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.     

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.     

庆祝《园艺学报》创刊50 周年

今年是《园艺学报》创刊发行50周年。50年来,《园艺学报》坚持为学术交流服务,为促进学科发展作贡献的办刊原则,以"科学性;创新性;对生产和科研有参考启迪作用"的标准,收录和发表了大量高水平的论文,记载了几代科技工作者呕心沥血创新之作,反映了中国园艺科学技术和园艺产业的发展历程。     

NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells

AIM:To study the mechanisms of nicotine-induced expression of intercellular adhesion molecule-1(ICAM-1).METHODS:Related luciferase reporter gene plasmids were constructed with molecular cloning techniques;above plasmids and intracontrol plasmid pSV-β-gal were co-transfected into human umbilical vein endothelial cells(HUVECs) with eukaryotic gene transfection techniques; the relative luciferase activities were detected in the transfected HUVECs.RESULTS:Series of luciferase reporter gene containing different sequences of human ICAM-1 promotor and site-directed mutants of NF-κB and Sp-1 in promotor were successfully constructed; Nicotine could increase the expression of luciferase reporter gene plasmid containing-579 bp(pGL3E-579/+36),-230 bp(pGL3E-230/+36) and mutated Sp-1 version(pGL3E-Sp-1-MU)(P＜0.05 vs control) of ICAM-1 promotor in the transfected HUVECs, whereas deletion derivative (pGL3E-134/+36) and mutation (pGL3E- NF-κB -MU) of downstream NF-κB site of ICAM-1 promotor prevent nicotine-induced increase in expression of luciferase reporter gene plasmid.CONCLUSION:NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.     

Inhibitory effect of anti-miR-196a on invasion and migration of human pancreatic cancer PANC-1 cells

AIM:To investigate the expression of miR-196a in different pancreatic cancer cell lines and to observe the effect of anti-miR-196a on the biological behaviors of human pancreatic cancer PANC-1 cells. METHODS:The expression of miR-196a in the pancreatic cancer cells was examined by real-time quantitative PCR. Anti-miR-196a was chemically synthesized and transfected into PANC-1 cells by Lipofectamine 2000. The cell proliferation was measured by CCK-8 assay. The apoptosis was determined by flow cytometry. Matrigel invasion assay was performed to examine the migration and invasion of the tumor cells. The wild-type and mutant-type NF-κB inhibitor α(NFKBIA) 3'UTR luciferase reporter vectors were constructed. The relative activity of Renilla luciferase was detected to confirm the binding site of miR-196a on NFKBIA mRNA. RESULTS:The expression of miR-196a in human pancreatic cancer cell lines was significantly higher than that in human pancreatic ductal epithelial H6c7 cells. The expression of anti-miR-196a in miR-196a group was down-regulated. After transfected with miR-196a, no change of cell proliferation and apoptosis was observed, but the abilities of invasion and migration were reduced(P<0.01). Compared with negative control, wild-type NFKBIA3'UTR or mutant-type NFKBIA 3'UTR, cotransfection of anti-miR-196a and wild-type NFKBIA 3'UTR significantly increased the relative activity of Renilla luciferase. CONCLUSION:miR-196a is one of the oncomiRs and may be a target microRNA of human pancreatic cancer for gene therapy.     

Association analysis between two SNPs in SNCA and PARK16 genes and Parkinson disease in a Han Chinese population in Liaoning area

AIM: To investigate the genotypic frequency of rs3857059 in SNCA gene and rs16856139 in PARK16 gene for determining the potential genetic risk factors of Parkinson disease (PD) in a Han Chinese population in Liaoning area of China. METHODS: The genomic DNA from 213 PD patients and 214 matched controls was amplified in the multiplex PCR system and subsequently genotyped by digestion with endonuclease Pvu II. Genetic parameter and association studies were carried out with SPSS 13.0 and PLINK 1.07 software. RESULTS: We accurately detected all genotypes in the 2 loci with PCR-restriction fragment length polymorphism (RFLP) techniques. The gene frequency of G allele in the rs3857059 locus was higher in PD group than that in control group with statistical significance (χ²= 7.592,P<0.01, OR=0.677, 95% CI=0.517~0.888). The T allele frequency in the rs16856139 locus was lower in PD group than that in control group and statistical result revealed a significant difference (χ²=11.511, P<0.01, OR=0.390, 95% CI=0.227~0.669). CONCLUSION: The 2 SNPs investigated in SNCA and PARK16 genes are likely to play roles as common risk factors for PD disease in the Han Chinese population.     

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.     

Androgen responsive element decoy DNA inhibits the promoter of prostate specific antigen and induces apoptosis of LNCaP cells

AIM:To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS:Firstly, pGL3-PSA luciferase expression vector containing 640bp-promoter fragment of PSA gene was constructed. Then, a 23-mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3-PSA and ARE decoy DNA were cotransfected into PC3-M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS:The reporter assay showed that the activity of luciferase was significantly reduced in the ARE decoy-transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy-transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION:The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.     

Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.     

Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.