Effect of P53 binding site on regulation of human NOD8 gene

AIM: To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene. METHODS: Using the method of bioinformatics, we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees. NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector p NOD8 (760 bp)-EGFP-C2/ mp NOD8 (750 bp)-EGFP-C2. The constructed plasmids p NOD8 (760 bp)-EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were transiently transfected into the cell line HEK293 by JetPei^TM and treated with pifithrin alpha (PFT-α,an inhibitor of P53) at different concentrations for 24 h. The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting. In addition, chromatin immunoprecipitation (ChIP) assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid p NOD8 (760 bp)-EGFP was transfected into HEK293 cells. RESULTS: The plasmids p NOD8 (760 bp) -EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis. The results of ChIP confirmed that P53 bound to the promoter of NOD8 . The mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2 (P<0.05). Furthermore, the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8 (750 bp)-EGFP- NOD8 compared with the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 . Meanwhile, PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 in a concentration-dependent manner, and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression (P<0.01). As expected, the protein expression of NOD8 in pNOD8 (760 bp)-EGFP- NOD8 group significantly increased compared with that in pNOD8 -C2 group, the protein expression of NOD8 in mp NOD8 (750 bp)-EGFP- NOD8 group or pNOD8 (760 bp)-EGFP- NOD8 + PFT-α group was dramatically decreased compared with that in p NOD8 (760 bp) -EGFP- NOD8 group. CONCLUSION: The results suggest that the P53 binding site is critical for the regulation of NOD8 gene and there is positive feedback regulation between P53 binding site and NOD8 , which may maintain efficient balance between defense and self-inflicted injury in response to the invasion of pathogen.     

Cholesterol-sensitive action sites in promoter of prohibitin gene

AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200　bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer.     

Effects of rs5418 site variation in SLC2A4 promoter region on gene expression

AIM: To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2,facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter region of SLC2A4 gene was amplified by PCR. Mutant and wild-type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recombinant expression vectors with different alleles was detected by a dual-luciferase reporter assay system. RESULTS: The 716-bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3-SLC2A4-prom(A) and pGL3-SLC2A4-prom(G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418-A alleles produced significantly higher relative luciferase activity (19.49±4.41) than that with rs5418-G allele (13.04±4.45; P<0.05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the expression of SLC2A4 gene,thereby affecting the gene function.     

MicroRNA-3666 regulates expression of phosphatase and tensin homologue deleted on chromosome ten in leukemic cells

AIM: To explore the regulatory effect of microRNA-3666 (miR-3666) on the expression of its target gene phosphatase and tensin homologue deleted on chromosome ten (PTEN) in leukemic cells. METHODS: miR-3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR. miR-3666 targeting PTEN 3-untranslated region (3UTR) was predicted by TargetScan software. 3UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK2. The reporter activity was evaluated by the Dual-Luciferase Reporter Assay System after the luciferase promoter vector and miRNA were co-transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor (anti-miR-3666) or a synthetic control miRNA (anti-miR-C). The expression of PTEN protein in the above transfected K562 cells was determined by Western blotting. RESULTS: miR-3666 was up-regulated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells. The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666. Inhibition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K562 cells. CONCLUSION: miR-3666 is over-expressed in leukemic cells. The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN.     

Overexpressed human FAT10 protein induces the apoptosis in the starved HEK293 cells

AIM: To study the effect of human FAT10 on the apopotosis of HEK293 cells using flag-tagged human FAT10 protein.METHODS: The fragment of FAT10 gene was cloned into the pcDNA3-flag vector,which was identified by PCR,enzyme digestion and sequencing.The reconstructed plasmids were transfected into HEK293 cells.The expression of introduced FAT10 in the normal cultured and starved cells was detected respectively by Western blotting.XTT assay and DNA ladder method were used to analyze the effect of FAT10 on the apoptosis of starved HEK293 cells.RESULTS: The reconstructed plasmids were highly expressed in HEK293 cells with different expression mode at the mormal cultured and starved state.The livability of starved FAT10 overexpressed HEK293 cells was significantly lower than that of normal cultured cells.DNA ladder was observed in the starved FAT10 overexpressed cells,but not in the normal cultured cells.CONCLUSION: The eukaryotic expressed plasmids of flag-tagged FAT10 were constructed successfully,and highly expressed in HEK293.Overexpressed FAT10 enhances the apoptosis in the starved HEK293 cells.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Evaluation of tyrosinase gene's expression in HEK293 cells by magnetic resonance imaging

AIM:Tyrosinase gene was transfected into HEK293 cell as a reporter gene, it's property of synthesizing melanin, which can be examined by magnetic resonance imaging(MRI), is used to evaluate the tyrosinase gene's expression. The aim of this study was to search a way to evaluate the results of gene expression by MRI in vitro.METHODS:The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin. To observe the MRI signals of expressed melanin, the transfected cells were scanned by MRI sequences of T₁WI, T₁WI/SPIR and T₂WI. On the other hand, fontana stain was used to search for melanin granules in transfected cells, RT-PCR method was used to search for cDNA of tyrosinase gene.RESULTS:(1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthesize a large amount of melanin. The synthetic melanins of 10⁶ cells, which had been transfected 5μg, 10μg, 20μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MRI and appeared high signal in MRI T₁WI、T₁WI/SPIR、T₂WI sequences. The signal intensities of MRI imaging were related to the amounts of transfected plasmids positively. (2) The melanin granules could be found in HEK293 cells by Fontana stain. (3) The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.CONCLUSION:The fact that MRI could detect the synthet ic melanin of HEK293 cells, which controlled by expression of exogenous gene, demonstrates that medical imaging connecting with molecular biology technology can evaluate the result of gene expression in vitro.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.     

Repression of SFRP5 promoter activity by hepatitis B virus X protein

AIM: To investigate the molecular mechanism that hepatitis B virus X protein (HBx) inhibits the promoter activity of secreted frizzled-related protein 5 (SFRP5). METHODS: Serial truncated fragments of SFRP5 promoter were amplified and cloned into luciferase reporter vector pGL3-Basic. LO2 cells were transfected with recombinants, and then infected with the adenoviral vector expressing HBx protein (Ad-HBx) or the analogous adenovirus expressing green fluorescent protein (Ad-GFP) for control. The infection efficiency was examined under a fluorescence microscope and the activity of SFRP5 promoter was measured by luciferase assay. RESULTS: Truncated fragments of SFRP5 promoter were successfully constructed. Compared with the pGL3-Basic control, the luciferase activity was 6.32±0.04 of the -478 bp~+47 bp fragment, 5.79±0.32 of the -811 bp~+47 bp fragment, 3.59±0.34 of the -1 235 bp~+47 bp fragment, 3.86±0.39 of the -1 677 bp~+47 bp fragment and 3.26±0.42 of the -2 072 bp~+47 bp fragment, respectively. Overexpression of HBx inhibited the activity of SFRP5 promoter. The average inhibitory rates were 44% for -478 bp~+47b, 46% for -811 bp~+47 bp, 28% for -1 235 bp~+47 bp, 24% for -1 677 bp~+47 bp and 40% for -2 072 bp~+47 bp, respectively. CONCLUSION: HBx suppresses the activity of SFRP5 promoter, leading to down-regulation of SFRP5 expression.