562-HMGB1 cells or K562-pcDNA3.1 cells are successfully established, which provides a basis for exploring the roles and mechanisms of hmgb1 gene in leukemogenesis and development of leukemia.