Sp1 Promoter Research Articles

Multi-omics panoramic analysis ofHBV integration, transcriptional regulation, and epigenetic modifications in PLC/PRF/5 cell line.

The clearance or transcriptional silencing of integrated HBV DNA is crucial for achieving a functional cure in patients with chronic hepatitis Band reducing the risk of hepatocellular carcinoma development. The PLC/PRF/5cell line is commonly used as an in vitro model for studying HBV integration. In this study, we employed a range of multi-omics techniques to gain a panoramic understanding of the characteristics of HBV integration in PLC/PRF/5cells and to reveal the transcriptional regulatory mechanisms of integrated HBV DNA. Transcriptome long-read sequencing (ONT)was conducted to analyze and characterize the transcriptional activity of different HBV DNA integration sites in PLC/PRF/5cells. Additionally, we collected data related to epigenetic regulation, including whole-genome bisulfite sequencing (WGBS),histone chromatin immunoprecipitation sequencing (ChIP-seq),and assays for transposase-accessible chromatin using sequencing (ATAC-seq),to explore the potential mechanisms involved in the transcriptional regulation of integrated HBV DNA. Long-read RNA sequencing analysis revealed significant transcriptional differences at various integration sites in the PLC/PRF/5cell line, with higher HBV DNA transcription levels at integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome t (13:5). Combining long-read DNA and RNA sequencing results, we found that transcription of integrated HBV DNA generally starts downstream of the SP1, SP2, or XP promoters. ATAC-seq data confirmed that chromatin accessibility has limited influence on the transcription of integrated HBV DNA in the PLC/PRF/5cell line. Analysis ofWGBS data showed that the methylation intensity of integrated HBV DNA was highly negatively correlated with its transcription level (r = -0.8929, p = 0.0123). After AzaD treatment, the transcription level of integrated HBV DNA significantly increased, especially for the integration chr17, which had the highest level of methylation. Through ChIP-seqdata, we observed the association between histone modification of H3K4me3 and H3K9me3 with the transcription of integrated HBV DNA. Our findings suggest that the SP1, SP2 and XP in integrated HBV DNA, methylation level of surrounding host chromosome, and histone modifications affect the transcription of integrated HBV DNA in PLC/PRF/5cells. This provides important clues for future studies on the expression and regulatory mechanisms of integrated HBV.

The Dietary Flavonol Kaempferol Inhibits Epstein–Barr Virus Reactivation in Nasopharyngeal Carcinoma Cells

Kaempferol (KP, 3,4',5,7-tetrahydroxyflavone), a dietary flavonol, has anti-cancer, antioxidant, anti-inflammatory, antimicrobial, and antimutagenic functions. However, it is unknown whether kaempferol possesses anti-Epstein-Barr virus (EBV) activity. Previously, we demonstrated that inhibition of EBV reactivation represses nasopharyngeal carcinoma (NPC) tumourigenesis, suggesting the importance of identifying EBV inhibitors. In this study, Western blotting, immunofluorescence staining, and virion detection showed that kaempferol repressed EBV lytic gene protein expression and subsequent virion production. Specifically, kaempferol was found to inhibit the promoter activities of Zta and Rta (Zp and Rp) under various conditions. A survey of the mutated Zp constructs revealed that Sp1 binding regions are critical for kaempferol inhibition. Kaempferol treatment repressed Sp1 expression and decreased the activity of the Sp1 promoter, suggesting that Sp1 expression was inhibited. In conclusion, kaempferol efficiently inhibits EBV reactivation and provides a novel choice for anti-EBV therapy and cancer prevention.

Functional analysis of a viral promoter from a strawberry vein banding virus isolate from China

BackgroundPromoters are important factors affecting gene expression in cells. The driven activities of viral promoters were generally assessed to screen available promoters for transgenic and research and biotech industries. In this study, we cloned a full-length promoter from a Chinese isolate of strawberry vein banding virus (SVBV) and produced several deletion mutants for evaluation of applications in production of reporter proteins in stable transgenic plants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction. The effects of SVBV SP1, SP2, SP3, and SP4 on gene expression were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsTransient expression assays showed that the SVBV SP1 promoter and its three deletion mutants all expressed the reporter genes, albeit at very different levels. Interestingly, transcriptional activity driven by the SP1 promoter was much higher than that of the cauliflower mosaic virus (CaMV) 35S promoter. After stable transformation of the GUS gene into Nicotiana tabacum plants, SVBV SP1-driven transgene expression was approximately 2.6-fold higher than CaMV 35S promoter-driven transgene expression. In addition, GUS gene expression levels were enhanced by co-inoculation of the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing SVBV open reading frame (ORF) V or ORF VI.ConclusionsThe SVBV SP1 promoter from the Chinese isolate evaluated in this study could successfully drive transient and stable expression in plants, it was a stronger promoter than the CaMV 35S and FLt-US promoters and may be more useful for the production of stable transgenic plants.

SP1-induced lncRNA TUG1 regulates proliferation and apoptosis in islet cells of type 2 diabetes mellitus via the miR-188-3p/FGF5 axis.

To elucidate the role of TUG1 in the onset of type 2 diabetes mellitus (T2DM) and the potential mechanism. Relative levels of TUG1 and SP1 in high-fat diet animal model and high-glucose cell model were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and their correlation was analyzed. Potential binding sites in the promoter sequences of TUG1 and SP1 were predicted using the JASPAR. Their interaction was further confirmed by chromatin immunoprecipitation (ChIP) and Dual-Luciferase reporter assay. The influences of TUG1 on proliferative and apoptotic potentials in Min6 cells were examined by Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry, respectively. Subsequently, the interaction in the TUG1/miR-188-3p /FGF5 axis was similarly explored by Dual-Luciferase reporter assay. SP1 and TUG1 were downregulated in high-fat and high-glucose models, and they displayed a positive correlation. TUG1 bound E2 region in SP1 promoters. Knockdown of TUG1 inhibited proliferative rate and induced apoptosis in high-glucose-treated Min6 cells. Furthermore, the TUG1 / miR-188-3p /FGF5 axis was identified to be responsible for regulating Min6 cell functions. SP1 induces TUG1 downregulation in T2DM cell models, which further regulates proliferative and apoptotic potentials in islet cells by activating the miR-188-3p/FGF5 axis.

SP1-induced lncRNA LINC00689 overexpression contributes to osteosarcoma progression via the miR-655/SOX18 axis.

Many findings have demonstrated long noncoding RNAs (lncRNAs) as crucial regulatory molecules in the progression of osteosarcoma. The aim of this study was to explore the roles and mechanisms of LncRNA LINC00689 (LINC00689) in osteosarcoma. Differential levels of LINC00689 and miR-655 in osteosarcoma samples and cell lines were analyzed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The associations between LINC00689 expression and prognostic significance of osteosarcoma patients were analyzed using a series of statistical assays. Loss-of-function and gain-of-function assays were performed to investigate the role of LINC00689 in proliferation and metastasis in vitro. Bioinformatic assays, Luciferase report assays, and rescue assays were applied to illustrate the ceRNA mechanism network of LINC00689/miR-655/SOX18. We found that LINC00689 expression was distinctly upregulated in osteosarcoma specimens and cell lines. MiR-655 displayed a trend of remarkably decreased expression in osteosarcoma tissues. In addition, we showed that LINC00689 could specifically interact with the promoter of SP1 and activate LINC00689 transcription. Further clinical studies indicated that higher levels of LINC00689 were associated with advanced clinical stage, positively distant metastasis, and unfavorable clinical outcome. Functional studies revealed that the knockdown of LINC00689 suppressed the proliferation, migration, and invasion of osteosarcoma cells, and promoted apoptosis. Final mechanistic investigations confirmed that upregulation of LINC00689 competitively bound to miR-655 that prevented SOX18 from miRNA-mediated degradation, thus facilitating osteosarcoma progression. All our findings suggested that SP1-induced upregulation of LINC00689 promoted osteosarcoma progression by regulating miR-655/ SOX18 axis, which provided a novel insight for osteosarcoma tumorigenesis.

Interaction of Sp1 and APP promoter elucidates a mechanism for Pb2+ caused neurodegeneration

A ubiquitously expressed transcription factor, specificity protein 1 (Sp1), interacts with the amyloid precursor protein (APP) promoter and likely mediates APP expression. Promoter-interaction strengths variably regulate the level of APP expression. Here, we examined the interactions of finger 3 of Sp1 (Sp1–f3) with a DNA fragment containing the APP promoter in different ionic solutions using atomic force microscope (AFM) spectroscopy. Sp1–f3 molecules immobilized on an Si substrate were bound to the APP promoter, which was linked to the AFM tips via covalent bonds. The interactions were strongly influenced by Pb2+, considering that substituting Zn2+ with Pb2+ increased the binding affinity of Sp1 for the APP promoter. The results revealed that the enhanced interaction force facilitated APP expression and that APP overexpression could confer a high-risk for disease incidence. An increased interaction force between Sp1–f3 and the APP promoter in Pb2+ solutions was consistent with a lower binding free energy, as determined by computer-assisted analysis. The impact of Pb2+ on cell morphology and related mechanical properties were also detected by AFM. The overexpression of APP caused by the enhanced interaction force triggered actin reorganization and further resulted in an increased Young's modulus and viscosity. The correlation with single-force measurements revealed that altered cellular activities could result from alternation of Sp1–APP promoter interaction. Our AFM findings offer a new approach in understanding Pb2+ associated neurodegeneration.

HIF-1α Promotes the Metastasis of Esophageal Squamous Cell Carcinoma by Targeting SP1.

Background: In microenvironment of malignant tumors, Hypoxia-Inducible Factors (HIF), most importantly HIF-1α, play an important role in regulation of adaptive biological response to hypoxia, promoting angiogenesis and metastasis. However, the underlying mechanism that HIF-1α regulates metastasis needs to be further clarified.Methods: The expressions of HIF-1α and SP1 were detected in 182 samples of esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues by immunohistochemistry (IHC), and the correlation between the expression levels of HIF-1α and SP1 was analyzed. The expression of HIF-1α in ESCC cell lines TE1 and KYSE30 was then detected using qRT-PCR and western blot. The potential binding sites of HIF-1α on the SP1 promoter were analyzed using UCSC and JASPAR databases, verified by chromosomal immunoprecipitation (ChIP) assay and qRT-PCR. The effects of HIF-1α and SP1 on ESCC cell migration and invasion were then tested with Transwell and Matrigel experiments.Results: The expression of HIF-1α in cancer tissues is higher than adjacent normal tissues, and is correlated with metastasis, recurrence and poor prognosis. Upon silencing HIF-1α by siRNA, the invasion and migration ability of ESCC cells were significantly inhibited, which could be restored by the overexpression of SP1. Hypoxic conditions significantly increased the expression of HIF-1α and SP1 at both protein and mRNA levels in ESCC cells. HIF-1α enhanced SP1 transcription through binding to the promoter region. The expression of protein and mRNA levels of SP1 was decreased by silencing HIF-1α in cells. In contrast, overexpression of HIF-1α significantly increased the mRNA and protein levels of SP1. The expression of SP1 in ESCC was positively correlated with the protein expression of HIF-1α and poor prognosis.Conclusion: The results of our study indicate that HIF-1α promotes metastasis of ESCC by targeting SP1 in a hypoxic microenvironment. Further study on this mechanism may elucidate the possibility of HIF-1α and SP1 as new targets for the treatment of ESCC.

Characterization of IL-21-expressing recombinant hepatitis B virus (HBV) as a therapeutic agent targeting persisting HBV infection

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. In mouse models of HBV persistence, interleukin 21 (IL-21) has been identified as a potent inducer of viral clearance. Strict hepatotropism makes recombinant HBV (rHBV) vectors ideal for liver-targeting gene delivery. Previously, we established an rHBV vector termed 5c3c, which is highly replicative by itself, but requires HBV envelope proteins provided in trans to produce virions. 5c3c-based rHBV virions are capable of delivering cargo gene expression driven by HBV Sp1 promoter into infected hepatocytes. In this work, we explore the feasibility of using 5c3c-derived rHBV for liver-specific delivery of IL-21 as treatment of chronic HBV infection.Methods: 5c3c-derived rHBV replicons harboring mouse or human IL-21 genes (termed 5c3c-mIL-21 and 5c3c-hIL-21 respectively) were constructed and then tested for the production of rHBV virions in vitro and in vivo. 5c3c-mIL-21's anti-HBV effects were determined in chronic HBV mouse model. Furthermore, superinfection by rHBV virions was analysed using HBV-infected HepG2/NTCP cells and human liver chimeric mice.Results: 5c3c-mIL-21 and 5c3c-hIL-21 were efficiently replicative and produced enveloped virions when provided with envelope proteins, both in vitro and in vivo. In mouse model of HBV persistence, IL-21 expressed from injected 5c3c-mIL-21 replicon induced complete viral clearance. 5c3c-mIL-21 and 5c3c-hIL-21 virions could infect HepG2/NTCP cells and engender sustained IL-21 expression. Most importantly, IL-21-expressing rHBV virions could superinfect HBV-infected HepG2/NTCP cells and human hepatocytes in human liver chimeric mice, and engender sustained IL-21 expression and rHBV production.Conclusion: These data suggest the high potential of 5c3c-derived IL-21-expressing rHBV as a novel therapeutic against chronic HBV infection.

The regulation and interaction of colon cancer-associated transcript-1 and miR7-5p contribute to the inhibition of SP1 expression by solamargine in human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy with higher incidence in Southern China and Southeast Asia. Solamargine (SM), a steroidal alkaloid glycoside, has been shown to have anticancer properties. However, the underlying mechanism involved remains undetermined. In this study, we showed that SM inhibited the growth of NPC cells. Mechanistically, we found that solamargine decreased lncRNA colon cancer‐associated transcript‐1 (CCAT1) and increased miR7‐5p expression. There was a reciprocal interaction of CCAT1 and miR7‐5p. In addition, SM inhibited the expression of SP1 protein and promoter activity, which was strengthened by miR7‐5p mimics and inhibited by overexpressed CCAT1. MiR7‐5p could bind to 3'‐UTR of SP1 and attenuated SP1 gene expression. Exogenously expressed SP1 feedback resisted SM‐increased miR7‐5p expression and more importantly reversed SM‐inhibited growth of NPC cells. Finally, SM inhibited NPC tumor growth in vivo. Collectively, our results show that SM inhibits the growth of NPC cells through reciprocal regulation of CCAT1 and miR7‐5p, followed by inhibition of SP1 gene expression in vitro and in vivo. The interregulation and correlation among CCAT1, miR7‐5p and SP1, and the feedback regulatory loop unveil the novel molecular mechanism underlying the overall responses of SM in anti‐NPC.

Dopaminergic neuron injury in Parkinson's disease is mitigated by interfering lncRNA SNHG14 expression to regulate the miR-133b/ α-synuclein pathway.

This study explored the influence of long non-coding RNA (lncRNA) SNHG14 on α-synuclein (α-syn) expression and Parkinson’s disease (PD) pathogenesis. Firstly, we found that the expression level of SNHG14 was elevated in brain tissues of PD mice. In MN9D cells, the rotenone treatment (1μmol/L) enhanced the binding between transcriptional factor SP-1 and SNHG14 promoter, thus promoting SNHG14 expression. Interference of SNHG14 ameliorated the DA neuron injury induced by rotenone. Next, we found an interaction between SNHG14 and miR-133b. Further study showed that miR-133b down-regulated α-syn expression by targeting its 3’-UTR of mRNA and SNHG14 could reverse the negative effect of miR-133b on α-syn expression. Interference of SNHG14 reduced rotenone-induced DA neuron damage through miR-133b in MN9D cells and α-syn was responsible for the protective effect of miR-133b. Similarly, interference of SNHG14 mitigated neuron injury in PD mouse model. All in all, silence of SNHG14 mitigates dopaminergic neuron injury by down-regulating α-syn via targeting miR-133b, which contributes to improving PD.

SP1-mediated upregulation of lncRNA SNHG4 functions as a ceRNA for miR-377 to facilitate prostate cancer progression through regulation of ZIC5.

Long noncoding RNAs (lncRNAs) have been demonstrated to serve distinct roles in human tumorigenesis. Previous studies have found that lncRNA small nucleolar RNA host gene 4 (SNHG4) was dysregulated in several tumors. However, the expression, clinical significances, and action mechanisms of SNHG4 in prostate cancer (PCa) are still unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect SNHG4 expression in tissue samples and PCa cells. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine, clonogenic formation, wound-healing, and transwell invasion assays were, respectively, used to evaluate cell proliferation, colony formation ability, migration, and invasion. Flow cytometric analysis was applied to assess cell apoptosis. Chromatin immunoprecipitation assays were conducted to determine the binding between SP1 and SNHG4 promoter. Luciferase reporter assay, qRT-PCR, and western blot analysis were carried out to explore and confirm the interaction among SNHG4, miR-377, and ZIC5. SNHG4 was highly expressed in PCa and its upregulation was induced by transcription factor SP1. The high levels of SNHG4 were distinctly associated with tumor stage, lymph node metastasis, and reduced overall survival of patients with PCa. SNHG4 knockdown inhibited the growth, migration, and invasion of PCa cells. In addition, miR-377 was a target of SNHG4 and ZIC5 was a target gene of miR-377 in PCa. SNHG4 promoted ZIC5-mediated growth and metastasis through modulating miR-377. Our findings illuminate how SNHG4 formed a regulatory network to display a tumor-promotive effect in PCa and revealed that SNHG4 may be a novel therapeutic target and prognostic marker for patients with PCa.

The mechanism of SP1/p300 complex promotes proliferation of multiple myeloma cells through regulating IQGAP1 transcription

Our previous research had firstly shown that MM cells overexpressed IQGAP1 gene and activated Ras/Raf/MEK/ERK pathway. But the mechanism of IQGAP1 overexpression and IQGAP1 gene transcription regulation remains uncertain. The mechanism of IQGAP1 overexpression and transcriptional regulation of IQGAP1 gene in myeloma cells was explored in the study. Through bioinformatics analysis and prediction we predicted and screened transcription factor Sp1 as a possible upstream regulator of IQGAP1.The proliferation, cell cycle and downstream ERK1/2 and p-ERK1/2 proteins were detected after siRNA-IQGAP1 was transfected to myeloma cells. The expression of Sp1, p300, IQGAP1, p-ERK1/2 and ERK1/2 were detected after Sp1 and p300 were inhibited or overexpressed respectively. The dual-luciferase reporter system was used to detect the activity of IQGAP1 gene promoter. CHIP was used to detect the binding of the Sp1 and IQGAP1 promoter regions.CO-IP was used to explore the interaction between Sp1 and p300.The mRNA expression levels of Sp1,p300 and IQGAP1 of the myeloma patients were detected, and the correlation analysis of their mRNA expression levels were carried out. The results showed IQGAP1-siRNA inhibits cell proliferation, cell cycle, IQGAP1 expression and phosphorylation of ERK1/2 protein. Inhibition of Sp1 or p300 down-regulated ERK1/2 and IQGAP1 expression; overexpression of Sp1 or p300 up-regulated ERK1/2 and IQGAP1 expression; Sp1 and p300 had a positive regulation effect on IQGAP1.Over expression of Sp1 or p300 significantly increased activity of IQGAP1 gene promoter. The transcription factor Sp1 plays a regulatory role in the IQGAP1 promoter region. There is an interaction between Sp1 and p300 in myeloma cells. The mRNA expression levels of Sp1, IQGAP1 and p300 in MM samples showed a positive correlation. In summary IQGAP1 is required for cell proliferation in MM cells, and the transcription of Sp1/p300 complex regulates expression of IQGAP1 gene.

Long noncoding RNA SAMMSON promotes papillary thyroid carcinoma progression through p300/Sp1 axis and serves as a novel diagnostic and prognostic biomarker.

Accumulating evidence suggests that long noncoding RNA (lncRNA) plays a fundamental role in cancer progression. However, its biological function in papillary thyroid carcinoma (PTC) has not been fully elucidated. Here, we deciphered the essential role of lncRNA SAMMSON in PTC. SAMMSON was identified to be notably upregulated in PTC cells, tissues, and plasma, and could be used as an effective diagnostic and prognostic biomarker for PTC patients. Knockdown of SAMMSON significantly inhibited PTC cell proliferation and invasion in vitro as well as tumorigenicity and metastasis in vivo. Mechanistically, SAMMSON was transcriptionally elevated by oncogenic Sp1, in turn, upregulated SAMMSON was capable of acting as a scaffold to recruit p300 to increase H3K9ac and H3K27ac levels on Sp1 promoter region, leading to transcriptional activation of Sp1, thereby facilitating PTC progression. Taken together, our data demonstrate that SAMMSON is an oncogenic lncRNA and unveil the crucial role of SAMMSON/Sp1 positive feedback loop in tumorigenesis and aggressiveness of PTC.

Dopaminergic Neuron Injury in Parkinson Disease Is Mitigated by Interfering lncRNA SNHG14 Expression to Regulate the miR-133b/α-Synuclein Pathway

Background: Abundant α-synuclein (α-syn) protein accumulation is the pathological hallmark of Parkinson disease (PD) and it induces dopaminergic (DA) neuron apoptosis. This study explored the influence of long non-coding RNA (lncRNA) SNHG14 on α-syn expression and PD pathogenesis. Methods: PD model was established in mouse with a stereotaxic surgery. In the brain tissue of PD mice, the expression levels of SNHG14, miR-133b, α-syn and SP-1 protein were determined. Chromatin immunoprecipitation (ChIP) assay was performed to validate the binding between SP-1 and SNHG14 promoter. DA neuron viability was analyzed with CCk-8 assay. Interplay between SNHG14 and miR-133b was examined with RNA immunoprecipitation (RIP) and RNA pull-down assay. Regulatory relationship between miR-133b and α-syn was verified through dual luciferase reporter assay. Results: Compared with the sham group (n=7), the expression of SNHG14, αsyn and SP-1 protein was increased in PD mice model (n=7), while miR-133b was down-regulated. Binding between transcriptional factor SP-1 and SNHG14 promoter was enhanced by rotenone, and promoted SNHG14 expression. Interference of SNHG14 ameliorated the DA neuron injury induced by rotenone. Interaction between SNHG14 and miR-133b was identified in clonal mesencephalic DA cell line MN9D cells. MiR-133b negatively regulated α-syn expression by targeting its 3'-UTR of mRNA. SNHG14 regulated α-syn expression via targeting miR-133b. Interference of SNHG14 improved DA neuron activity that damaged by rotenone through miR-133b. Interference of SNHG14 mitigated neuron injury in PD mouse model. Conclusion: Interference of lncRNA SNHG14 expression mitigates dopaminergic neuron injury by down-regulating α-syn via targeting miR133b, which contributes to improving PD pathological state. Funding Statement: This study was supported by the grants from the Youth Entrepreneurship Foundation of the First Affiliated Hospital of Zhengzhou University. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

MiR-29b/Sp1/FUT4 axis modulates the malignancy of leukemia stem cells by regulating fucosylation via Wnt/\u03b2-catenin pathway in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is initiated and maintained by a unique, small subset of leukemia stem cells (LSCs). LSCs are characterized by unrestricted self-renewal and contribute to the malignancy of leukemia. Aberrant protein fucosylation is associated with AML progression. However, it is still less understood that the miR-29b/Sp1/FUT4 crosstalk involved in the fucosylation-mediated LSCs malignancy in AML.MethodsAML cell lines were sorted by magnetic microbeads to obtain the CD34 + CD38- sub-population. The key biomarkers for LSCs were identified by flow cytometry. Fucosyltransferase genes were screened by qRT-PCR, and FUT4 was focused. Effect of FUT4 on LSCs malignancy was determined by CCK8 assay, sphere formation assay, immunofluorescence staining, apoptosis and in vivo xenografts experiments. The linkage of FUT4 promoter and Sp1 was confirmed by dual-luciferase reporter gene assay. ChIP-PCR assay was used to show the directly binding of Sp1 and FUT4 promoter. Activity of Wnt//β-catenin pathway was determined by western blot. Overall survival curves were diagrammed by Kaplan-Meier analysis.ResultsHere, the expressional profiles of 11 fucosyltransferase genes were different comparing LSCs and non-LSCs of KG-1a and MOLM13 cells, whereas CD34 + CD38- cells exhibited higher expression of FUT4. Functionally, alteration of FUT4 in CD34 + CD38- cells modulated LSCs malignant behaviors both in vitro and in vivo. Transcriptional inhibitor actinomycin D (Act D) or translational inhibitor cycloheximide (CHX) prevented LSCs progression, and Sp1 was identified as the efficient regulator of FUT4 transcription. Moreover, miR-29b directly affected the binding of Sp1 and FUT4 promoter region, which further mediated LSCs proliferation, apoptosis and drug-resistance through fucosylated-CD44 via activation of Wnt/β-catenin pathway. Clinically, Sp1 and FUT4 were up-regulated and positively correlated with poor overall survival of AML patients.ConclusionThese data indicated that miR-29b/Sp1/FUT4 axis promoted the malignant behaviors of LSCs by regulating fucosylated CD44 via Wnt/β-catenin pathway. Identifying LSCs surface markers and targeting LSCs were important for the development of potential therapies in AML.

SP1-SYNE1-AS1-miR-525-5p feedback loop regulates Ang-II-induced cardiac hypertrophy.

Cardiac hypertrophy (CH) has become a huge threat to human health. Recent years, long noncoding RNAs (lncRNAs) have been studied in human diseases, including CH. According to bioinformatics analysis, 10 lncRNAs possibly involved in the progression of CH were screened out. Among which, lncRNA SYNE1 antisense RNA 1 (SYNE1-AS1) could be upregulated by Angiotensin II (Ang-II) in cardiomyocytes. Thus, we chose SYNE1-AS1 to do further study. To identify the biological function of SYNE1-AS1 in CH, SYNE1-AS1 was silenced in Ang-II-induced cardiomyocytes. Results of immunofluorescence staining demonstrated that increased cell surface area in Ang-II-induced cardiomyocytes was reduced by SYNE1-AS1 knockdown. Moreover, the hypertrophic responses were attenuated by SYNE1-AS1 knockdown. Mechanically, SYNE1-AS1 positively regulated Sp1 transcription factor (SP1) by sponging microRNA-525-5p (miR-525-5p). On the basis of previous reports, SP1 can transcriptionally activate lncRNAs. Therefore, we investigated the interaction between SP1 and SYNE1-AS1 promoter. Intriguingly, SYNE1-AS1 was activated by SP1. At last, rescue assays demonstrated the function of SP1-SYNE1-AS1 axis in CH. In conclusion, SP1-induced upregulation of lncRNA SYNE1-AS1 promoted CH via miR-525-5p/SP1 axis.

Curcumin enhances cisplatin sensitivity of human NSCLC cell lines through influencing Cu-Sp1-CTR1 regulatory loop

BackgroundCurcumin is a naturally occurring polyphenol which has been demonstrated to possess diverse biological activities. We previously reported that curcumin is a biologically active copper chelator with antitumor activity. Copper transporter 1 (CTR1) on the plasma membrane of eukaryotic cells mediates both copper as well as anticancer drug cisplatin uptake. PurposeThis study aims to investigate whether curcumin enhances cisplatin sensitivity of human non-small cell lung cancer (NSCLC) through influencing Cu-Sp1-CTR1 regulatory loop. MethodsThe combination effect of curcumin and cisplatin on cell proliferation and apoptosis was determined in vitro and in vivo. Platinum level in A549 cells and tumor tissue was measured by atomic absorption spectrometry (AAS). The binding ability of specificity protein 1 (Sp1) to CTR1 and Sp1 promoters was detected by ChIP assay and luciferase reporter assay system. ResultsHere we show that combined curcumin and cisplatin treatment markedly inhibited A549 cells proliferation and induced its apoptosis. Using a mouse model of A549 xenograft, we demonstrated that curcumin inhibits copper influx and increases uptake of platinum ion in tumor. Curcumin treatment enhances the binding of Sp1 to CTR1 and Sp1 promoters, thus induces CTR1 expression and chemosensitization to cisplatin treatment. This process is regulated by the Cu-Sp1-CTR1 regulatory loop. Moreover, the enhancement mediated by curcumin on cisplatin therapeutic efficacy in cultured human NSCLC cell lines (A549, H460, H1299) was dependent on CTR1. ConclusionsOur results demonstrated copper chelator curcumin enhances the benefits of platinum-containing chemotherapeutic agents and CTR1 could be a promising therapeutic target for non-small cell lung cancer treatment.

Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway.

The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of gene expression and replication of hepatitis B virus (HBV) is not fully understood. Previous reports have documented the induction of the expression of viral large surface protein (LHBs) by HNF1α through activating viral Sp1 promoter. Large amount of LHBs can block the secretion of hepatitis B surface antigen (HBsAg). Here we found that HNF1α overexpression inhibited HBV gene expression and replication in Huh7 cells, resulting in marked decreases in HBsAg, hepatitis B e antigen (HBeAg) and virion productions. In contrast, knockdown of endogenous HNF1α expression enhanced viral gene expression and replication. This HNF1α-mediated inhibition did not depend on LHBs. Instead, HNF1α promoted the expression of NF-κB p65 and slowed p65 protein degradation, leading to nuclear accumulation of p65 and activation of the NF-κB signaling, which in turn inhibited HBV gene expression and replication. The inhibitor of the NF-κB signaling, IκBα-SR, could abrogate this HNF1α-mediated inhibition. While the dimerization domain of HNF1α was dispensable for the induction of LHBs expression, all the domains of HNF1α was required for the inhibition of HBV gene expression. Our findings identify a novel role of HNF1α in the regulation of HBV gene expression and replication.

Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-activated Receptor-β/δ Modulation of SP1 Promoter Occupancy

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparβ/δ+/+ mice but not in Pparβ/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARβ/δ, was up-regulated in Pparβ/δ+/+ liver following ethanol exposure, but not in Pparβ/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARβ/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARβ/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARβ/δ attenuates ethanol-induced Cyp2b10 expression in Pparβ/δ+/+ liver but not in Pparβ/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARβ/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARβ/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

Imperfect Symmetry of Sp1 and Core Promoter Sequences Regulates Early and Late Virus Gene Expression of the Bidirectional BK Polyomavirus Noncoding Control Region.

Polyomaviridae currently comprise more than 70 members, including 13 human polyomaviruses (PyVs), all of which share a bidirectional genome organization mediated by the NCCR, which determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centered around TATA and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by the directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised toward EVGR expression, which can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag, which is the major EVGR protein, drives viral genome replication, renders subsequent LVGR expression independently of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation, and virulence in their natural hosts.