MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression / 华西口腔医学杂志

OBJECTIVE@#This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo.@*METHODS@#1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection.@*RESULTS@#The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo.@*CONCLUSIONS@#MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.

Construction of Dual-luciferase reporter gene vector of 3'UTR of the COL4A3 gene and validation of its targeting relationship with miR-299 / 中华显微外科杂志

Objective To construct a dual-luciferase reporter gene vector and validate the targeting relation ship between miR-299 and the COL4A3 gene,laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene.Methods This study was made from March,2018 to December,2018.Firstly,the potential binding sites between miR-299 and COL4A3-3'UTR were pre dicted using bioinformatics.Then,the wild and mutant COL4A3-3'UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors.The vectors were validated by enzyme digestion and gene sequencing.Finally,the cells were resuscitated,amplified,transfected and divided into 4 groups:COL4A3-WT+miR-299/NC group,COL4A3-WT+miR-299-inhibitor/NC-inhibitor group,COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group.Each group contains 3 holes,respectively.Luciferase activity in each group was determined using a dual-luciferase assay kit.The statistical analysis was conducted and differences between groups were compared by t test.Probabilities lower than 5％(P＜0.05) were considered statistically significant.Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully.Luciferase assay demonstrated that in wild COL4A3 gene,luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38％) compared with the NC group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In wild COL4A3 gene treated with inhibitor,luciferase activity increased in the miR-299-inhibitor group (The average R/F value was 153.98％) compared with the NC-inhibitor group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In mutant COL4A3 gene treated with inhibitor,no obvious statistical differences in luciferase activity were found between miR-299 transfection group (The average R/F value was 102.09％),miR-299-inhibitor group (The average R/F value was 108.51％) and NC group (The average R/F value was 104.70％),NC-inhibitor group (The average R/F value was 105.13％) and/9＞0.05.Conclusion The dual-luciferase reporter gene vector of the 3'UTR of the COL4A3 gene is constructed successfully.In addition,dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3'UTR of the COL4A3 gene.

Screening of antioxidant components in Scutellariae Radix through mediating Nrf2-ARE signaling pathway based on estrogen receptor / 中国药理学通报

Aim: To establish ARE dual-luciferase reporter assay system and used it to identify the antioxidant substance of Scutellaria baicalensis Georgi. Methods: 293T cells were transiently co-transfected with ARE luciferase reporter plasmid PGL 4. 37 and sea kidney luciferase reporter plasmid PRL-TK. Three major active ingredients of Scutellaria baicalensis Georgi such as scutellarin, baicalein, baicalin and/or estrogen receptor (ER) specific inhibitor were added to Nrf2-ARE luciferase reporter assay system to detect whether they exerted antioxidant effect through the estrogen receptor affecting the Nrf2-ARE signaling pathway. Results: Baicalin (100 μmol · L-1) could obviously activate Nrf2-ARE pathway in 293T cells, and the induced expression was(1. 56 ±0. 01) times that of blank group (P < 0. 01). After pre-administration of ER specific inhibitor, the induced expression decreased to (1. 02 ±0. 23) times, and the antioxidant effect disappeared. After pre-administration of ER and Nrf2-ARE pathway specific inhibitor respectively, ROS in HaCaT cells injured by UVB significantly increased and and SOD was markedly down-regulated by baicalin. Conclusion Baicalin plays antioxidant activity through mediating Nrf2-ARE signaling pathway based on estrogen receptor.

Construction of dual luciferase reporter plasmid of HIPK3 3′UTR and verification its targeting miR-146 / 基础医学与临床

Objective To construct a dual luciferase reporter vector containing the 3′untranslated region(3′UTR) of HIPK3 gene and verify the relationship between HIPK3 and miR-146.Methods The binding sites of miR-146 and HIPK3 genes were predicted by miRDB database and DIANA TOOLS database. The 3′UTR region sequences of HIPK3 genes and its mutants were respectively inserted into the luciferase report plasmid psiCHECK-2 to construct a wild-type and a mutant recombinant dual luciferase report plasmid. The 293T cells were divided into 6 groups and transfected with 1) HIPK3-WT+NC negative control;2)HIPK3-WT+miR-146a mimics;3)HIPK3-WT+miR-146b mimics;4)HIPK3-MU+NC negative control; 5)HIPK3-MU+miR-146a mimics and 6)HIPK3-MU+ miR-146b mimics respectively. After 48 hours, the luciferase activity was detected.Results HIPK3-WT and HIPK3-MU re-combinant plasmid were successfully constructed. When HIPK3-WT recombinant plasmids and miR-146b mimics were transfected into 293T cells, the luciferase activity was decreased (P<0.05). Conclusions miR-146a does not have a target relationship with HIPK3 gene,whereas miR-146b can regulate the 3′UTR of HIPK3 gene.

Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines / 生物工程学报

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

Effects of the polymorphisms in ABCG1 gene on the transcription activity and the susceptibility with coronary artery disease / 中华急诊医学杂志

Objective To investigate the effects of the polymorphisms in the promoter of ATP binding cassette transporter (ABCG1) on the transcription activity,and the relationship of the polymorphisms with the susceptibility to coronary artery disease (CAD).Methods A case-control study was conducted,217 CADpatients and 142 controls were enrolled in this study.Thesingle nucleotide polymorphisms (SNPs) in the promoter of ABCG1 were identified by sequencing.The promoter haplotypes of ABCG1 were determined with allele specific primer sequencing or Gene cloning sequencing.The transcription activity of the promoter haplotypes were evaluated with dual luciferase reporter system.The frequency of SNPs and haplotypes were analyzed between CAD group and the control group,premature CAD and non-premature CAD group,as well as multivessel lesion and single vessel lesion group.The frequency distribution was compared between two groups with x2 test or Fisher exact test.The difference of the luciferase activity was compared between groups by t-test or one-way analysis of variance.Results Only 3 SNPs were found in ABCG1 promoter sequence of about 1 000 bp upstream of the transcription start site,which are-384 (A/G),-204 (A/C) and-134 (T/G),respectively.The 3 SNPs are in strong linkage disequilibrium,Tajima's D =2.655 (P ＜ 0.01),which constituted 3 haplotypes.There was no significant difference in SNPs and haplotype frequency between the CAD group and the normal control group,and the severity of vascular disease and the early onset of coronary heart disease were not associated with the polymorphisms in ABCG1 promoter.There was no significant difference in the transcriptional activity of the three constitutive promoter haplotypes,but the transcriptional activity was notably elevated as the GAT haplotype was mutated into GAG (P ＜ 0.05).Conclusions The 3 SNPs identified in ABCG1 promoter region A did not alter the promoter activity.There was no significant correlation between the frequency distribution of SNPs and promoter haplotypes and the susceptibility to CAD.

Identification of targeting relationship between mouse lncRNA-H19 and miR-199 a-5 p by dual-luciferase reporter assay / 中国病理生理杂志

AIM:To validate the association between long noncoding (lncRNA)-H19 and microRNA-199a-5p (miR-199a-5p) through the dual-luciferase reporter gene system by construction of a luciferase reporter vector containing the gene of lncRNA-H19.METHODS:The potential complementary binding sites of lncRNA-H19 and miR-199a-5p were predicted by RegRNA 2.0.The H19 gene or its mutant ( Mut) fragment was cloned into luciferase reporter vector psi-CHECK-2.Restriction enzyme analysis and sequence analysis were used to identify whether the recombinant plasmids of the H19 and H19-Mut were successfully constructed .miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics neg-ative control or miR-199a-5p inhibitor negative control was co-transfected into the 293T cells with the luciferase reporters containing H19 or H19-Mut.Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups in order to verify the relationship between lncRNA-H19 and miR-199a-5p.RESULTS:The results of double enzyme diges-tion and DNA sequencing showed that the sequence of luciferase reporter vector was correct .The results of dual-luciferase reporter assay indicated that the H 19 reporter gene luciferase activity significantly decreased in miR-199a-5p mimics group by 49%(P<0.01), and the H19 reporter gene luciferase activity was obviously upregulated in miR-199a-5p inhibitor group compared with miR-199a-5p mimics group ( P<0.01).However, miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics negative control and miR-199a-5p inhibitor negative control showed no effect at H 19-Mut reporter gene.CONCLUSION:lncRNA-H19 binds to miR-199a-5p to exert an inhibitory effect at transcriptional level .

Effects of puerarin on proliferation of osteoblasts and Runx2-targeting miRNAs / 中国药理学通报

Aim To study proliferation capacity of cell and the target relationship between microRNA and Runx2 after effect of puerarin on osteoblasts MC3 T3-E1 . Methods The proliferation capacity of cell was detected by MTT after effect of puerarin on osteoblasts MC3 T3-E1 . The vitality of osteoblasts was detected by activity of alkaline phosphatase. The expression level of mRNA and protein of Runx2 were detected by real-time quantitative PCR and Western blot. The result of miRNA expression spectrum was compared with the predicted result to determine the Runx2-targeting miR-NAs. The expression levels of miRNAs possiby targeted to Runx2 were detected by real-time quantitative PCR. The RhoE 3′UTR vector and RhoE mut 3′UTR vector were constructed. miRNA-204 mimics and miRNA-204 NC were synthetised. The target genes were verified by dual luciferase report gene assay. Results After osteo-blasts treated with puerarin, proliferation capacity and activity of cells were enhanced , expression levels of mRNA and protein of Runx2 were both increased , the expression levels of miRNA-204 and miRNA-344 f-5 p were declined, the expression levels of miRNA-2861 was increased,the expression levels of miRNA-23a-5p, miRNA-770-5 p and miRNA-871-5 p showed no obvious change. According to the results of dual luciferase re-porter gene method after cell transfection of 48 h, only set of 3′UTR Runx2+mimics the miRNA-204 of fluo-rescein protein expression level decreased significantly, showing only the miRNA-204 inhibits Runx2 3′UTR report gene expression. Conclusion Puerarin pro-motes the proliferation of osteoblasts and regulates the miRNAs which possibly target to Runx2 .

Construction of pGL3-TNF-α3′UTR luciferase reporter gene and tanshinone compounds screening / 中国药理学通报

Aim To screen the potential inhibitors of post-transcriptional activity of pro-inflammatory media-tor TNF-α from the lipophilic constituents in Chinese Medicine Salvia miltiorrhiza Bunge ( Danshen) , we es-tablished dual luciferase reporter gene system pGL3-TNF-α3′UTR ( 3′untranslated region ) co-transfected with Renilla control gene. Methods Complementary DNA ( cDNA) template was obtained from human um-bilical vein endothelial cells ( HUVECs ) . The full length DNA of TNF-α 3′-UTR was amplified through PCR, and then connected the luciferase reporter vector pGL3-control after enzyme digestion. pGL3-TNF-α 3′UTR constructs were co-transfected with pSVRenilla into the mononuclear macrophages RAW264. 7 cells. The relative activity of reporter genes was measured by dual luciferase reporter ( DLR ) assay system after the stimulus of lipopolysaccharide ( LPS ) in presence or absence of tanshinones compounds. Results The pGL3-TNF-α3′UTR luciferase reporter gene was suc-cessfully constructed. The cloning DNA fragment and sequence were both consistent with the GENBANK da-tabase. LPS significantly induced the relative reporter activityof RAW264 . 7 cells transfected with pGL3-TNF-α 3′UTR. Among four tanshinones compounds, we found only cryptotanshinone could significantly de-crease LPS-induced relative reporter activity. Conclu-sion The pGL3-TNF-α 3′UTR construct combined with DLR assay system was successfully established, which can be used to discover the agents such as cryp-totanshinone that regulate the post-transcription of TNF-α in treatment of inflammatory and malignant dis-eases.

The identification of human microRNA335′s predicated target gene CCL1 1,CCL26 and SOX4 / 重庆医学

Objective To identify the targeted-regulating relationship between human MicroRNA335 (hsa-miR-335 )and CCL1 1,CCL26 and SOX4.Methods The potential fragments of hsa-miR-335 target genes CCL1 1,CCL26 and SOX4 were predicted by the bioinformatics analyzing tools online.The 3′untranslated regions(3′UTR)of the CCL1 1,CCL26 and SOX4 were connected to the eukaryotic expression vectors pMIR REPORT.The constructs of pMIR-REPORT-CCL1 13′UTR,pMIR-REPORT-CCL26 3′UTR,pMIR-REPORT-SOX4 3′UTR and positive control were co-transfected with Pre-miRTM miRNA335 Precursor or negative control into 293 T7/1 7 cell line by lipofectamine 2000,respectively.Both Firefly and Renilla luciferase activity were detected by dual luciferase reporter assay system.Results Compared with the negative control group,luciferase assay revealed that has-miR-335 could significantly diminish luciferase activity from SOX4 reporter vector (P 0.05).Conclusion The results suggested that hsa-miR-335 targeted regu-lated SOX4,but not targeted CCL1 1 and CCL26.

Spectrum efficiency capture technology screening anti-inflammatory and anti-asthmatic components of Shedanchenpi oral liquid / 中国药学杂志

OBJECTIVE: To develop a simple and dual-target method based on ultra-performance liquid chromatography/quadru-pole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and β2-adrenergic receptor) luciferase reporter assay system for rapid determination of various bioactive compounds of the traditional Chinese medicine preparation Shedanchenpi oral liquid. METHODS: Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and β2-adrenergic receptor activity laciferase reporter assay system and simultaneously identified according to the time-of-flight mass spectrometry data. RESULTS: Synephrine had the most obvious inhibition on the activation effect of β2-AR. Cholalic acid, glycocholic acid, taurochenodeoxycholic acid, and taurocholic acid activation displayed the most significant inhibitory effect on the activation effect of NF-κB. CONCLUSION: Dual bioactivity-integrated online spectrum efficiency capture technology is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.

Constructing a p53-fused dual luciferase reporter and verifying its function / 中南大学学报(医学版)

Objective:To construct a p53-fused dual luciferase reporter and to test whether this reporter can mimic wild-type p53 activities in a high-throughput screen.Methods:A restriction endonuclease site was added to each terminus and the stop codon of the wild-type full-length p53 open reading frame (ORF) was removed by PCR. A restriction endonuclease site was added to each terminus and the start codon of the ifrelfy luciferase ORF was removed by PCR. The two modified ORFs were inserted upstream of the IRES-induced renilla luciferase ORF in a CMV-derived vector. hTe p53 fusion protein was expressed in cells to test its MDM2-mediated degradation, subcellular localization, and induction of p53-responsive promoter. Results:hTe p53-fused dual luciferase reporter was successfully constructed. Atfer transfection into the host cells, the reporter expressing the p53 fusion protein that was degraded by oncoprotein MDM2, was mainly located inside the nucleus, and induced the p53-responsive promoter, respectively. Conclusion:hTe p53-fused dual luciferase reporter (p53FL/IRES/RL) can identify modulators of P53 protein level in a high-throughput screen of genetic or chemical libraries.

The negative effects of Trim34α on activation of luciferase reporter gene containing NF-κB promoter / 中华微生物学和免疫学杂志

Objective To investigate the effects of Trim34α on the activation of luciferase reporter gene containing NF-κB promoter induced by adaptor proteins TAB2. Methods The total RNA was isolated from HeLa cells. After amplification with RT-PCR, the target sequences were cloned into 5'-Flag-pcDNA3.1 (+) vector. The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing. It was transfected into HEK293T cells to detected Trim34α expression by Western blot. Simultaneously, the effects of Trim34α on the NF-κB activation induced by TAB2 were determined by dual-luciferase reporter assay. Results Restriction enzyme digestion, colony PCR and sequencing confirmed the vector was constructed successfully, furthermore it expressed Trim34α protein in HEK293T cells. Moreover, trim34α could form high-molecular-weight oligomeric protein, and here we called it trimsome. Interestingly, dual-luciferase assay showed that Trim34α could effectively block TAB2-induced NF-κB activation. Conclusion Trim34α was involved in negative regulation of TAB2-induced NF-κB activation and could form high-molecular-weight oligomer.

Influence of two kinds of SV40 poly (A) signals on upstream gene expression / 第二军医大学学报

Objective: To investigate the regulatory effects of two kinds of SV40 poly(A) signals on the upstream gene expression in three cell lines, so as to provide theoretical evidence for selection of poly(A) of vectors. Methods: A dual luciferase reporter vector Dual-Luc was constructed, and two SV40 poly(A) signal sequences were inserted in the downstream of the R-Luc gene separately. Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293, L-02, and HeLa cells. The relative quantities of the target gene (F-Luc) to control gene (R-Luc) were measured by Dual-Glo™ Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: The two Dual-Luciferase vectors (Dual-Luc2 and Dual-Luc3) were successfully constructed. Dual-Glo™ Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells(P>0.05), 6.16±0.39 and 3.83±0.39 in L-02 cells(P<0.05), and 1.21±0.10 and 0.66±0.02 in HeLa cells(P<0.05), respectively. The results of RT-PCR were consistent with those of luciferase assay. Conclusion: The two kinds of SV40 poly(A) signals have different regulatory effects on the upstream gene expression in different cell lines. SV40 poly(A) signals regulate the upstream gene expression at the transcriptional stage.

Effect of ginsenoside Rg1 on the transcriptional activation of NF-κB induced by H_2O_2 in 293T cell / 中国免疫学杂志

Objective:To observe the influence of ginsenoside Rg1 on transcriptional activation of NF-κB induced by hydrogen peroxide (H_2O_2) in 293T cell,and probe into the antioxidant mechanism of ginsenoside Rg1.Methods:In the experiment,cells was exposed to H_2O_2 after pretreatment with Rg1,cell proliferation and cytotoxicity studies were detected by MTT and Trypan blue.The quantities of generation of intracellular reactive oxygen species (iROS) was analyzed by flow cytometric analysis measured with fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA).NF-κB-responsive element-luciferase reporter gene was transfected and dual-luciferase cis-reporting systems were used to assay the transcriptional activity of NF-κB under the stimulated circumstance of oxidative stress induced by H_2O_2.Results:The results of MTT showed that ginsenoside Rg1 apparently protected the proliferation of 293T cell,which were repressed by H_2O_2 (P＜0.05).The results by trypan blue showed that H_2O_2 stimulated substantial cytotoxicity.This effect was markedly attenuated by treatment with ginsenoside Rg1.Oxidant production,measured as the fluorescence of dichlorofluorescein,was significant increased by 40%-50% through H_2O_2 stimulation.The decrease in iROS generation was significant blocked by 35%-40% through Rg1 and antioxidant.The relative luciferase reporter assay of NF-κB was apparently improved by H_2O_2-induced(P＜0.05),but Ginsenoside Rg1 significantly repressed the relative value of luciferase (P＜0.05).Conclusion:Ginsenoside Rg1 has the obvious protective function from the damage of oxidative stress damage,whose possible mechanism is to eliminate excessive free radicals of the cells effectively,to reduce transcriptional activation of nuclear factor kappa B(NF-κB),and subsequently to suppress the NF-κB circuit activation.

Construction of pGL3-Basic-SREBP-1c-promoter reporter gene vector and detection of its function / 基础医学与临床

Objective To construct human SREBP-1c-promoter reporter gene vector and to detect its function.Methods Human blood genome DNA was extracted and pGL3-Basic-SREBP-1c-promoter reporter gene vector was constructed.Furthermore,the function of SREBP-1c-promoter was confirmed by dual-luciferase reporter assay.ResultspGL3-Basic-SREBP-1c-promoter reporter gene vector was successfully constructed and the promoter activity was obviously repressed by co-transfection FoxO1.Overexpression FoxO1 inhibited the SREBP-1c protein expression.Conclusion FoxO1 repressed the SREBP-1c protein expression through inhibition the SREBP-1c transcription.

Effect of -1 SNP on CCNH promoter activity / 基础医学与临床

Objective To study the effect of-1 site single nucleotide polymorphism(SNP) on CCNH gene promoter transcription activity.Methods PCR and site-directed mutagenesis technology were used to construct CCNH basic promoter and-1G mutate promoter.Dual-Luciferase Reporter assay system was used to detect the transcription activity of constructed promoter.Results In AD293 cells,the activity of-1G mutate type promoter was significantly lower than that of wild type-1T promoter(P

Role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in esophageal carcinoma cells / 中国免疫学杂志

Objective:To identify the role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in human esophageal carcinoma cells.Methods:Esophageal carcinoma EC109 cells were transfected with expressing vectors CMV-Sp1 or CMV-Sp3,and the effect of Sp1 and Sp3 over-expression on ezrin mRNA and protein expression was determined by real time RT-PCR and Western blot analysis.Furthermore,EC109 cells were cotransfected with the ezrin promoter-directed luciferase reporter vector and control vector pRL-TK along with transcription factor expression vector.The roles of Sp1 and Sp3 in ezrin promoter activation and whether this activation occurred through the Sp1 binding site,-75/-69,were analyzed by dual-luciferase reporter assay system.Results:Over-expression of transcription factors Sp1 and Sp3 significantly increased the expression of ezrin mRNA and protein and the ezrin promoter activity in EC109 cells.Sp1 and Sp3 enhanced the promoter activity through different binding sites and only Sp1 did that through the-75/-69 site.Conclusion:Sp1 and Sp3 can regulate ezrin expression in EC109 cells.