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.     

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。     

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.     

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.     

Hypermethylation of SFRP genes in esophageal squamous cell cancer

AIM: To investigate the promoter methylation of secreted frizzled-related protein(SFRP) genes in esophageal squamous cell cancer(ESCC). METHODS: The methods of methylation-specific PCR(MS-PCR) and RT-PCR were applied to examine the CpG methylation of the SFRP promoter and the mRNA expression of SFRP genes,respectively, in 78 samples of ESCC and corresponding adjacent non-cancer tissues. The protein expression of β-catenin was determined by immunohistochemistry.RESULTS: In the ESCC tissues, the frequencies of promoter methylation in SFRP1 , SFRP2 , SFRP4 and SFRP5 genes were 65.4%(51/78), 69.2%(54/78), 62.8%(49/78) and 52.6%(41/78),respectively, significantly higher than those in the adjacent tissues(P<0.01). The hypermethylation of these genes had no correlation with clinical stage and pathological classification in ESCC tissues(P>0.05). The frequency of simultaneous methylation of the 4 genes was correlated with the clinical stage(P<0.05). The positive rates of mRNA expression of the 4 genes in ESCC tissues were 42.3%(33/78), 46.2%(36/78), 50.0%(39/78) and 39.7%(31/78), respectively lower than those in the adjacent tissues(P<0.01). The mRNA expression of SFRP genes and the ectopic expression of β-catenin were correlated with the methylation frequency of SFRP genes(P<0.01).CONCLUSION: Promoter methylation of SFRP1 , SFRP2 , SFRP4 and SFRP5 genes was a frequent event in ESCC, indicating a contribution to the pathogenesis of ESCC through aberrant canonical Wnt/β-catenin signaling pathway. Combination analysis of methylation status in SFRP genes may has definite value on estimating prognosis of ESCC.     

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.     

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.     

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.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL)，and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

Identification and functional characterisation of a novel anther-specific LTP promoter from Brassica campestris ssp. chinensis

The pollen development-related gene msLTP from Brassica campestris ssp. chinensis belongs to the lipid transfer protein (LTP) gene family. The promoter of the msLTP gene was isolated by thermal asymmetric interlaced PCR (TAIL-PCR). The functional elements of the promoter (named Bc15) were analysed. Some anther/pollen-specific elements, such as the GGTT box and the GTGA box, were identified in the sequence. In an attempt to confirm the promoter activity, a series of 5′-truncated constructs was fused with the GUS reporter gene to functionally analyse the key regulatory regions of the promoter. Transient expression analysis showed high GUS activity in onion epidermal cells containing the region from ?731 to ?1 bp. The truncated construct bearing the ?731 to ?1 bp fragment was stably introduced into Arabidopsis. Histochemical analysis of the transgenic plants showed distinct and specific GUS expression at different stages of anther/pollen development. Transverse sections of flower buds further indicated that the Bc15 promoter activates reporter gene expression in the anther specifically. These findings elucidated the molecular mechanisms of LTP gene regulation during pollen development, and suggested a potential application of the Bc15 promoter in engineering male sterility for hybrid production.