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E2F family participates in most human malignancies by activating the transcription of the cell cycle-related genes. Whereas, as a specifical atypical member of this family, E2F7 was described as a repressor against its downstream genes and exerted oscillatory and controversial functions in cancers. Our previous study identified a molecular interaction promoting hepatocellular carcinoma (HCC) growth induced by SOX4 and Anillin. Meanwhile, we preliminarily identified SP1 as the upstream activator of SOX4. Intriguingly, we observed that the repressive E2F7 presents a remarkable high expression in HCC, and is positively correlated and involved in the same pathway with the potentially SP1/SOX4/Anillin axis. However, their exact interaction or mechanism controlling tumor progress between these genes has not been illustrated. Thus, we focused on this point in this study and attempted to improve the potential regulating axis in HCC cell proliferation and tumor growth for promoting tumor prevention and control. The expression profile of E2F7 in HCC tissues and tumor cells was detected along with the related candidate genes, through real-time quantitative polymerase chain reaction assay, the Western blot analysis, and the immunohistochemistry assay, combined with bioinformatics analysis of the HCC information from the the Cancer Genome Altas and Gene Expression Omnibus data sets. The correlation between E2F7 and HCC patients' clinicopathologic features was explored. Gain-of and loss-of-function assays were conducted both in vitro and in vivo along with the rescue experiment, for revealing the relative genes' functions in HCC progress. The ChIP and the dual-luciferase reporter assays were performed to verify the transcriptional regulating profile between E2F7 and SP1/SOX4/Anillin axis. E2F7 was upregulated in HCC and significantly correlated with SP1/SOX4/Anillin axis. High E2F7 expression is associated with dismal clinicopathologic features and poor survival of the patients. E2F7 depletion potently impaired SP1/SOX4/Anillin expression and significantly inhibited HCC growth. Furthermore, intensive exploration demonstrated that E2F7 preserves high SP1 levels by abrogating miR-383-5p in a transcriptional way. Atypical E2F7 is an important repressive transcription factor commonly upregulated in the HCC environment. E2F7 facilitates HCC growth by repressing miR-383-5p transcription and sequentially promoting SP1/SOX4/Anillin axis. Our findings provide us with probable targets for HCC prevention and therapeutic treatment.

Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from −273 to −10 base pairs upstream of the transcription start site identified by 5'‐RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB‐downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression., Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT.

Transcription factor SP1 could manipulate pathways involved in ovarian cancer progression. LncRNAs are involved in SP1‐mediated tumorigenesis. LncRNA DANCR could promote metastasis of ovarian cancer. However, the regulatory function and involvement of SP1‐induced lncRNA DANCR in ovarian cancer remain elusive. Data from this study showed that SP1 was up‐regulated in ovarian cancer tissues and cells (CAOV3, SKOV3, A2780), and SP1 could bind to the promoter region of DANCR through chromatin immunoprecipitation and leuciferase activity assays. Therefore, DANCR was transcriptionally induced by SP1 in ovarian cancer tissues and cells (CAOV3, SKOV3, A2780). Functionally, reduced expression of DANCR suppressed cell viability, migration and invasion of CAOV3, while enhanced DANCR expression contributed to SKOV3 growth. Over‐expression of SP1 reversed the suppressive effects of DANCR interference on ovarian cancer progression. In conclusion, SP1‐induced DANCR contributed to oncogenic potential of ovarian cancer, suggesting a promising therapeutic target for ovarian cancer.

Long noncoding RNAs (lncRNAs) have drawn growing attention due to their regulatory roles in various diseases, including tumors. Recently, lncRNA FOXD3 antisense RNA 1 (FOXD3-AS1) was shown to be overexpressed in colon adenocarcinoma and glioma, exerting oncogenic functions. However, its expression and effects in cervical cancer (CC) remained unknown. In this research, our group first reported that the levels of FOXD3-AS1 were distinctly elevated in CC samples and cell lines. The distinct upregulation of FOXD3-AS1 was associated with lymphatic invasion, distant metastasis, and International Federation of Gynecology and Obstetrics stage, and also predicted poor clinical results of CC patients. Next, transcription factor SP1 was demonstrated to resulting in the upregulation of FOXD3-AS1 in CC. Functional assays indicated that knockdown of FOXD3-AS1 distinctly suppressed CC progression via affecting cell proliferation, cell apoptosis, and metastasis. Moreover, mechanistic studies suggested that FOXD3-AS1 acted as an endogenous sponge by directly binding miR-296-5p, resulting in the suppression of miR-296-5p. In addition, we also reported that high mobility group A, a direct target of miR-296-5p, could mediate the tumor-promotive effects that FOXD3-AS1 displayed. Overall, our present study might help to lead a better understanding of the pathogenesis of CC, provide a novel possible tumor biomarker, and probe the feasibility of lncRNA-directed treatments for CC.

Toxoplasma gondii excreted‐secreted antigens (ESA) could result in adverse outcomes of pregnancy including abortion, stillbirth, foetal infection or teratogenesis in mice during early stage of pregnancy. Defective generation or function of regulatory T cells (Tregs) may account for those adverse pregnancy outcomes. Forkhead box p3 (Foxp3), which is the key transcriptional factor of Tregs, modulates its development and maintains inhibitory function. We previously demonstrated that ESA inhibited Foxp3 expression by attenuating transforming growth factor β RII/Smad2/Smad3/Smad4 pathway. In this study, we propose to study the role of ESA on the activity of Foxp3 promoter and explore potential mechanisms. We demonstrated that ESA suppressed Foxp3 promoter activity using dual‐luciferase reporter assay. ESA functioned at −443/−96 region of Foxp3 promoter to suppress its activity using truncated fragments of Foxp3 promoter. Further analysis revealed that suppressive role of ESA on Foxp3 promoter activity is related to specificity protein 1 (SP1). Transfection of expression plasmid of pcDNA3.1‐SP1 could restore the down‐regulation of Foxp3 induced by ESA. In conclusion, this study provides a new mechanism by which ESA could inhibit the Foxp3 promoter activity via SP1.

Long noncoding RNAs (lncRNAs) were identified as a vital part in the development and progression of cancer in recent years. Colorectal neoplasia differentially expressed (CRNDE), a lncRNA, functions as an oncogene in some malignant neoplasias, but its role in the progression of osteosarcoma (OS) is still poorly understood. To dissect the difference in the expression of CRNDE, quantitative real-time polymerase chain reaction was utilized to evaluate it in OS tissues and cell lines (U2OS, MG63, and MNNG/HOS) compared with that in the adjacent normal tissues/osteoblast cells (hFOB1.19). The role of CRNDE in OS lines was assessed using Cell Counting Kit-8, colony formation, 5-ethynyl-2'-deoxyuridine staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, flow cytometry, Transwell assays, and Western blot, respectively. The results demonstrated that the expression of CRNDE was high in OS tissues and cell lines, and partly induced by SP1. CRNDE knockdown attenuated OS cell proliferation and invasion and induced apoptosis and G0/G1 arrest. Moreover, the expression of mesenchymal markers N-cadherin, Vimentin and Snail were downregulated, while the expression of epithelial markers E-cadherin and ZO-1 were conversely upregulated due to CRNDE knockdown. The mechanistic investigations showed that CRNDE promoted glycogen synthase kinase-3β phosphorylation to activate the Wnt/β-catenin pathway. The results suggested that lncRNA CRNDE indeed contributed to OS proliferation, invasion, and epithelial-mesenchymal transition, working as an oncogene, demonstrating that lncRNA CRNDE may be a valid therapeutic target for the OS.

Bone metastasis is associated with cancer‐related death in patients with prostate cancer (PCa). Long noncoding RNAs (lncRNAs) play critical roles in tumor progression of PCa. Nevertheless, the biological function of lncRNAs in PCa bone metastasis remains unclear. PCAT7 was identified as a bone metastasis‐related lncRNA via analyzing TCGA dataset. Meanwhile, PCAT7 was found to be elevated in primary PCa tissues with bone metastasis and associated with bone metastasis status and poor prognosis of patients with PCa. Functionally, our results reveal that PCAT7 overexpression promotes PCa bone metastasis in vivo, as well as migration, invasion, and EMT of PCa cells in vitro; on the contrary, PCAT7 knockdown has an inverse effect. Mechanistically, PCAT7 activates TGF‐β/SMAD signaling by upregulating TGFBR1 expression via sponging miR‐324‐5p. In turn, TGF‐β signaling forms a positive feedback loop with PCAT7 via SMAD3/SP1 complex‐induced PCAT7 upregulation. Finally, the clinical positive correlation between PCAT7 and TGFBR1 and TGF‐β signaling activity, and the negative association with miR‐324‐5p are further demonstrated in PCa tissues and clinical primary PCa cells. This study reveals a novel mechanism that is responsible for the constitutive activation of TGF‐β signaling in PCa bone metastasis, implying that PCAT7 can act as a potential therapeutic target against bone metastasis of PCa via disrupting the constitutive active loop between PCAT7 and TGF‐β signaling., lncRNA PCAT7 is upregulated in primary prostate cancer tissues with bone metastasis. PCAT7 activates TGF‐β/SMAD signaling by upregulating TGFBR1 expression via sponging miR‐324‐5p. In turn, TGF‐β signaling forms a positive feedback loop with PCAT7 via SMAD3/SP1 complex‐induced PCAT7 upregulation. This feedback loop promotes prostate cancer bone metastasis.

Background As a classic oral drug used in diabetic patients, metformin has exhibited an anticancer role in many types of cancers in recent years. Here, we aimed to investigate the role of metformin in the growth of breast cancer and its novel targets. Methods A cell viability assay was utilized to examine the inhibitory effect of metformin on proliferation of breast cancer cells. Western blotting and RT-PCR assays were used to detect the regulation of metformin on the expression of oncogenic HMGA2. The luciferase reporter vector of HMGA2 promoter was constructed. A luciferase reporter gene assay was performed to analyze the effect of metformin on HMGA2 promoter activity in breast cancer cells. Chromatin immunoprecipitation (ChIP) assay was performed to show the binding of Sp1 to HMGA2 promoter in breast cancer cells with or without metformin treatment. The function of metformin-regulated HMGA2 in breast cancer growth was tested using a cell viability assay. Results Cell proliferation was obviously inhibited in breast cancer cells treated with different concentrations of metformin. The level of mRNA and protein of HMGA2 was significantly reduced by metformin in the cells. Mechanistically, metformin was able to inactivate the HMGA2 promoter through downregulating transcription factor Sp1 in the cells. In terms of function, treatment with metformin suppressed the proliferation of breast cancer cells and overexpressed HMGA2 reversed the inhibition of cell proliferation mediated by metformin. Conclusion Metformin resists the growth of breast cancer through targeting Sp1/HMGA2 signal.

MicroRNAs (miRNAs) have been shown to have complicated implications in the pathogenesis of Parkinson's disease (PD). However, the role of miR-29c and the underlying mechanism in the development of PD remain not well understood. In this work, the MPTP-treated mice or MPP+ -intoxicated SH-SY5Y cells were established as an in vivo or in vitro PD model. Then the specific agomir of miR-29c was employed to examine its biological function on PD progress. We found that miR-29c was down-expressed but SP1 was high-expressed in substantia nigra pars compacta (SNpc) of MPTP-induced PD mice. Overexpression of miR-29c attenuated dopaminergic neuron loss and α-synuclein accumulation in SNpc of PD mice. Furthermore, the increments of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and TUNEL-positive apoptotic cells in MPTP-treated mice were ameliorated by miR-29c. Similarly, in SH-SY5Y cell models of PD, we also found that miR-29c inhibited inflammatory cytokine production, reduced apoptotic rate and suppressed pro-apoptotic regulator activity. In addition, the increased expression of SP1 in PD models was found to be inhibited by miR-29c. Luciferase reporter assay confirmed that SP1 was complementary with miR-29c. Knockdown of SP1 with siRNA restored α-synuclein accumulation, inflammation and apoptosis in MPP+ -induced SH-SY5Y cells. Collectively, this current work presents that miR-29c may directly target SP1 to protect against the neuroinflammatory and apoptotic responses in PD, providing a potential biomarker for PD diagnosis and treatment.

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.

JNK activity has been implicated in the malignant proliferation, invasion and drug-resistance of glioma cells (GCs), but the molecular mechanisms underlying JNK activation are currently unknown. Here, we reported that MKK7, not MKK4, directly activates JNK in GCs and exerts oncogenic effects on tumor formation. Notably, MKK7 expression in glioma tissues was closely correlated with the grade of the glioma and JNK/c-Jun activation. Mechanistically, MKK7 transcription critically depends on the complexes formed by HDAC4 and the transcriptional factors SP1 and Krüppel-like factor-5 (KLF5), wherein HDAC4 directly deacetylates both SP1 and KLF5 and synergistically upregulates MKK7 transcription through two SP1 sites located on its promoter. In contrast, the increases in acetylated-SP1 and acetylated-KLF5 after HDAC4 inhibition switched to transcriptionally suppress MKK7. Selective inhibition of HDAC4 by LMK235, siRNAs or blockage of SP1 and KLF5 by the ectopic dominant-negative SP1 greatly reduced the malignant capacity of GCs. Furthermore, suppression of both MKK7 expression and JNK/c-Jun activities was involved in the tumor-growth inhibitory effects induced by LMK235 in U87-xenograft mice. Interestingly, HDAC4 is highly expressed in glioma tissues, and the rate of HDAC4 nuclear import is closely correlated with glioma grade, as well as with MKK7 expression. Collectively, these findings demonstrated that highly expressed MKK7 contributes to JNK/c-Jun signaling-mediated glioma formation. MKK7 transcription, regulated by SP1 and KLF5, critically depends on HDAC4 activity, and inhibition of HDAC4 presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c-Jun signaling in GCs.

Non-small-cell lung cancer (NSCLC) is a kind of lung cancer with high incidence and poor outcomes all over the world. Studies have validated that the upregulation of long noncoding RNA LINC00657 is related to several cancers. Nevertheless, the underlying regulatory mechanism of LINC00657 in NSCLC has not been well elucidated. In the present study, quantitative reverse-transcription polymerase chain reaction (RT-qPCR) revealed that LINC00657 level was apparently elevated in NSCLC cells. Loss-of function assays demonstrated that LINC00657 silence retarded cell proliferation and migration in NSCLC cells. Moreover, the chromatin immunoprecipitation result identified the transcription factor SP1 could bind with LINC00657 promoter, and RT-qPCR proved SP1 positively regulated LINC00657 expression in NSCLC cells. In addition, the mechanistic investigations unveiled that LINC00657 was an endogenous sponge of miR-26b-5p and therefore boosted the expression of copper metabolism MURR1 domain-containing 8 (COMMD8), one of the targets of miR-26b-5p. Besides, miR-26b-5p could negatively regulate LINC00657 or COMMD8 in NSCLC cells. With the application of rescue assays, we uncovered that overexpression of COMMD8 partly mitigated the impairment of LINC00657 repression on NSCLC cell proliferation and migration. Together, our study illustrated that SP1-stimulated LINC00657 promoted NSCLC progression through targeting miR-26b-5p/COMMD8 axis, offering a novel potential therapeutic target for NSCLC.

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.

Influenza virus cross-species transmission is restricted by the host, but viruses overcome this restriction by accumulating mutations which allow them to adapt to a new host. Among the many factors which facilitate virus host adaptation, polymerase basic protein 2 (PB2) 627 plays an important role, although the underlying molecular mechanism has not been fully understood. In a previous study, we found that histone H1.2 (encoded by HIST1H1C) regulates human or avian influenza virus replication in different ways, indicating that it might be involved in virus host adaptation. Herein, we found that HIST1H1C expression, phosphorylation and methylation levels are decreased when infected with H1N1 influenza virus and increased when infected with H5N1 influenza virus. Overexpressing the eight gene segments of the influenza virus, we found that only PB2 significantly affects HIST1H1C expression and modifications. Since the 627 site is different between the H5N1 and H1N1 influenza viruses we constructed PB2-627E (avian variant) and PB2-627K (human variant) mutant viruses, and observed that the effects of the wild-type and the mutant viruses on HIST1H1C expression and modifications are the opposite of one another. Further analysis showed that influenza virus PB2-627 regulates HIST1H1C expression via Sp1, and specifically that PB2-627K down-regulates Sp1 and HIST1H1C while PB2-627E up-regulates Sp1 and HIST1H1C. In addition, HIST1H1C can feedback regulate DNA-dependent protein kinase and euchromatic histone-lysine N-methyltransferase 1/2, leading to altered HIST1H1C phosphorylation and methylation levels, and affecting influenza virus replication accordingly. In summary, this study illustrates the mechanism of PB2-627E/K-mediated regulation of influenza virus host adaptation.

Cytochrome P450 1A1 (CYP1A1) is abundant in the kidney, liver, and intestine and is involved in the phase I metabolism of numerous endogenous and exogenous compounds. Therefore, exploring the regulatory mechanism of its basal expression in humans is particularly important to understand the bioactivation of several procarcinogens to their carcinogenic derivatives. Site-specific mutagenesis and deletion of the transcription factor binding site determined the core cis-acting elements in the human CYP1A1 proximal and distal promoter regions. The proximal promoter region [overlapping xenobiotic-responsive element (XRE) and GC box sequences] determined the basal expression of CYP1A1. In human hepatocellular carcinoma cells (HepG2) with aryl hydrocarbon receptor (AhR) or specificity protein 1 (Sp1) knockdown, we confirmed that AhR and Sp1 are involved in basal CYP1A1 expression. In HepG2 cells overexpressing either AhR or Sp1, AhR determined the proximal transactivation of basal CYP1A1 expression. Via DNA affinity precipitation assays and ChIP, we found that AhR bound to the promoter and recruited Sp1 to transactivate CYP1A1 expression. The coordinated interaction between Sp1 and AhR was identified to be DNA mediated. Our work revealed a basal regulatory mechanism of an interesting human gene by which AhR interacts with Sp1 through DNA and recruits Sp1 to regulate basal CYP1A1 expression.

Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA‐mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin‐induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor‐1α (HIF‐1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR‐125b‐5p and miR‐146a‐5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF‐1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co‐expressed and up‐regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF‐1α significantly inhibited SP1 and ROBO4, whereas SP1 down‐regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR‐125b‐5p and miR‐146a‐5p were down‐regulated by hyperglycaemia and/or hypoxia. Up‐regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR‐125b‐5p targeted SP1 and ROBO4, and miR‐146a‐5p targeted HIF‐1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF‐1α/SP1/ROBO4 or up‐regulation of miR‐125b‐5p/miR‐146a‐5p. In general, our results identified a novel mechanism that miR‐125b‐5p/miR‐146a‐5p targeting HIF‐1α/SP1‐dependent ROBO4 expression could retard DR progression.

Objective DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. Methods DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Results Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4's promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4's promoter decreased the transactivation of SP1 on KLF4. Conclusion We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4's promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1's binding and transcriptional regulation on KLF4.

Mounting evidence suggests the vital roles of long noncoding RNA (lncRNAs) in the glioma. However, the role of LINC00511 in gliomagenesis is still uncovered. Here, in this study, we aim to investigate the effects of LINC00511 on the glioma cancer phenotype and its deepgoing mechanism. Results indicated that LINC00511 was up‐regulated in glioma tissues and cell lines, moreover its overexpression positively correlated with the poor prognosis and advanced pathological stages. For the upstream regulation, LINC00511 was epigenetically up‐regulated by transcription factor specificity protein 1 (SP1). Gain and loss of functional experiments demonstrated that LINC00511 promoted the proliferation and invasion of glioma cells in vitro. The knockdown of LINC00511 repressed the tumour growth in vivo. Mechanistically, LINC00511 positively regulated the CCND2 expression via competitively sponging with miR‐124‐3p. Overall, our finding illuminates that LINC00511 is induced by SP1 and accelerates the glioma progression through targeting miR‐124‐3p/CCND2 axis, constructing the SP1/LINC00511/miR‐124‐3p/CCND2 axis.

Mannan-binding lectin (MBL) acts as a soluble pattern recognition molecule in the innate immune system, which is primarily produced by the liver. MBL deficiency occurs with high frequency in the population and is reported to be associated with susceptibility to several liver diseases. In the present study, we investigated the pathophysiological role of MBL in acetaminophen (APAP)-induced hepatotoxicity. After APAP treatment, MBL-deficient (MBL-/- ) mice had significantly higher mortality and aggravated hepatic necrosis as well as elevated serum lactate dehydrogenase and alanine aminotransferase levels compared to control mice. The enhanced hepatotoxicity in MBL-/- mice was associated with increased concentration of APAP toxic metabolisms. Furthermore, we demonstrated here that genetic ablation of MBL resulted in excessive reactive oxygen species (ROS) production and enhanced c-Jun N-terminal kinase (JNK) activation, leading to up-regulated specificity protein 1 (SP1) nuclear expression, thus promoted CYP2E1 hepatic expression and consequently exacerbated APAP-induced liver injury in mice. Importantly, we have validated that MBL protected against APAP toxicity in human HepaRG cells in vitro with the same mechanism. Our study revealed an unexpected function of MBL in drug metabolism, thus providing new insight into the drug-induced liver injury in patients with MBL deficiency.

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiβ mRNA stability and expression. Positively regulation of rhogdiβ mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiβ mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIβ axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.

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.

Gastric cancer is the fourth most common cancer and ranks as the second leading cause of cancer-related deaths across the world. The combination therapy of surgery with chemotherapeutic drugs, that is, mitomycin C (MMC), is becoming a major strategy for patients with advanced gastric cancer. However, drug resistance is a major factor that limits the effectiveness of chemotherapy, which ultimately leads to the failure of cancer chemotherapy. X-ray repair cross complementing group 1 (XRCC1), a scaffold protein of the base excision repair process, has been implicated in the development of tumor chemoresistance. Thus, this study aimed to explore whether XRCC1 expression could be regulated, its role in gastric AGS cancer cells treated with MMC, and the underlying mechanism. The results of this study demonstrate that XRCC1 expression could be upregulated in AGS cells treated with MMC, and this upregulation could subsequently reduce the cytotoxicity of MMC in AGS cells. Furthermore, MMC-upregulated XRCC1 expression was regulated by MAPK signaling through activating the transcription factor Sp1. These results indicate the role of XRCC1 in the development of drug resistance to MMC in gastric AGS cells. Elucidating the mechanism concerning the MAPKs and transcription factor Sp1 may provide another notion for the development of a clinical chemotherapy strategy for gastric cancers in the future.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Increasing evidence has suggested high-fat diet (HFD) is an independent risk factor for myocardial ischemia/reperfusion (MI/R) injury. Long noncoding RNAs (lncRNAs) recently attracted much attraction in the study of MI/R injury. However, the functional questions of specific lncRNAs in HFD-induced MI/R injury have not been well elucidated. Uc.48+ is a lncRNA from a transcribed ultraconserved region (T-UCR) of human, mouse, and rat genomes. Here, we explored the aggravating role of uc.48+and identified purinergic P2X7 receptor (P2X7R) as a downstream regulator of uc.48+ in HFD-induced MI/R vulnerability. We demonstrated uc.48+ expression was upregulated, accompanied by the corresponding upregulation of P2X7R in HFD I/R myocardium and HFD-induced MI/R vulnerability. Overexpression of uc.48+enhanced, whereas silencing of uc.48 + decreased the expression of P2X7R, cardiomyocyte apoptosis, and MI/R injury. The functional relevance of uc.48+ regulated P2X7R expression and the subsequent NF-κB signaling to promote cardiomyocyte apoptosis was supported by inhibition of P2X7R with its specific antagonist (A438079) as well as the inhibitor of NF-κB signaling (pyrrolidine dithiocarbamate, PDTC) in H9c2 hypoxia/reoxygenation (H/R) cells transfected with pcDNA3.0-uc.48 + plasmid, and RNA immunoprecipitation (RIP) suggested uc.48+ could interact with transcription factor Sp1. Importantly, Sp1 inhibitor (mithramycin, MIT) was found to suppress uc.48+ -induced P2X7R expression and the NF-κB signaling and cardiomyocyte apoptosis. Our findings provide a potential novel mechanism through which uc.48+ boosts cardiomyocyte apoptosis and MI/R vulnerability to HFD. Thus, uc.48+ is a novel regulator of HFD-induced MI/R injury; targeting uc.48+ may be a novel therapeutic approach of MI/R vulnerability to HFD.

Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL-21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL-21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL-21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL-21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR-302c is able to negatively regulate IL-21 expression by binding directly to its target site in the 3'-untranslated region. Moreover, after using fresh human CD4-positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL-21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL-21 expression.

Background There is abundant evidence to indicate that HBXIP functions as an oncoprotein and transcription co-activator during the development and promotion of cancers. In multiple cancers, ZEB1 serves as a transcription activator to regulate gene expression. We explored the roles of ZEB1 in HBXIP-induced breast cancer growth. Methods HBXIP regulation of ZEB1 was evaluated by reverse transcription PCR and immunoblotting. The stimulation of ZEB1 promoter by HBXIP and/or Sp1 was tested using luciferase reporter gene analysis. The alteration of cell proliferation mediated by HBXIP-induced ZEB1 was tested using methyl-thiazolyl-tetrazolium and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation analysis. ZEB1 and HBXIP expression in human breast cancer tissues was analyzed using quantitative real-time PCR. The relationship between HBXIP and ZEB1 was confirmed by Pearson's correlation coefficient. Results We observed dose-dependent upregulation of ZEB1 by HBXIP in breast cancer cells. HBXIP can activate the ZEB1 promoter by interacting with transcription factor Sp1. Cell viability and EdU incorporation analysis showed that HBXIP could drive cell proliferation by enhancing ZEB1 in breast cancer. Using quantitative real-time PCR, ZEB1 overexpression and a positive relationship between ZEB1 and HBXIP were observed in clinical breast cancer samples. Conclusion Oncogenic HBXIP controls the transcription regulation of ZEB1 by co-activating Sp1, thereby accelerating breast cancer growth.

Bevacizumab (Bv) can be used synergistically with fluoropyrimidine‐based chemotherapy to treat colorectal cancer. Whether and how it affects the delivery of fluoropyrimidine drugs is unknown. The present study aimed to explore the effect of Bv on the delivery of 5‐fluorouracil (5‐FU) to tumors and the underlying mechanism from metabolic perspective. Bv enhanced the anti‐tumor effects of 5‐FU in LoVo colon cancer xenograft mice and increased the 5‐FU concentration in tumors without affecting hepatic 5‐FU metabolism. Interestingly, Bv remarkably upregulated thymidine phosphorylase (TP) in tumors, which mediated the metabolic activation of 5‐FU. Although TP is reported to promote angiogenesis and resistance, the combination of Bv and 5‐FU resulted in anti‐angiogenesis and vessel normalization in tumors, indicating that the elevated TP mainly contributed to the enhanced response to 5‐FU. Bv also induced TP upregulation in LoVo cancer cells. Treatment with vascular endothelial growth factor receptor 2 (VEGFR2) antagonist apatinib and VEGFR2 silencing further confirmed TP upregulation. Bv and apatinib both enhanced the cytotoxicity of 5‐FU in LoVo cells, but there was no synergism with adriamycin and paclitaxel. We further demonstrated that the effect of Bv was dependent on VEGFR2 blockade and specificity protein 1 activation via MDM2 inhibition. In summary, Bv enhanced the accumulation of 5‐FU in tumors and the cytotoxicity of 5‐FU via TP upregulation. We provide data to better understand how Bv synergizes with 5‐FU from metabolic perspective, and it may give clues to the superiority of Bv in combination with fluoropyrimidine drugs compared to other chemotherapeutic drugs in colon cancer.

We previously reported that gentisic acid (2,5‐dihydroxybenzoic acid) is the third most abundant phenolic component of Dendropanax morbifera branch extracts. Here, we investigated its effects on cardiac hypertrophy and fibrosis in a mouse model of pressure overload and compared them to those of the beta blocker bisoprolol and calcium channel blocker diltiazem. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC). Beginning 2 weeks after this procedure, the mice were given daily intraperitoneal injections of gentisic acid (100 mg/kg/d), bisoprolol (5 mg/kg/d) or diltiazem (10 mg/kg/d) for 3 weeks. Cardiac hypertrophy was evaluated by the heart weight‐to‐body weight ratio, the cardiomyocyte cross‐sectional area after haematoxylin and eosin staining, and echocardiography. Markers of cardiac hypertrophy and fibrosis were tested by reverse transcription‐quantitative real‐time polymerase chain reaction, western blotting and Masson's trichrome staining. The suppressive effects of gentisic acid treatment on TAC‐induced cardiac hypertrophy and fibrosis were comparable to those of bisoprolol administration. Cardiac hypertrophy was reversed and left ventricular septum and posterior wall thickness were restored by gentisic acid, bisoprolol and diltiazem treatment. Cardiac hypertrophic marker gene expression and atrial and brain natriuretic peptide levels were decreased by gentisic acid and bisoprolol, as were cardiac (interstitial and perivascular) fibrosis and fibrosis‐related gene expression. Cardiac hypertrophy‐associated upregulation of the transcription factors GATA4 and Sp1 and activation of extracellular signal‐regulated kinase 1/2 were also negated by these drugs. These results suggest that gentisic acid could serve as a therapeutic agent for cardiac hypertrophy and fibrosis.

Monoamine oxidase B (MAO B) inhibitors, which inhibit dopamine decomposition by antagonizing MAO B activity, are approved and widely used for clinical treatment of Parkinson's disease (PD). Nonetheless, the mechanism of the abnormally increased MAO B activity in PD is still unclear. Previous research showed transcription factor specificity protein 1 (SP1) directly regulates MAO B activity by binding the SP1 binding sequence in MAO B promoter. In our study, we first observed that the SP1 protein level and SP1 binding activity in the MAO B promoter were increased in 1-methyl-4-phenylpyridinium (MPP+ ) neurotoxin-induced SH-SY5Y cells. Inhibition of SP1 by pretreatment with SP1 inhibitor mithramycin A (MMA) attenuated the abnormal increase in SP1 binding activity and the MAO B protein level to basal levels. Then, we investigated the neuroprotective effects of SP1 inhibition. In SH-SY5Y cell models of PD, preincubation with MMA or knockdown by SP1-specific small interfering RNA showed potent protection against MPP+ -induced apoptosis via SP1. In a male C57BL/6 mouse model of PD, MAO B activity and MPP+ concentrations in mouse brain following injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were increased, whereas the elevated MAO B activity was decreased after pre-injection of MMA. Moreover, MMA ameliorated MPTP-induced loss of dopaminergic neurons in the substantia nigra pars compacta and mouse behavioral impairments. Altogether, our study suggests that SP1 is a principal factor regulating increases in MAO B activity, and SP1 inhibition produces neuroprotective effects in PD models through decreases in MAO B activity, which may be a new neuroprotective therapeutic strategy for PD treatment.

Background and aims The role of Ras guanine nucleotide-releasing protein 1 (RasGRP1) in tumourigenesis has been a subject of debate, and its functions and clinical significance in hepatocellular carcinoma (HCC) remain unknown. Here, we evaluated the expression of RasGRP1 in HCC and determined how it contributes to HCC cell proliferation. Methods RasGRP1 expression was measured by quantitative polymerase chain reaction (qPCR) and Western blotting of 24 paired HCC tissues and para-tumour tissues. RasGRP1 expression was confirmed by immunohistochemical analysis of a tissue microarray from 1 independent cohort. Overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method, and risk factors that contributed to OS or DFS were identified using Cox regression analysis. The biologic relevance of RasGRP1 was examined by small interfering RNAs and an exogenous plasmid construct. Chromatin immunoprecipitation assays were performed to examine the binding of Sp1 to the RasGRP1 promoter. Results Increased RasGRP1 expression was associated with tumour size (P = .004), tumour-node-metastasis stage (P = .032), and Barcelona Clinic Liver Cancer stage (P = .002). RasGRP1 overexpression was an independent prognostic factor in HCC patients. RasGRP1 downregulation inhibited cell proliferation, whereas RasGRP1 overexpression promoted cell proliferation. Moreover, specificity protein 1 bound to the RasGRP1 promoter and promoted RasGRP1 transcription. In addition, RasGRP1 overexpression enhanced activation of the c-Raf pathway. Conclusions RasGRP1 is upregulated in HCC and promotes HCC cell proliferation. Thus, RasGRP1 may be a novel therapeutic target for HCC.

Basic helix-loop-helix family member e40 (BHLHE40) is located in 3p26.1 and acts as a transcriptional repressor of the circadian rhythm by suppressing the expression of the clock genes and clock-controlled genes. Recent research indicated that BHLHE40 may be involved in regulating tumor cell progression. However the mechanism by which BHLHE40 regulates the invasion and metastasis of tumor cells is unclear. Our in vitro assays showed that BHLHE40 promoted tumor cell invasion while BHLHE40 silencing by siRNA suppressed tumor cell invasion of MCF-7 cells. BHLHE40 suppressed the mRNA and protein expression of CLDN1 CLDN4 and CDH1 and promoted the expression of SNAI1 and SNAI2. Reporter assays demonstrated that BHLHE40 suppressed CLDN1 transcription but not through direct binding to the E-box motifs in the CLDN1 promoter. Further studies demonstrated BHLHE40 suppressed CLDN1 transcription by preventing the interaction between SP1 and a specific motif within the promoter region of CLDN1. BHLHE40 could not further suppress CLDN1 transactivation after SP1 siRNA transfection that is, BHLHE40-induced suppression of CLDN1 relied on SP1. Furthermore our data indicated that SP1 was a major regulator of CLDN1 transcription by binding to a specific motif that was located at -233 to -61 bp upstream of the transcription start site. Immunoprecipitation and co-localization data revealed an interaction between BHLHE40 and SP1. By constructing deletion mutants we found that the BHLH and Orange regions are both essential for the BHLHE40-SP1 interaction. BHLHE40 probably acts as an inhibitory nuclear cofactor or perhaps recruits other inhibitory cofactors to inhibit the SP1-mediated CLDN1 transactivation. These results suggest that BHLHE40 facilitates cell invasion and may be used as a novel target for breast cancer prevention and treatment.

Metastasis-associated lung adenocarcinoma transcript 1 (malat1) is an oncogenic long non-coding RNA (lncRNA) which has been proven to be associated with various types of tumors. Transcription factor specificity protein 1 (SP1) is overexpressed in many types of cancers. Previously, we observed that malat1 expression level is regulated by SP1 in lung cancer. In the present study, we found that transfection of expression construct of malat1 5' end fragment M5 enhances stability and transcriptional activity of SP1. Various SP1 target genes are also upregulated following overexpression of malat1 M5 in lung adenocarcinoma cells. We also showed that malat1 M5 interacts with the C-terminal domain of SP1 by RNA immunoprecipitation (RIP) assay coupled with UV cross-linking. Malat1-SP1 association results in increase of SP1 stability. In turn, SP1 promotes malat1 transcription, thus forming a positive feedback loop. In conclusion, our data show that in lung adenocarcinoma cells, malat1 interacts with SP1 protein and promotes SP1-mediated transcriptional regulation of SP1 target genes.

DHX15 plays a role in leukaemogenesis and leukaemia relapse. However, the mechanism underlying the transcriptional regulation of DHX15 in ALL has not been elucidated. Our present study aimed to explore the functional promoter region of DHX15 and to investigate the transcription factors controlling the transcription of this gene. A luciferase assay performed with several truncated constructs identified a 501‐bp region as the core promoter region of DHX15. Site‐directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that ETS1 and SP1 occupied the DHX15 promoter. Furthermore, knockdown of ETS1 and SP1 resulted in suppression of DHX15, whereas the overexpression of these genes led to up‐regulation of DHX15. Interestingly, in samples obtained from patients with ALL at diagnosis, both ETS1 and SP1 correlated positively with DHX15 expression. Additionally, differences in methylation of the DHX15 core promoter region were not observed between the patients and controls. In conclusion, we identified the core promoter region of DHX15 and demonstrated that ETS1 and SP1 regulated DHX15 expression in ALL.

Transforming growth factor‐beta (TGF‐β) functions as a potent proliferation inhibitor and apoptosis inducer in the early stages of breast cancer, yet promotes cancer aggressiveness in the advanced stages. The dual effect of TGF‐β on cancer development is known as TGF‐β paradox, and the remarkable functional conversion of TGF‐β is a pivotal and controversial phenomenon that has been widely investigated for decades. This phenomenon may be attributed to the cross talk between TGF‐β signaling and other pathways, including EGF receptor (EGFR) signaling during cancer progression. However, the underlying mechanism by which TGF‐β shifts its role from a tumor suppressor to a cancer promoter remains elusive. In this study, TGF‐β is positively correlated with EGFR expression in breast cancer tissues, and a functional linkage is observed between TGF‐β signaling and EGFR transactivation in breast cancer cell lines. TGF‐β promotes the migration and invasion abilities of breast cancer cells, along with the increase in EGFR expression. EGFR is also essential for TGF‐β‐induced enhancement of these abilities of breast cancer cells. Canonical Smad3 signaling and ERK/Sp1 signaling pathways mediate TGF‐β‐induced EGFR upregulation. Hence, our study provided insights into a novel mechanism by which TGF‐β supports breast cancer progression.

Aberrant HGF/Met signaling promotes tumor migration, invasion, and metastasis through both autocrine and non-autocrine mechanisms; however, the molecular downstream signaling mechanisms by which HGF/Met induces metastasis are incompletely understood. We here report that Ezrin expression is stimulated by HGF and correlates with activated HGF/Met, indicating that HGF/Met signaling regulates the expression of Ezrin. We show that HGF/Met signaling activates the transcription factor Sp1 through the MAPK pathway, and activated Sp1 can in turn directly bind to the promoter of Ezrin gene and regulate its transcription. Notably, knockdown of Ezrin expression by shRNAs inhibits the metastasis induced by either HGF/Met autocrine or non-autocrine signaling in syngeneic wildtype and HGF transgenic mouse hosts. We also used small molecule drugs in preclinical mouse models to confirm that Ezrin is one of the downstream molecules mediating HGF/Met signaling-induced metastasis in melanoma. We conclude that Ezrin is a key downstream factor involved in the regulation of HGF/Met signaling-induced metastasis and demonstrate a link between Ezrin and HGF/Met/MAPK/Sp1 activation in the metastatic process. Our data indicate that Ezrin represents a promising therapeutic target for patients bearing tumors with activated HGF/Met signaling.

Maintaining zinc homeostasis is an important property of all organisms. In the yeast Saccharomyces cerevisiae, the Zap1 transcriptional activator is a central player in this process. In response to zinc deficiency, Zap1 activates transcription of many genes and consequently increases accumulation of their encoded proteins. In this report, we describe a new mechanism of Zap1-mediated regulation whereby increased transcription of certain target genes results in reduced protein expression. Transcription of the Zap1-responsive genes RTC4 and RAD27 increases markedly in zinc-deficient cells but, surprisingly, their protein levels decrease. We examined the underlying mechanism further for RTC4 and found that this unusual regulation results from altered transcription start site selection. In zinc-replete cells, RTC4 transcription begins near the protein-coding region and the resulting short transcript leader allows for efficient translation. In zinc-deficient cells, RTC4 RNA with longer transcript leaders are expressed that are not efficiently translated due to the presence of multiple small open reading frames upstream of the coding region. This regulation requires a potential Zap1 binding site located farther upstream of the promoter. Thus, we present evidence for a new mechanism of Zap1-mediated gene regulation and another way that this activator protein can repress protein expression.

Our most recent studies demonstrate that RhoGDIβ is able to promote human bladder cancer (BC) invasion and metastasis in an X‐link inhibitor of apoptosis protein‐dependent fashion accompanied by increased levels of matrix metalloproteinase (MMP)‐2 protein expression. We also found that RhoGDIβ and MMP‐2 protein expressions are consistently upregulated in both invasive BC tissues and cell lines. In the present study, we show that knockdown of RhoGDIβ inhibited MMP‐2 protein expression accompanied by a reduction of invasion in human BC cells, whereas ectopic expression of RhoGDIβ upregulated MMP‐2 protein expression and promoted invasion as well. The mechanistic studies indicated that MMP‐2 was upregulated by RhoGDIβ at the transcriptional level by increased specific binding of the transcription factor Sp1 to the mmp‐2 promoter region. Further investigation revealed that RhoGDIβ overexpression led to downregulation of miR‐200c, whereas miR‐200c was able directly to target 3′‐UTR of jnk2 mRNA and attenuated JNK2 protein translation, which resulted in attenuation of Sp1 mRNA and protein expression in turn, inhibiting Sp1‐dependent mmp‐2 transcription. Collectively, our studies demonstrate that RhoGDIβ overexpression inhibits miR‐200c abundance, which consequently results in increases of JNK2 protein translation, Sp1 expression, mmp‐2 transcription, and BC invasion. These findings, together with our previous results showing X‐link inhibitor of apoptosis protein mediating mRNA stabilization of both RhoGDIβ and mmp‐2, reveal the nature of the MMP‐2 regulatory network, which leads to MMP‐2 overexpression and BC invasion.

Berberine (BBR), one of active alkaloid found in the rhizome, exhibited anti‐cancer properties. We have showed that BBR inhibited growth of non‐small cell lung cancer (NSCLC) cells through mitogen‐activated protein kinase (MAPK)‐mediated increase in forkhead box O3a (FOXO3a). However, the in‐depth mechanism underlying the anti‐tumor effects still remained to be elucidated. Herein, we further confirmed that BBR not only induced cell cycle arrest, but also reduced migration and invasion of NSCLC cells. Mechanistically, we observed that BBR reduced 3‐phosphoinositide‐dependent protein kinase‐1 (PDPK1) and transcription factor SP1 protein expressions. Exogenously expressed SP1 overcame BBR‐inhibited PDPK1 expression. Moreover, BBR inhibited DNA methyltransferase 1 (DNMT1) gene expression and overexpressed DNMT1 resisted BBR‐inhibited cell growth. Intriguingly, overexpressed PDPK1 antagonized BBR‐inhibited SP1 and DNMT1 expressions. Finally, metformin enhanced the effects of BBR both in vitro and in vivo. Collectively, we observe that BBR inhibits proliferation of NSCLC cells through inhibition of SP1 and PDPK1; this results in a reduction of DNMT1 expression. The interplay of PDPK1 and SP1 contributes to the inhibition of DNMT1 in response to BBR. In addition, there is a synergy of BBR and metformin. This study uncovers a new mechanism of BBR in combination with metformin for NSCLC‐associated therapy.

Single zinc finger domain containing proteins are very few in number. Of numerous zinc finger proteins in eukaryotes, only three of them like GAGA, Superman and DNA binding by one finger (Dof) have single zinc finger domain. Although few zinc finger proteins have been described in eubacteria, no protein with single C4 zinc finger has been described in details in anyone of them. In this article, we are describing two novel C-terminal C4 zinc finger proteins-Msmeg_0118 and Msmeg_3613 from Mycobacterium smegmatis. We have named these proteins as Mszfp1 (Mycobacterial Single Zinc Finger Protein 1) and Mszfp2 (Mycobacterial Single Zinc Finger Protein 2). Both the proteins are expressed constitutively, can bind to DNA and regulate transcription. It appears that Mszfp1 and Mszfp2 may activate transcription by interacting with RNA polymerase.

Licochalcone A (LCA), isolated from the root of Glycyrrhiza inflata, are known to have medicinal effect such as anti-oxidant, anti-bacterial, anti-viral, and anti-cancer. Though, as a pharmacological mechanism regulator, anti-cancer studies on LCA were not investigated in human breast cancer. We investigated the anti-proliferative and apoptotic effect of LCA in human breast cancer cells MCF-7 and MDA-MB-231 through MTS assay, PI staining, Annexin-V/7-AAD assay, mitochondrial membrane potential assay, multi-caspase assay, RT-PCR, Western blot analysis, and anchorage-independent cell transformation assay. Our results showed the little difference between two cells, as MCF-7 cell is both estrogen/progesterone receptor positive, there were only effect on Sp1 protein level, but not in mRNA level. Adversely, estrogen/progesterone/human epidermal growth factor receptor 2 triple negative, MDA-MB-231 showed decreased Sp1 mRNA, and protein levels. To confirm the participation of Sp1 in breast cancer cell viability, siRNA techniques were introduced. Both cells showed dysfunction of mitochondrial membrane potential and mitochondrial ROS production, which reflects it passed intracellular mitochondrial apoptosis pathway. Additionally, LCA showed the anti-proliferative and apoptotic effect in breast cancer cells through regulating Sp1 and apoptosis-related proteins in a dose- and a time-dependent manner. Consequently, LCA might be a potential anti-breast cancer drug substitute. J. Cell. Biochem. 118: 4652-4663, 2017. © 2017 Wiley Periodicals, Inc.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.

Nasopharyngeal carcinoma (NPC), a disease common in the South-East Asian population, has high lymph node metastatic ability. Melatonin, an endogenously produced substance present in animals, plants, fungi, and bacteria, has oncostatic activity via several mechanisms. The molecular mechanisms involved in melatonin-mediated tumor inhibitory potential are not completely defined. Here, we show that melatonin treatment inhibits TPA-induced cell motility by regulating the matrix metalloproteinase-9 (MMP-9) expression in NPC. We also identified the signaling cascade through which melatonin inhibits MMP-9 expression; this involves melatonin regulating the binding activity of the transcription factor specificity protein-1 (SP-1)-DNA. Our mechanistic analysis further reveals that the c-Jun N-terminal kinase/mitogen-activated protein kinase pathway is involved in the melatonin-mediated tumor suppressor activity. Furthermore, the findings indicate a functional link between melatonin-mediated MMP-9 regulation and tumor suppressing ability and provide new insights into the role of melatonin-induced molecular and epigenetic regulation of tumor growth. Thus, we conclude that melatonin suppresses the motility of NPC by regulating TPA-induced MMP-9 gene expression via inhibiting SP-1-DNA binding ability. The results provide a functional link between melatonin-mediated SP-1 regulation and the antimetastatic actions of melatonin on nasopharyngeal carcinoma.

The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene-specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter-specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP-associated factor TAF4. We herein report the structural details of the glutamine-rich domains (Q-domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q-domains of Sp1 and four Q-domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q-domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well-accepted concept of "coupled folding and binding."

Cellular stress leads to an upregulation of gene transcription. We asked if there is a specificity in the activation of the stress-responsive transcription factors Nrf2, ATF4, and AP-1/c-Jun, or if activation of these proteins is a redundant cellular answer toward extracellular stressors. Here, we show that oxidative stress, induced by stimulation of the cells with the oxidant arsenite, strongly activated gene transcription via the stress-responsive element (StRE), while phorbol ester or tunicamycin, activators of AP-1/c-Jun or ATF4, respectively, activated AP-1 or nutrient-sensing response element-mediated transcription. Preincubation of the cells with N-acetyl-cysteine or overexpression of thioredoxin selectively attenuated arsenite-induced upregulation of StRE-regulated transcription. Expression of either dominant-negative or constitutively active mutants of Nrf2, ATF4, or c-Jun confirmed that distinct transcription units are regulated by these transcription factors. Physiological stimuli involving the activation of either Gαq-coupled designer receptors or the protein kinases c-Jun N-terminal protein kinase or p38 strongly stimulated transcription via AP-1/c-Jun, with minimal effects on Nrf2 or ATF4-responsive promoters. Thus, activation of transcription by extracellular signaling molecules shows specificity at the level of the chemical nature of the signaling molecule, at the level of the intracellular transduction process, and at the level of signal-responsive transcription factors. J. Cell. Biochem. 118: 127-140, 2017. © 2016 Wiley Periodicals, Inc.

Obesity is characterized by an accumulation of excessive body fat and can be diagnosed by a variety of measures, such as BMI. However, in some obese individuals, oxidative stress is also thought to be an important pathogenic mechanism of obesity-associated metabolic syndrome. Oxidative stress increases the lipid peroxidation product, 4-hydroxynonenal (4-HNE), which is one of the most abundant and active lipid peroxides. Within the adipose tissue, adipocytes are derived from adipose tissue-derived stromal cells (ADSCs), which play a key role in the generation and metabolism of adipose tissue. Additionally, obesity is associated with low-grade inflammation. Specific microRNAs (miRNAs) that regulate obesity-associated inflammation are largely dysregulated in metabolic syndrome (MS). In this study, we aim to confirm whether 4-HNE and miRNAs play a role in the regulation of TNF-α gene transcription. We enrolled six obese individuals who were referred to Harbin Medical University (Heilongjiang, China) and six nonobese control participants. Plasma 4-HNE levels of the 12 subjects were determined by ELISA. Using qRT-PCR, we measured ETS1, miR-29b, SP1, and TNF-α levels in subcutaneous white adipose tissue (WAT). Furthermore, we examined the relationship between ETS1 and TNF-α using a luciferase reporter assay and a ChIP assay. Our results suggest that ETS1 promotes TNF-α gene transcription in adipocytes. In addition, we demonstrated that 4-HNE promotes TNF-α gene transcription through the inhibition of the miR-29b → SP1 → TNF-α pathway and promotion of the ETS1 → TNF-α pathway. Anat Rec, 299:1145-1152, 2016. © 2016 Wiley Periodicals, Inc.

Radioresistance remains a major problem in the treatment of patients suffering from nasopharyngeal carcinoma (NPC). A better understanding of the mechanisms of radioresistance may generate new strategies to improve NPC patients' responses to therapy. This study was designed to investigate the effect of microRNA on the radiosensitivity of NPC cells. A microRNA microarray indicated that miR‐24 was downregulated in NPC cell lines and tissues. Furthermore, cell proliferation was suppressed and radiosensitivity increased when miR‐24 was ectopically expressed in NPC cells. Specificity protein 1 (SP1) was additionally verified as a direct functional target of miR‐24, which was found to be involved in cell viability as well as the radiosensitivity of NPC cells. In conclusion, the results of this study suggest that the miR‐24/SP1 pathway contributed to the reduction in radioresistance in human NPC and that it may thus represent a therapeutic target.

The regulation of vascular endothelial growth factors C (VEGF-C) and D (VEGF-D), and their receptor VEGFR3 gene and protein expression by all-trans-retinoic acid (atRA) in A549 lung cancer cells, was investigated. We showed that atRA treatment increased VEGF-C, VEGF-D, and VEGFR3 protein and mRNA contents in dose-dependent manner. atRA-mediated increase of both ligands and receptor expression correlated with the elevated level of retinoic acid receptor α (RARα) expression, while the level of another atRA receptor, peroxisome proliferator-activated receptor β/δ (PPARβ/δ), was decreased. We demonstrated that the classical counterpart of RARα, retinoid X receptor α (RXRα), was down-regulated in both cytoplasm and nucleus of A549 cells upon atRA addition. On the contrary, the nuclear quantity of another possible RARα counterpart, transcription factor Sp1, was increased after atRA treatment.

Despite the important role played by the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier regulation and the implication of both nmMLCK and vascular endothelial growth factor (VEGF) in the pathogenesis of acute respiratory distress syndrome (ARDS), the role played by nmMLCK in VEGF-induced vascular permeability is poorly understood. In this study, the role played by nmMLCK in VEGF-induced vascular hyperpermeability was investigated. Human lung endothelial cell barrier integrity in response to VEGF is examined in both the absence and the presence of nmMLCK small interfering RNAs. Levels of nmMLCK messenger RNA (mRNA), protein, and promoter activity expression were monitored after VEGF stimulation in lung endothelial cells. nmMYLK promoter activity was assessed using nmMYLK promoter luciferase reporter constructs with a series of nested deletions. nmMYLK transcriptional regulation was further characterized by examination of a key transcriptional factor. nmMLCK plays an important role in VEGF-induced permeability. We found that activation of the VEGF signaling pathway in lung endothelial cells increases MYLK gene product at both mRNA and protein levels. Increased nmMLCK mRNA and protein expression is a result of increased nmMYLK promoter activity, regulated in part by binding of the Sp1 transcription factor on triggering by the VEGF signaling pathway. Taken together, these findings suggest that MYLK is an important ARDS candidate gene and a therapeutic target that is highly influenced by excessive VEGF concentrations in the inflamed lung.

To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs).The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays.Expression levels of miR-138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR-138 is able to repress the expression of COL2A1 by directly targeting Sp-1 and hypoxia-inducible factor 2α (HIF-2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR-138 in the RNA-induced silencing complex and confirmed binding of Sp-1 to the COL2A1 promoter region in HACs.We propose that an evolutionary pressure helps to suppress expression levels of miR-138 in human cartilage, thus enabling expression of appropriate tissue-specific matrix genes. Inhibition of miR-138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.