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Inhibitor of DNA binding 1 (ID1) has an aberrantly high expression in multiple cancer tissues, including colon cancer, lung cancer, breast cancer, and so on, which is closely related to cancer aggressiveness and poor clinical outcomes in cancer patients. It has been reported that ID1 maintains colorectal cancer cells (CRCs) stemness traits and contributes to the CRC drug resistance. While, the biological molecular mechanisms have not been fully elucidated. In this research, we found that ID1 upregulates octamer binding transcription factor (OCT4) protein level as well as OCT4 signaling pathway via Western blot, gene set enrichment analysis (GSEA), dual-luciferase reporter assay, and real-time PCR. Through the in vitro sphere formation assay, we found that overexpression of OCT4 reverses the inhibitory effect of knocking down ID1 on CRC sphere formation ability. With the help of JASPAR and GEPIA database, we predicted a novel transcriptional repressor—forkhead box D3 (FOXD3) of OCT4. Finally, by using co-immunoprecipitation (Co-IP), confocal and real-time PCR, we demonstrated that ID1 interacts with FOXD3 to inhibit its transcriptional repression activity and therefore to upregulate OCT4 transcription and OCT4 signaling pathway. In conclusion, this study provides a new theoretical basis for the regulation mechanism of colon cancer stem cells, and the newly found protein-protein interaction of ID1-FOXD3 provides a potential drug target for the therapy of CRC.
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Objective: To identify the transcriptional activity and expression profile of PcMYB1, encoding a new R2R3-MYB transcription factor from Polygonum cuspidatum, and evaluate the biological functions of PcMYB1 in transgenic Arabidopsis thaliana. Methods: The yeast one-hybrid system assay was conducted to test the transcriptional activity of PcMYB1. The tissue-specific expression profiles of PcMYB1 and the gene expression of P. cuspidatum leaves in response to UV-C irradiation were analyzed by RT-PCR analysis. To assess the biological functions of PcMYB1, the gene was expressed in A. thaliana under the control of CaMV 35S promoter. To obtain information about the lignin composition, cross-sections of the basal part of the inflorescence stem of wild-type and transgenic A. thaliana plants were treated with Wiesner staining, and the lignin content was measured by acetyl bromide method. RT-PCR analysis was used to determine expression levels of the genes encoding the enzymes of lignin biosynthesis. Results: After expression of reporter and effector constructed in yeast, β-galactosidase assays showed that the transcriptional activation activity of VP16 domain was reduced markedly when fused to PcMYB1 protein, indicating that PcMYB1 has transcriptional repression activities. Expression pattern analysis showed that PcMYB1 was widely expressed in all tissues examined, but predominantly in leaves. PcMYB1 showed a peak of transcription at 6 h post UV-C treatment. The transgenic lines with reduction in height was 24.07% shorter than the wild-type plants. Wiesner staining of lignin in stem cross-sections revealed the typical intense red stain of secondary cell walls in wild-type plants, but less intense staining was detected in transgenic plants, and lignin accumulation was significantly decreased (about 14.81%) in transgenic plants stems. The expression of genes involved in the lignin biosynthetic pathway, including AtC4H, AtC3H, AtF5H, AtCOMT, and AtCAD, were down-regulated in transgenic lines compared to wild-type plants. Conclusion: Taken together, this study provided the evidence for the biological functions of PcMYB1 as a negative regulator of lignin pathway.
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Objective To investigate the effect of sterol regulating element binding protein (SREBP1c) and its ac-tive form (SREBP1cm) on human protein kinase R-like endoplasmic reticulum kinase (PERK).Methods Re-porter victors of PERK promoter and its truncations were constructed with pGL 3-basic and co-transfected with internal reference pRL-SV40 into cell and luciferase activity was detected .pcDNA3.1 ( -)-SREBP1c or pcDNA3.1 ( -)-SREBP1cm was co-transfected with PERK promoter transcriptional activity core regions and the detection of dual -lu-ciferase reporter gene was used to analyze the regulation of SREBP 1c/1cm on PERK promoter transcriptional activity . The expression level of PERK mRNA and protein were detected by RT-PCR and Western blot .Results PERK pro-moter and truncations were successfully constructed into pGL 3-basic, and PERK promoter core area of transcription-al activity had determined;Dual-luciferase report gene showed that SREBP 1c inhibited PERK promoter transcrip-tional activity and SREBP1cm promoted PERK promoter transcriptional activity .RT-PCR and Western blot showed that SREBP1c decreased PERK mRNA and protein expression , but SREBP1cm increased PERK mRNA and protein expression, which was consistent with the detection of dual-luciferase report gene .Conclusions SREBP1c and SREBP1cm have a opposite regulation effect on PERK promoter transcriptional activity and its expression .
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Objective To establish a quick method to probe the environmental estrogen-like compounds via fluorescent imaging analysis in high content analysis (HCA) technology.Methods HCA assays were performed to quantitatively in-vestigate the effect of collected environmental pollutants , including bisphenol A , nonylphenol , 1,2-phenylenediamine , 4-aminophenol, resorcinol, 3-aminophenol, 1,4-phenylenediamine and 2,5-diaminotoluene on nuclear granules formation ( Foci-formation) of estrogen receptor α( ERα)-EGFR( enhanced green fluorescent protein ) fusion-protein, which was se-lected as a research model in this study .The results were confirmed by the ERαtranscriptional activity by luciferase as-says.Results Compounds 1,2-phenylenediamine, 4-aminophenol, resorcinol, 3-aminophenol, 1,4-phenylenediamine and 2,5-diaminotoluene sulfate could not induce the ERα-EGFR nuclear granule formation.17-β-Estradiol, bisphenol A, or nonylphenol enhanced ERα-EGFR nuclear granule formation in a dose-dependent manner .The EC50 value was (4.17 ± 0.41) nmol/L, (1.48 ±1.79) μmol/L,or (3.70 ±0.78) μmol/L, respectively.The minimum detectable concentration was 1 nmol/L (17-β-estradiol)and 300 nmol/L (bisphenol A, nonylphenol).In luciferase tests, 17-β-estradiol, bisphe-nol A, or nonylphenol increased ERαtranscriptional activity in a dose-dependent manner ,and the EC50 value was (4.46 ±0.56) nmol/L, (2.31 ±0.21) μmol/L, or (6.60 ±0.94) μmol/L, respectively.The minimum detectable concentration was 3 nmol/L (17-β-estradiol), 300 nmol/L (bisphenol A),and 1 μmol/L (nonylphenol).Conclusion As an efficient method for ERαagonist identification , HCA assays based on the cell image phenotypes analysis can be used in quick recog -nition of environmental compounds with ER agonist-like activity.In all experimental compounds , only bisphenol A and non-ylphenol have a clear ER agonist-like activity .
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Prostate cancer is the most frequently diagnosed cancer. Although prostate tumors respond to androgen ablation therapy at an early stage, they often acquire the potential of androgen-independent growth. Elevated transcriptional activity of androgen receptor (AR) and/or signal transducer and activator of transcription-3 (STAT3) contributes to the proliferation of prostate cancer cells. In the present study, we examined the effect of resveratrol, a phytoalexin present in grapes, on the reporter gene activity of AR and STAT3 in human prostate cancer (LNCaP-FGC) cells stimulated with interleukin-6 (IL-6) and/or dihydrotestosterone (DHT). Our study revealed that resveratrol suppressed the growth of LNCaP-FGC cells in a time- and concentration-dependent manner. Whereas the AR transcriptional activity was induced by treatment with either IL-6 or DHT, the STAT3 transcriptional activity was induced only by treatment with IL-6 but not with DHT. Resveratrol significantly attenuated IL-6-induced STAT3 transcriptional activity, and DHT- or IL-6-induced AR transcriptional activity. Treatment of cells with DHT plus IL-6 significantly increased the AR transcriptional activity as compared to DHT or IL-6 treatment alone and resveratrol markedly diminished DHT plus IL-6-induced AR transcriptional activity. Furthermore, the production of prostate-specific antigen (PSA) was decreased by resveratrol in the DHT-, IL-6- or DHT plus IL-6-treated LNCaP-FGC cells. Taken together, the inhibitory effects of resveratrol on IL-6- and/or DHT-induced AR transcriptional activity in LNCaP prostate cancer cells are partly mediated through the suppression of STAT3 reporter gene activity, suggesting that resveratrol may be a promising therapeutic choice for the treatment of prostate cancer.
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Humans , Dihydrotestosterone , Genes, Reporter , Interleukin-6 , Prostate , Prostate-Specific Antigen , Prostatic Neoplasms , Receptors, Androgen , Transducers , VitisABSTRACT
Aim To investigate the NIH3T3/STAT3CA cell proliferation ability and the STAT3 transcriptional activity affected by PTPMeg2 . Methods MTT assay and xenograft nude mice model were used to investigate the NIH3 T3/STAT3 CA cell proliferation inhibited by PTPMeg2 in vitro and in vivo. Co-immunoprecipitation assay was used to measure the interaction between PT-PMeg2 and STAT3CA. STAT3 transcriptional activity was measured by dual-luciferase assay. Results The NIH3 T3/STAT3 CA cell proliferation ability was signifi-cantly inhibited by PTPMeg2 in vitro and in vivo com-pared with the control group ( P <0.05 ) . The tran-scriptional activity was increased by PTPMeg2 , but not the PTPMeg2 mutant (PTPMeg2C515S) and the ShPT-PMeg2 . Conclusion PTPMeg2 plays a role in inhibi-ting the proliferation ability of NIH3 T3/STAT3 CA cells through inhibiting the STAT3 transcriptional activity.
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Peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a nuclear receptor superfamily; members of which play key roles in the control of body metabolism principally by acting on adipose tissue. Ligands of PPARgamma, such as thiazolidinediones, are widely used in the treatment of metabolic syndromes and type 2 diabetes mellitus (T2DM). Although these drugs have potential benefits in the treatment of T2DM, they also cause unwanted side effects. Thus, understanding the molecular mechanisms governing the transcriptional activity of PPARgamma is of prime importance in the development of new selective drugs or drugs with fewer side effects. Recent advancements in molecular biology have made it possible to obtain a deeper understanding of the role of PPARgamma in body homeostasis. The transcriptional activity of PPARgamma is subject to regulation either by interacting proteins or by modification of the protein itself. New interacting partners of PPARgamma with new functions are being unveiled. In addition, post-translational modification by various cellular signals contributes to fine-tuning of the transcriptional activities of PPARgamma. In this review, we will summarize recent advancements in our understanding of the post-translational modifications of, and proteins interacting with, PPARgamma, both of which affect its transcriptional activities in relation to adipogenesis.
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Gene Expression Regulation , Homeostasis , Models, Genetic , PPAR gamma/genetics , Protein Processing, Post-Translational , Sumoylation , Transcription Factors/metabolism , UbiquitinationABSTRACT
Objective To clone the human mucin (MUC)5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. Methods The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software.After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced.By promoter deletion analysis, 3 promoter segments with diferent lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors.Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-кB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Results Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end.The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully.Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P＜0.05 vs. contro1) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. Conclusion This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression.There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.
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AIM: To construct HBx eukaryotic expression vector pEYFP-C1-X and eukaryotic expression vector pGL3-P4 driven by P4 promoter of human IGF-II gene and to investigate the effect of HBx on the transcription activity of IGF-II gene P4 promoter. METHODS: HBx gene and P4 promoters were cloned into pEYFP-C1 and pGL3-basic vectors respectively by gene recombination techniques to construct recombinant plasmids pEYFP-C1-X and pGL3-P4. HepG2 cells were transfected with pEYFP-C1-X and the resistant cell clones were selected by G418. Then methylated pGL3-P4 was transiently transfected into the above cell clones, and the transcription activity of P4 promoter was determined by dual-luciferase reporter assay system. RESULTS: (1) Aim fragments HBx gene and P4 promoter that were cloned were 465 bp and 1 246 bp, respectively and the DNA sequences were accordant with GenBank data confirmed by restricted enzyme digestion and sequencing. (2) HepG2-EYFP-X cells that expressed HBx protein were obtained. (3) Luciferase activity of methylated P4 promoter in the HepG2-EYFP-X was more than that of control cell HepG2-EYFP (P<0.01). CONCLUSION: HBx may enhance the transcription activity of the P4 promoter.
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DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-AZA-CdR on the development of preimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR (0.2, 1.0, or 5.0 μmol/L). Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0 μmol/L) or 4-cell embryos (5.0 μmol/L), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0 μmol/L) or 3/4-cell (5.0 μmol/L) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0 or 5.0 μmol/L) than that in control embryos (P < 0.05). These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An aunexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae (p < 0.05) in a dose- dependent manner, whereas no significant change was detected in 2-or 4-cell embryos (P > 0.05). The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to bc mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.
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The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.
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PURPOSE: Until recently, breast cancer carcinogenesis has not been fully understood, but the roles of estrogen receptors(ERs) and growth factor receptors(like HER2) were known to be important. Growth factors have been shown to synergize in the E2 signaling pathway, although the actual molecular mechanism remains largely unknown. To investigate the effect of HER2 overexpression on the ERE(estrogen responsive element)-mediated transcriptional activity of the ERs, this study was designed. METHODS: NIH3T3 cells, T6-17 cells (NIH3T3 cells with stably transfected with HER2), and MCF-7 cells were maintained in dextran-coated charcoal stripped 10% Dulbecco's Modified Eagle Medium (DMEM). Transient transfection of constructs (pcDNA3-ER alpha, pcDNA3-ER beta, pERE-luc, pAP-1-luciferase, and pcDNA-HER2) into each cells was performed using the Lipofectamine PLUS(TM) system. Reporter gene assays using ERE-luciferase or AP-1-luciferase were used to measure the ER transcriptional activities after treatment with estradiol (E2) and tamoxifen. RESULTS: Reporter gene assay using ERE-luciferase in both ER alpha and ER beta, showed much less responsiveness to estrogen in HER2 overexpressing T6-17 cells than in NIH3T3 cells, but there was no remarkable difference after treatment with tamoxifen. The AP-1-mediated transcriptional activity was increased in ER beta after tamoxifen treatment, but it disappeared in HER2-expressing T6-17 cells. The responsiveness to estrogen in HER2-transfected MCF-7 cells was also slightly less than in the control MCF-7 cells, and the ERE-mediated transcriptional activity of estrogen in MCF-7 cells was decreased, in a dose-dependent manner, after HER2 transfection. CONCLUSION: Coexpression of HER2 and ER seems to make cells less responsive to estrogen stimulation, and decrease the ERE-mediated transcriptional activity in both ER alpha and ERbeta. These results suggest that the expression of HER2 reduces the estrogen dependency in cell growth and eventually induces estrogen independent-growth.
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Breast Neoplasms , Carcinogenesis , Charcoal , Eagles , Estradiol , Estrogen Receptor alpha , Estrogen Receptor beta , Estrogens , Genes, Reporter , Intercellular Signaling Peptides and Proteins , MCF-7 Cells , Tamoxifen , TransfectionABSTRACT
Background and purpose:Gene therapy is a novel approach for the treatment of the patients with breast cancer. One of the effective ways is to direct transgenic expression to specific tissues or tumors with the use of tissue-specific-promoters (TSP). hTERT (human telomerase reverse transcriptase) is highly expressed in many types of cancers including breast cancer. Thus, we hypothesized that the hTERT promoter targeting with gene therapy vectors could be exploited for breast cancer. In this study, we amplified hTERT gene promoter and cloned it into the reporter vector pEGFP and pGL3-Basic. Afterwards, the specific transcription of hTERT promoter in MCF7 cells was evaluated. Methods:hTERT gene minimal promoter was PCR amplified and cloned into the reporter plasmid pEGFP-1 and pGL3-Basic.The constructs pEGFP/TERT and pGL3/TERT were transfected into MCF7 breast cancer cells and HBL100 human epithelial cells, respectively.The expression of EGFP and luciferase were investigated, respectively..Results:pEGFP/TERT and pGL3 /TERT bearing hTERT gene promoter were constructed. The specific expression of EGFP was detected in MCF7 cells while little expression of EGFP was seen in HBL100 cells.In accordance with EGFP, luciferase driven by hTERT also showed specific and high activity in MCF7 cell (RLU/U: 33784), which is 15 times higher than in HBL100 (RLU/U: 2400).Conclusions:The high transcriptional activity of hTERT gene promoter in MCF7 cell indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.
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The dynamic changes of the transcriptional activities of the nuclei of the rat liver and skeletal muscle cells were observed in the 6th, 12th, 24th, 48th, and 72nd hours after the animals were inflicted with 30% TBSA third degree burns.It was found that the incorporation of [3H]-UTP into the liver cell nuclei in vitro increased significantly in the 6th and 12th postburn hours. The peak of elevation occurred in the 6th hour postburn (from 7408 ?690 cpm/50?g DNA of normal to 10175 ? 1227 cpm/50?g DNA, P