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Background Parkinson disease is a neurodegenerative disease and is characterized by resting tremor, dementia, and gait disorder. Previous studies have indicated that long noncoding RNA participates in the regulation of the pathogenesis of Parkinson disease. The study aimed to reveal the effects of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) on 1-methyl-4-phenylpyridine (MPP+)–induced human neuroblastoma cell injury and the underlying mechanism. Methods The expressions of NEAT1, microRNA (miR)-519a-3p, and transcription factor specific protein 1 (SP1) were detected by quantitative real-time polymerase chain reaction. The protein expressions of SP1 and inflammation-related factors were determined by Western blot. Cell viability was determined by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was investigated by flow cytometry analysis. The targeting relationship between miR-519a-3p and NEAT1 or SP1 was predicted by starBase online database and verified by a dual-luciferase reporter assay. Results NEAT1 and SP1 expressions were significantly upregulated, whereas miR-519a-3p was downregulated in MPP+-treated neuroblastoma cells in a dose- and time-dependent manner when compared with control groups. NEAT1 knockdown restrained MPP+-induced repression of cell viability and promotion of cell apoptosis and inflammation. Additionally, NEAT1 served as a sponge of miR-519a-3p and regulated MPP+-caused cell injury by interacting with miR-519a-3p. Also, SP1, a target gene of miR-519a-3p, rescued miR-519a-3p–mediated actions under MPP+ treatment. Importantly, NEAT1 stimulated SP1 expression through interaction with miR-519a-3p. Conclusions NEAT1 silencing protected against MPP+-induced neuroblastoma cell injury by regulating the miR-519a-3p/SP1 pathway. This finding provides a novel direction for the development of therapeutic strategies for Parkinson disease.

Pregnancy and VEGF (vascular endothelial growth factor) stimulate uterine artery endothelial cell (UAEC) hydrogen sulfide production via selectively upregulating CBS (cystathionine β-synthase) but not CSE (cystathionine γ-lyase) expression. This study was conducted to determine the mechanisms by which VEGF utilizes to stimulate pregnancy-dependent upregulation of CBS and hydrogen sulfide production in human UAEC. The proximal human CBS promoter contains 4 Sp1 (specificity protein 1; a/b/c/d) sites and 1 YY1 (Yin Yang 1) site; luciferase assays using reporter genes driven by human CBS promoter with a series of 5′-deletions identified a promoter sequence (−574 to −394) containing Sp1d and the YY1 sites critical for basal and VEGF-stimulated CBS promoter activation. VEGF stimulated pregnancy-dependent recruitment of Sp1 to Sp1d and YY1 to YY1 and also recruited YY1 to Sp1c and increased Sp1/YY1 association in pregnant human UAEC, suggesting formation of a Sp1/YY1 complex at the Sp1c site. Endothelial Sp1 and YY1 proteins were significantly greater in pregnant than nonpregnant human uterine artery. VEGF stimulated pregnancy-dependent Sp1 and YY1 protein expression in vitro. Treatment with Sp1 and YY1 siRNAs completely blocked Sp1/YY1-mediated pregnancy-dependent CBS protein upregulation and hydrogen sulfide production by VEGF in human UAEC. VEGF did not trans -activate CSE promoter or increase CSE expression, and Sp1/YY1 knockdown did not affect CSE expression in human UAEC. Thus, pregnancy augments EC Sp1 and YY1 expression and promotes the recruitment of Sp1/YY1 to their DNA-binding sequences in proximal human CBS promoter to upregulate CBS transcription, underlying a novel mechanism to mediate VEGF-stimulated pregnancy-dependent endothelial hydrogen sulfide production in the human uterine artery.

Background We aimed to investigate the functions and underlying mechanism of lncRNA SNHG1 in bone differentiation and angiogenesis in the development of osteoporosis. Methods The differential gene or proteins expressions were measured by qPCR or western blot assays, respectively. The targeted relationships among molecular were confirmed through luciferase reporter, RIP and ChIP assays, respectively. Alkaline phosphatase (ALP), alizarin red S (ARS) and TRAP staining were performed to measure the osteoblast/osteoclast differentiation of BMSCs. The viability, migration and angiogenesis in BM-EPCs were validated by CCK-8, clone formation, transwell and tube formation assays, respectively. Western blot and immunofluorescence detected the cytosolic/nuclear localization of β-catenin. Ovariectomized (OVX) mice were established to confirm the findings in vitro. Results SNHG1 was enhanced and miR-181c-5p was decreased in serum and femoral tissue from OVX mice. SNHG1 directly inhibited miR-181c-5p to activate Wnt3a/β-catenin signaling by upregulating SFRP1. In addition, knockdown of SNHG1 promoted the osteogenic differentiation of BMSCs by increasing miR-181c-5p. In contrast, SNHG1 overexpression advanced the osteoclast differentiation of BMSCs and inhibited the angiogenesis of BM-EPCs, whereas these effects were all reversed by miR-181c-5p overexpression. In vivo experiments indicated that SNHG1 silencing alleviated osteoporosis through stimulating osteoblastogenesis and inhibiting osteoclastogenesis by modulating miR-181c-5p. Importantly, SNHG1 could be induced by SP1 in BMSCs. Conclusions Collectively, SP1-induced SNHG1 modulated SFRP1/Wnt/β-catenin signaling pathway via sponging miR-181c-5p, thereby inhibiting osteoblast differentiation and angiogenesis while promoting osteoclast formation. Further, SNHG1 silence might provide a potential treatment for osteoporosis. Graphic abstract

RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1−/− cells enhances the TCA flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1−/− deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance., RIPK1 is critical for normal development and cell death. Here, the authors identify a metabolic role for RIPK1 in aspartate homeostasis, as increased aspartate levels in RIPK1-deficient cells inhibits starvation-induced autophagy by ULK1.

Loss of polarity protein Par3 promotes breast cancer tumorigenesis and metastasis. The underlying molecular mechanisms of Par3 down-regulation and related prognostic significance in breast cancer remain unclear. Here, we discovered that Par3 down-regulation was associated with shorter relapse-free survival in Luminal A subtype of breast cancer. Par3 knockdown promoted breast cancer cells migration and invasion. Importantly, we identified that transcription factor Sp1 bound to PARD3 promoter region and induced Par3 expression. Breast cancer patients with low Sp1 showed significantly worse RFS and low expression level of Par3. Par3 over-expression partially reversed Sp1 knockdown induced migration and invasion. Together, decreased Sp1 level mediates Par3 down-regulation, which correlated with poor prognosis of ER + breast cancer patients, via reduced binding with PARD3 promoter.

BackgroundBrain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke.MethodsUsing a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia.ResultsWe observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice.ConclusionsThese findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

Meningioma has the second highest incidence among primary intracranial tumors. There is no effective treatment or targeted therapy for meningioma except excision and radiotherapy. Sp1 (specificity protein 1) is a transcription factor that regulates tumor growth, but the literature describing the relationship between Sp1 and meningioma is scarce. The purpose of this study was to investigate the relationship between Sp1 expression and the clinicopathological parameters in meningioma by immunohistochemical staining.We collected samples from 74 patients from 2008-2012 in Kaohsiung Medical University Hospital, with staging and prognosis of the pathological section of the meningioma.Our results show that Sp1 expression was significantly associated with World Health Organization grade, malignancy, and recurrence in patients with meningioma.High expression of Sp1 in patients with meningioma was linked to poor prognosis of recurrence-free time. Therefore Sp1 may be a candidate biomarker of prognosis and therapy target in meningioma.

Muscle regeneration after damage or during myopathies requires a fine cooperation between myoblast proliferation and myogenic differentiation. A growing body of evidence suggests that microRNAs play critical roles in myocyte proliferation and differentiation transcriptionally. However, the molecular mechanisms underlying the orchestration are not fully understood. Here, we showed that miR-130b is able to repress myoblast proliferation and promote myogenic differentiation via targeting Sp1 transcription factor. Importantly, overexpression of miR-130b is capable of improving the recovery of damaged muscle in a freeze injury model. Moreover, miR-130b expression is declined in the muscle of muscular dystrophy patients. Thus, these results indicated that miR-130b may play a role in skeletal muscle regeneration and myopathy progression. Together, our findings suggest that the miR-130b/Sp1 axis may serve as a potential therapeutic target for the treatment of patients with muscle damage or severe myopathies.

We previously reported that dysregulation of histone deacetylase 2 (Hdac2) was associated with the prognosis of sudden sensorineural hearing loss. However, the underlying molecular mechanisms are poorly understood. In the present study, we developed an acute hearing loss animal model in guinea pigs by infusing lipopolysaccharides (LPS) into the cochlea and measured the expression of Hdac2 in the sensory epithelium. We observed that the level of Hdac2 was significantly decreased in the LPS-infused cochleae. The levels of apoptosis-inhibition genes Bcl-2 and Bcl-xl were also decreased in the cochlea and correlated positively with the levels of Hdac2. Caspase3 or TUNEL-positive spiral ganglion neurons, hair cells, and supporting cells were observed in the LPS-infused cochleae. These in vivo observations were recapitulated in cell culture experiments. Based on bioinformatics analysis, we found miR-204-5p was engaged in the regulation of Hdac2 on Bcl-2. Molecular mechanism experiments displayed that miR-204-5p could be regulated by Hdac2 through interacting with transcription factor Sp1. Taken together, these results indicated that the Hdac2/Sp1/miR-204-5p/Bcl-2 regulatory axis mediated apoptosis in the cochlea, providing potential insights into the progression of acute hearing loss. To our knowledge, the study describes a miRNA-related mechanism for Hdac2-mediated regulation in the cochlea for the first time., Graphical Abstract, Dysregulation of histone deacetylase 2 (Hdac2) was associated with the prognosis of sudden sensorineural hearing loss. However, the underlying molecular mechanisms are poorly understood. This study revealed a role of Hdac2/miR-204-5P/Bcl-2 axis in the apoptosis of cochlear cells and provided a potential insight into the pathogenesis of acute hearing loss.

A synthetic peptide that blocks the interaction between the metastasis-enhancing calcium-binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA-MB-231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)-14 (MT1-MMP). Mechanistic analysis of the downregulation of MMP-14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)-κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP-14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP-14 expression levels in MDA-MB-468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xeno-graft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP-14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

Nasopharyngeal carcinoma is a type of otolaryngological malignancy with high incidence. Long non-coding RNAs (lncRNAs) are closely related to nasopharyngeal carcinoma. LncRNA AFAP1-AS1 (AFAP1-AS1) has been found to play important roles in nasopharyngeal carcinoma progression and poor prognosis. However, the mechanism underlying AFAP1-AS1 in regulating nasopharyngeal carcinoma is still unclear. In current study, AFAP1-AS1 was found to be up-regulated in nasopharyngeal carcinoma tissues and cells. AFAP1-AS1 overexpression and knockdown were conducted in nasopharyngeal carcinoma cells. The results proved that AFAP1-AS1 promoted the survival and migration of nasopharyngeal carcinoma cells. Additionally, specificity protein 1 (SP1) was enhanced in nasopharyngeal carcinoma tissues and cells, and induced AFAP1-AS1 expression. The interaction between AFAP1-AS1 and miR-497-5p was confirmed. AFAP1-AS1 was demonstrated to regulate CELF1, a target gene of miR-497-5p. Further functional analysis revealed that AFAP1-AS1 knockdown attenuated SP1-induced nasopharyngeal carcinoma progression. These results indicate that SP1-induced AFAP1-AS1 facilitates nasopharyngeal carcinoma progression by regulating miR-497-5p/CELF1 pathway, which provides a new target for nasopharyngeal carcinoma treatment.

Glioma is a common life-threatening tumor with high malignancy and high invasiveness. LncRNA ZFPM2 antisense RNA 1 (ZFPM2-AS1) was confirmed to be implicated in numerous tumors, while its biological function and mechanism have not been thoroughly understood in glioma. The gene expression was measured by RT-qPCR. Cell proliferation, cell cycle, and cell apoptosis of glioma cells were validated by CCK-8, colony formation, flow cytometry and TUNEL assays. The effect of ZFPM2-AS1 on tumor growth was verified by in vivo assay. The exploration on ZFPM2-AS1-mediated mechanism was carried out via ChIP, luciferase reporter, and RIP assays. In the present study, ZFPM2-AS1 was demonstrated as a highly-expressed lncRNA in glioma tissues and cells. ZFPM2-AS1 silencing suppressed cell proliferation and cell cycle, but facilitated cell apoptosis. In addition, the inhibitive effect of silenced ZFPM2-AS1 was also observed in tumor growth. Furthermore, we found that SP1 interacted with ZFPM2-AS1 promoter to transcriptionally activate ZFPM2-AS1 expression. Moreover, ZFPM2-AS1 was identified as a competing endogenous RNA (ceRNA) for miR-515-5p to target SOD2. Rescue assays verified that SOD2 overexpression partially abolished the suppressive impact of ZFPM2-AS1 silencing on glioma cell growth. In conclusion, this study corroborated the regulatory mechanism of SP1/ZFPM2-AS1/miR-515-5p/SOD2 axis in glioma, indicating that targeting ZFPM2-AS1 might be an effective way to treat glioma.

Background Circular RNAs (circRNAs) are endogenous non-coding RNAs, which are associated with various biological processes, including microRNA (miRNA) interaction, protein binding and regulatory splicing. circRNA_0005529 (circ_0005529) is derived from vacuolar protein sorting 33 homologue B (VPS33B), and its biological role in gastric cancer (GC) has not been examined. In this study, the expression and location of circ_0005529 and microRNA-527 (miR-527) were determined by qRT-PCR and fluorescence in situ hybridization (FISH). Cell proliferation and cell migration were determined by MTT, EdU incorporation, colony formation, wound scratch and transwell assays. In addition, immunohistochemistry and western blotting were performed to determine the expressions of specificity protein 1 (Sp1), PCNA, c-myc, E-cadherin and N-cadherin. Western blotting and luciferase reporter assay were performed to study the interaction between circ_0005529 and miR-527 or miR-527 and Sp1. The functional effects of circ_0005529 on GC through regulating Sp1 were further evaluated using xenograft and metastatic mouse models in vivo. Results Our results showed that circ_0005529 was upregulated in GC tissues and cells, and had promoting effects on cell proliferation and cell migration. Mechanism analysis suggested that circ_0005529 could bind to microRNA-527 (miR-527) and reduce its expression. The interaction between miR-527 and Sp1 in GC was systematically studied. In addition, the results indicated that Sp1 upregulation could rescue the effects on cell proliferation and migration caused by circ_0005529. Moreover, the inhibitory effects of circ_0005529 downregulation on GC growth and metastasis were evaluated in mouse models. These findings suggested that the axis of circ_0005529/miR-527/Sp1 may serve as a promising treatment target for GC diagnosis and treatment. Conclusions These findings suggested that the signal axis of circ_0005529/miR-527/Sp1 may has the potential to be explored as a novel therapeutic target for GC diagnosis and treatment. Graphical abstract Mechanism diagram: During GC development, overexpressed circ_0005529 sponged miR-527 and then upregulated the expression of Sp1. Subsequently, epithelial-mesenchymal transition (EMT), cell proliferation and cell migration were promoted, which ultimately facilitated the tumor metastasis

Background: MIR155HG is a long non-coding RNA (lncRNA) that has been shown to be dysregulated in a range of tumor types, but the functions of this lncRNA in melanoma remain to be explored. Objectives: We explored the functions of lncRNA MIR155HG in melanoma progression. Methods: The expression of miR155HG was analyzed in clinical melanoma. Bioinformatics analysis was performed to assess the potential tumor-related functions of miR155HG. The interaction of miR155HG and SP1 and the inhibition of PSIP1 by miR-485-3p were analyzed by ChIP, luciferase reporter experiments, and the biological effects in melanoma were explored by colony formation assays, EdU cell proliferation assays, Transwell analysis, and intracranial melanoma mouse model. Results: Herein, we found that MIR155HG was markedly upregulated in melanoma cell lines and tissues. We further determined that the SP1 transcription factor was responsible for driving MIR155HG upregulation in melanoma. Elevated MIR155HG levels were linked to decreased overall survival (OS) in melanoma patients, and we further determined that MIR155HG expression was an independent predictor of melanoma patient prognosis. When MIR155HG was knocked down in melanoma cells, this impaired their proliferative, migratory, and invasive activity. By using predictive bioinformatics analyses, we identified miR-485-3p as a microRNA (miRNA) capable of binding to both MIR155HG and the 3’ UTR of PSIP1. Conclusion: Together, these results suggest that MIR155HG is capable of promoting melanoma cell proliferation via the miR-485-3p/PSIP1 axis. These novel findings provide new insights into the development of melanoma, potentially highlighting future avenues for therapeutic intervention.

Background: Mounting evidences have shown the importance of lncRNAs in carcinogenesis and cancer progression. LBX2-AS1 is identified as an oncogenic lncRNA that is abnormally expressed in gastric cancer and lung cancer samples. This study aims to explore the potential role of LBX2-AS1 in regulating proliferation and EMT in glioma, and the underlying mechanism. Methods: Relative levels of LBX2-AS1 in glioma samples and cell lines were detected by qRT-PCR and FISH. In vivo and in vitro regulatory effects of LBX2-AS1 on proliferation and EMT were examined in the xenograft glioma model and glioma cells. The interaction between SP1 and LBX2-AS1 was assessed by ChIP. Through bioinformatic analyses, dual-luciferase reporter assay, RIP and Western blot, the regulation of LBX2-AS1 and miR-491-5p on the target gene LIF was identified. Results: LBX2-AS1 was upregulated in glioma samples and cell lines, and its transcription was promoted by binding to the transcription factor SP1. As a lncRNA mainly distributed in the cytoplasm, LBX2-AS1 sponge miR-491-5p to further upregulate LIF. The subsequent activated LIF/STAT3 signaling was responsible for promoting proliferation and EMT in glioma. Conclusion: LBX2-AS1 is upregulated by SP1 in glioma, which promotes the progression of glioma by targeting the miR-491-5p/LIF axis. In view of this, LBX2-AS1 is suggested as a novel diagnostic biomarker and therapeutic target of glioma.

Lung cancer is the second most common tumor and has the highest mortality rate. Both novel therapeutic targets and approaches are needed to improve the overall survival of patients with lung cancer. MicroRNA-320a-3p belongs to the miR-320a family and has been reported as a tumor suppressor in multiple cancers. However, its definitive role and precise mechanism in the progression of lung cancer remain unclear. In this study, we developed a new type of gold nanorod modified with polyethyleneimine that targets cancer-specific nanoparticles by RGD peptide, which could condense miRNA to self-assemble supramolecular nanoparticles. The designed nanoparticles can achieve integrin αvβ3-targeted cancer therapy, realize photosensitive therapy by laser irradiation and attain gene-targeted therapy by miRNAs. These nanoparticles could deliver miR-320a into lung cancer cells specifically and efficiently. Moreover, we demonstrated that Au-RGD-miR-320a nanoparticles combined with laser irradiation dramatically inhibited the proliferation and metastasis, and enhanced the apoptosis of lung cancer, both in vitro and in vivo. In terms of the mechanism, miR-320a inhibits Sp1 expression by directly binding to the 3'UTR of Sp1, and it eventually enhanced the expression of PTEN and inhibited the expression of matrix metallopeptidase 9 (MMP9). These findings provide a new and promising anticancer strategy via the use of Au-RGD-miR-320a nanoparticles, and identify miR-320a/Sp1 as a potential target for future systemic therapy against lung cancer.

ObjectiveMicrobiota disorder promotes chronic inflammation and carcinogenesis. High glycolysis is associated with poor prognosis in patients with colorectal cancer (CRC). However, the potential correlation between the gut microbiota and glucose metabolism is unknown in CRC.Design18F-FDG (18F-fluorodeoxyglucose) PET (positron emission tomography)/CT image scanning data and microbiota PCR analysis were performed to measure the correlation between metabolic alterations and microbiota disorder in 33 patients with CRC. Multiple colorectal cancer models, metabolic analysis and Seahorse assay were established to assess the role of long non-coding RNA (lncRNA) enolase1-intronic transcript 1 (ENO1-IT1) in Fusobacterium (F.) nucleatum-induced glucose metabolism and colorectal carcinogenesis. RNA immunoprecipitation and chromatin immunoprecipitation sequencing were conducted to identify potential targets of lncRNA ENO1-IT1.ResultsWe have found F. nucleatum abundance correlated with high glucose metabolism in patients with CRC. Furthermore, F. nucleatum supported carcinogenesis via increasing CRC cell glucose metabolism. Mechanistically, F. nucleatum activated lncRNA ENO1-IT1 transcription via upregulating the binding efficiency of transcription factor SP1 to the promoter region of lncRNA ENO1-IT1. Elevated ENO1-IT behaved as a guider modular for KAT7 histone acetyltransferase, specifying the histone modification pattern on its target genes, including ENO1, and consequently altering CRC biological function.ConclusionF. nucleatum and glucose metabolism are mechanistically, biologically and clinically connected to CRC. Targeting ENO1 pathway may be meaningful in treating patients with CRC with elevated F. nucleatum.

Recently, we identified that the atypical protein kinase C isoform ι (PKCι) enhances the expression of Yes-associated protein 1 (YAP1) to promote the tumorigenesis of pancreatic adenocarcinoma harboring mutant KRAS (mu-KRAS). To advance our understanding about underlying mechanisms, we analyze the transcription of YAP1 in pancreatic cancer cells and reveal that transcription factor specificity protein 1 (Sp1) is upregulated by PKCι and subsequently binds to multiple sites in YAP1 promoter to drive the transactivation of YAP1 in pancreatic cancer cells carrying mu-KRAS. The bioinformatics analysis further substantiates that the expression of PKCι, Sp1 and YAP1 is correlated and associated with the stages and prognosis of pancreatic tumors. Moreover, our apoptotic detection data demonstrate that combination of PKCι and Sp1 inhibitors at subtoxic doses displays synergistic effects on inducing apoptosis and reversing the immunosuppression of pancreatic cancer cells, establishing the combination of PKCι and Sp1 inhibitors as a promising novel therapeutic approach, or an adjuvant strategy to potentiate the antitumor effects of other immunotherapeutic agents in pancreatic cancer treatment.

MicroRNA-7 (miR-7) has been identified as a tumor suppressor in non-small cell lung cancer (NSCLC) and a radiosensitivity regulator. Numerous studies have revealed that specific protein 1 (SP1) plays a critical role in the tumorigenesis of various types of cancers and regulates radiosensitivity and tumor suppressor p53-binding protein 1 (TP53BP1), which plays an essential role in DNA repair. However, it is not clear whether miR-7 has a regulatory effect on SP1 and TP53BP1 in NSCLC. In the present study it was revealed that miR-7 directly binds to the 3′UTR of SP1, thereby suppressing SP1 expression to regulate radiosensitivity. Overexpression of miR-7 and SP1 and knockdown of miR-7 and SP1 were performed using lentiviral transfection. Protein and mRNA abundance of SP1 and TP53BP1 were determined using western blotting and RT-qPCR, respectively, while miR-7 binding to SP1 was validated using a luciferase reporter assay. Biological function analysis indicated that miR-7 negatively regulated SP1 and inhibited cell proliferation, migration, and invasion when combined with radiation. It was also revealed that the expression of TP53BP1 was positively regulated by SP1 or negatively regulated by miR-7. In conclusion, SP1 was a target of miR-7, and the decreased expression of SP1 resulting from miR-7 overexpression in NSCLC was vital for improving radiosensitivity in NSCLC cells. Moreover, SP1 expression was detected in 95 paired NSCLC and adjacent normal tissues, and it was determined that SP1 was significantly upregulated in NSCLC tissues and that its upregulation was correlated with the degree of tissue differentiation. Thus, SP1 and/or miR-7 may be potential molecular targets in NSCLC radiotherapy.

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.

GDF15 has been recently recognized as a tumor-suppressive gene. However, the underlying mechanism by which GDF15 affects breast carcinogenesis is not well understood. Here, we showed that the inhibitory effect of GDF15 on cell proliferation was dependent on the nuclear localization of the protein. Dynamic translocation of GDF15 into the nucleus altered expression of a number of genes, including KISS-1, and resulted in inhibition of cell growth and invasive behavior. Using KISS-1 promoter-driven luciferase reporter and chromatin immunoprecipitation assays, we demonstrated that, in highly malignant breast cancer cells, GDF15 directly interacts with specific protein-1 (Sp1) at the Sp1-binding sites of the KISS-1 promoter, leading to upregulated KISS-1 expression. Our study indicates that nuclear GDF15 could serve as a transcriptional coactivator to mediate the expression of particular genes to reduce cell proliferation.

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.

The biological effect and molecular mechanism of miR‐5188 have not been thoroughly investigated. The study aims at elucidating the role of miR‐5188 in glioma progression. Human glioma cell lines and tissues were used for functional and expression analysis. Cellular and molecular techniques were performed to explore the functions and mechanisms of miR‐5188 in glioma. In our investigation, we demonstrated that miR‐5188 promoted cell proliferation, the G1/S transition of the cell cycle, migration and invasion in glioma and reduced the lifespan of glioma‐bearing mice. miR‐5188 directly targeted FOXO1 and activated PI3K/AKT‐c‐JUN signalling, which enhanced miR‐5188 expression. Moreover, the c‐JUN transcription factor functionally bound to the miR‐5188 promoter region, forming the positive feedback loop. The feedback loop promoted glioma progression through activating the PI3K/AKT signalling, and this loop is augmented by the interaction between SP1 and c‐JUN. Moreover, it was also found that the miR‐5188/FOXO1 axis is facilitated by SP1‐activated PI3K/AKT/c‐JUN signalling. In glioma samples, miR‐5188 expression was found to be an unfavourable factor and was positively associated with the mRNA levels of SP1 and c‐JUN, whereas negatively associated with the mRNA levels of FOXO1. Our investigation demonstrates that miR‐5188 could function as a tumour promoter by directly targeting FOXO1 and participating in SP1‐mediated promotion of cell growth and tumorigenesis in glioma.

Phloretin is a flavonoid existed in various plants and has been reported to possess anticarcinogenic activity. However, the anticancer mechanism of phloretin in prostate cancer (PCa) remains unclear. Here, ourin vitroandin vivoexperimental data demonstrate that phloretin inhibits the phosphorylation and the activation of EGFR and then inhibits its downstream PI3K/AKT and MEK/ERK1/2 pathways in PCa cells. Inhibition of these two pathways further decreases expression of Sp1 by inhibitingSp1gene transcription, induces degradation of Sp1 protein by inhibiting GSK3βphosphorylation, suppresses nucleolin-enhanced translation of Sp1 mRNA by inhibiting nucleolin phosphorylation, and directly inactivates transcription activity of Sp1. Inhibition of Sp1 subsequently decreases the expression of Sp3/4, VEGF, and Survivin and then upregulates apoptosis-related proteins and downregulates cell cycle-related proteins in PCa cells. Finally, phloretin treatment in PCa cells induces cell growth inhibition and apoptosis, suggesting that phloretin may be an effective therapy compound in the treatment of prostate cancer.

Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA‐326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound‐healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR‐326 expression. Human OS tissues showed lowly expressed miR‐326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR‐326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR‐326 gene. miR‐326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR‐326 gene by promoting histone deacetylation; miR‐326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

Significance Bone marrow failure (BMF) syndromes are inherited life-threatening conditions characterized by low blood cell production and predisposition to cancer. In this study we report a germ line frameshift variant in the Sp1 transcription factor in a family of patients with BMF and acute leukemia. Sp1 is ubiquitously expressed in human tissues and regulates transcription for blood cell lineage specification. Dissecting the molecular function of this SP1 variant revealed a hypermorphic effect, triggering superactivation of Sp1-mediated transcription in the patients’ blood. To our knowledge, this is the first report of a naturally occurring germ line variant in SP1 that alters transcriptional networks and disrupts hematopoiesis in humans., Inherited bone marrow failure (BMF) syndromes are a heterogeneous group of diseases characterized by defective hematopoiesis and often predisposing to myelodysplastic syndrome (MDS) and acute myelogenous leukemia. We have studied a large family consisting of several affected individuals with hematologic abnormalities, including one family member who died of acute leukemia. By whole-exome sequencing, we identified a novel frameshift variant in the ubiquitously expressed transcription factor specificity protein 1 (SP1). This heterozygous variant (c.1995delA) truncates the canonical Sp1 molecule in the highly conserved C-terminal DNA-binding zinc finger domains. Transcriptomic analysis and gene promoter characterization in patients’ blood revealed a hypermorphic effect of this Sp1 variant, triggering superactivation of Sp1-mediated transcription and driving significant up-regulation of Sp1 target genes. This familial genetic study indicates a central role for Sp1 in causing autosomal dominant transmission of BMF, thereby confirming its critical role in hematopoiesis in humans.

AIMS: In cardiomyocytes, there is microRNA (miR) in the mitochondria that originates from the nuclear genome and matures in the cytoplasm before translocating into the mitochondria. Overexpression of one such miR, miR-181c, can lead to heart failure by stimulating reactive oxygen species (ROS) production and increasing mitochondrial calcium level ([Ca(2+)](m)). Mitochondrial calcium uptake 1 protein (MICU1), a regulatory protein in the mitochondrial calcium uniporter complex, plays an important role in regulating [Ca(2+)](m). Obesity results in miR-181c overexpression and a decrease in MICU1. We hypothesize that lowering miR-181c would protect against obesity-induced cardiac dysfunction. METHODS AND RESULTS: We used an in vivo mouse model of high-fat diet (HFD) for 18 weeks and induced high lipid load in H9c2 cells with oleate-conjugated bovine serum albumin in vitro. We tested the cardioprotective role of lowering miR-181c by using miR-181c/d(−/−) mice (in vivo) and AntagomiR against miR-181c (in vitro). HFD significantly upregulated heart levels of miR-181c and led to cardiac hypertrophy in wild-type mice, but not in miR-181c/d(−/−) mice. HFD also increased in ROS production and pyruvate dehydrogenase activity (a surrogate for [Ca(2+)](m)), but the increases were alleviated in miR-181c/d(−/−) mice. Moreover, miR-181c/d(−/−) mice fed a HFD had higher levels of MICU1 than did wild-type mice fed a HFD, attenuating the rise in [Ca(2+)](m). Overexpression of miR-181c in neonatal ventricular cardiomyocytes (NMVM) caused increased ROS production, which oxidized transcription factor Sp1 and led to a loss of Sp1, thereby slowing MICU1 transcription. Hence, miR-181c increases [Ca(2+)](m) through Sp1 oxidation and downregulation of MICU1, suggesting that the cardioprotective effect of miR-181c/d(−/−) results from inhibition of Sp1 oxidation. CONCLUSION: This study has identified a unique nuclear-mitochondrial communication mechanism in the heart orchestrated by miR-181c. Obesity-induced overexpression of miR-181c increases [Ca(2+)](m) via downregulation of MICU1 and leads to cardiac injury. A strategy to inhibit miR-181c in cardiomyocytes can preserve cardiac function during obesity by improving mitochondrial function. Altering miR-181c expression may provide a pharmacologic approach to improve cardiomyopathy in individuals with obesity/type 2 diabetes.

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.

Cardiac hypertrophy (CH) is a common cardiac disease and is closely associated with heart failure. Protocadherin 17 (PCDH17) was reported to aggravate myocardial infarction. Present study was designed to illustrate the impact of PCDH17 and the mechanism of PCDH17 expression regulation in CH. CH model in vivo and in vitro was established by transverse aortic constriction (TAC) and Ang‐II treatment. Hypertrophy was evaluated in PMC and H9c2 cells by examining cell surface area and hypertrophic markers. Results demonstrated that PCDH17 was up‐regulated in CH in vivo and in vitro. PCDH17 knock‐down alleviated hypertrophic response in Ang‐II‐induced cardiomyocytes. By means of ENCORI database and a series of mechanism assays, miR‐322‐5p and miR‐384‐5p were identified to interact with and inhibit PCDH17. Next, lncRNA SNHG14 (small nucleolar RNA host gene 14) was validated to sponge both miR‐322‐5p and miR‐384‐5p to elevate PCDH17 level. The subsequent rescue assays revealed that miR‐322‐5p and miR‐384‐5p restored SNHG14 depletion‐mediated suppression on hypertrophy in Ang‐II‐induced cardiomyocytes. Besides, Sp1 transcription factor (SP1) was verified as the transcription factor activating both SNHG14 and PCDH17. Both SNHG14 and PCDH17 reversed SP1 knock‐down‐mediated repression on hypertrophy in Ang‐II‐induced cardiomyocytes. Together, present study first uncovered ceRNA network of SNHG14/miR‐322‐5p/miR‐384‐5p/PCDH17 in Ang‐II‐induced cardiomyocytes.

Background Ovarian cancer is the most lethal gynecologic cancer. Chemoresistance, especially platinum-resistance, is closely related to metastasis of ovarian cancer, however, the molecular basis by which links chemoresistance and metastasis remains vague. Disordered arachidonic acid (AA) metabolism has been shown to play an important role in the advanced ovarian cancer. This study aimed to explore the underlying mechanism involving eicosanoid metabolism that controlling chemoresistance and metastasis of ovarian cancer. Methods Cisplatin (DDP)-resistant SKOV3 (SKOV3-R) cells were constantly induced. Ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was performed to determine the AA metabolism in SKOV3 and SKOV3-R cells. Half maximal inhibitory concentration (IC50) and percentage of cell viability were tested using cell counting kit 8 (CCK-8). Realtime quantitative PCR (qPCR) and immunohistochemistry (IHC) were used to evaluate indicated genes and proteins respectively. Bioinformatic analysis and chromatin immunoprecipitation (ChIP) were performed to predict and identify the co-transcription factor of interest genes. Tumor growth and metastasis in the liver were assessed with nude mice by subcutaneously injection of SKOV3-R cells. Results SKOV3-R cells expressed higher multidrug resistance-associated proteins (MRPs) MRP1 and MRP4. They showed enhanced metastatic ability and produced increased AA-derived eicosanoids. Mechanistically, MRPs, epithelial mesenchymal transition (EMT) markers Snail and Slug, as well as key enzymes involved in AA-metabolism including 12-lipoxygenase (12LOX) were transcribed by the mutual transcription factor SP1 which was consistently upregulated in SKOV3-R cells. Inhibition of SP1 or 12LOX sensitized SKOV3-R cells to DDP and impaired metastasis in vitro and in vivo. Conclusion Our results reveal that SP1-12LOX axis signaling plays a key role in DDP-resistance and metastasis, which provide a new therapeutic target for ovarian cancer.

Background Growing studies have suggested the dysregulation of long non-coding RNAs (lncRNAs) in several tumors, including osteosarcoma (OS). However, limited studies report metastasis-associated lncRNAs in OS. Our present study aimed to explore the roles of lncRNA LINC00514 (LINC00514) in OS. Materials and methods The LINC00514 expression was measured using qPCR assays in OS tissues and cell lines. The clinical significance of LINC00514 expression in OS patients was analyzed using chi-square test, Kaplan-Meier assays and multivariate analysis. The possible effects of LINC00514 in tumor cellular progression were determined using a series of functional assays. The mechanisms of LINC00514 action were explored through bioinformatics, luciferase reporter assays and RT-PCR assays. The mechanisms involved the upregulation of LINC00514 expression in OS were determined using luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Results We showed that LINC00514 expressions were distinctly upregulated in both OS tissues and cell lines, especially in advanced cases. High levels of LINC0051 were positively correlated with advanced tumor stages, distant metastasis, and reduced survival of patients with OS. Functional experiments indicated that silencing of LINC00514 suppressed the ability of cell growth, colony formation and metastasis, whereas promoted cell apoptosis in vitro. Mechanistic investigation revealed that LINC00514 could directly bind to miR-708 and effectively serve as a ceRNA for miR-708. In addition, LINC00514 was upregulated by the transcription factor SP1. Conclusion Our findings revealed SP1-induced upregulation of LINC00514 as an oncogene in OS through competitively binding to miR-708, suggesting that there are potential diagnostic and treatment values of LINC00514 in OS.

Background Glioblastoma is associated with poor prognosis and high mortality. Although the use of first-line temozolomide can reduce tumor growth, therapy-induced stress drives stem cells out of quiescence, leading to chemoresistance and glioblastoma recurrence. The specificity protein 1 (Sp1) transcription factor is known to protect glioblastoma cells against temozolomide; however, how tumor cells hijack this factor to gain resistance to therapy is not known. Methods Sp1 acetylation in temozolomide-resistant cells and stemlike tumorspheres was analyzed by immunoprecipitation and immunoblotting experiments. Effects of the histone deacetylase (HDAC)/Sp1 axis on malignant growth were examined using cell proliferation–related assays and in vivo experiments. Furthermore, integrative analysis of gene expression with chromatin immunoprecipitation sequencing and the recurrent glioblastoma omics data were also used to further determine the target genes of the HDAC/Sp1 axis. Results We identified Sp1 as a novel substrate of HDAC6, and observed that the HDAC1/2/6/Sp1 pathway promotes self-renewal of malignancy by upregulating B cell-specific Mo-MLV integration site 1 (BMI1) and human telomerase reverse transcriptase (hTERT), as well as by regulating G2/M progression and DNA repair via alteration of the transcription of various genes. Importantly, HDAC1/2/6/Sp1 activation is associated with poor clinical outcome in both glioblastoma and low-grade gliomas. However, treatment with azaindolyl sulfonamide, a potent HDAC6 inhibitor with partial efficacy against HDAC1/2, induced G2/M arrest and senescence in both temozolomide-resistant cells and stemlike tumorspheres. Conclusion Our study uncovers a previously unknown regulatory mechanism in which the HDAC6/Sp1 axis induces cell division and maintains the stem cell population to fuel tumor growth and therapeutic resistance.

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: Atopic dermatitis (AD) is recognized as a common inflammatory skin disease and frequently occurred in Asian and Black individuals. Objective: Since the limitation of dataset associated with human severe AD, this study aimed to screen potential novel biomarkers involved in mild AD. Methods: Expression profile data (GSE75890) were obtained from the database of Gene Expression Omnibus. Using limma package, the differentially expressed genes (DEGs) between samples from AD and healthy control were selected. Furthermore, function analysis was conducted. Meanwhile, the protein-protein interaction (PPI) network and transcription factor (TF)-miRNA-target regulatory network were constructed. And quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the expressions patterns of key genes. Results: In total, 285 DEGs including 214 upregulated and 71 downregulated genes were identified between samples from two groups. The upregulated DEGs were mainly involved in nine pathways, such as hematopoietic cell lineage, pertussis, p53 signaling pathway, staphylococcus aureus infection, and cell cycle, while tight junction was the only pathway enriched by the downregulated DEGs. Cyclin B (CCNB)1, CCNB2, cyclin A (CCNA)2, C-X-C motif chemokine ligand (CXCL)10, and CXCL9 were key nodes in PPI network. The TF-miRNA-target gene regulatory network focused on miRNAs such as miR-106b, miR-106a, and miR-17, TFs such as nuclear factor kappa B subunit 1, RELA proto-oncogene, Sp1 transcription factor, and genes such as matrix metallopeptidase 9, peroxisome proliferator activated receptor gamma , and serpin family E member 1. Moreover, the upregulation of these genes, including CCNB1, CCNB2, CCNA2, CXCL10, and CXCL9 were confirmed by qRT-PCR. Conclusion: CCNB1, CCNB2, CCNA2, and CXCL9 might be novel markers of mild AD. miR-106b and miR-17 may involve in regulation of immune response in AD patients. (Ann Dermatol 32(2) 130∼ 140, 2020)

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.

Background: Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D 2 , a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D 2 /DP1 axis in T cells on age-related hypertension. Methods: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4 + T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. Results: The prostaglandin D 2 /DP1 axis was downregulated in CD4 + T cells from older humans and aged mice. DP1 deletion in CD4 + T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4 + T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4 + T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway–mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4 + T cells attenuated age-related hypertension in CD4 + T cell–specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. Conclusions: The prostaglandin D 2 /DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L–mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

Hearts often undergo abnormal remodelling and hypertrophic growth in response to pathological stress. Long non-coding RNAs (LncRNAs) can change cardiac function and participate in regulation of cardiac hypertrophy. The present study aims to identify the role of AK045171 in cardiac hypertrophy and the underlying mechanism in hypertrophic cascades. Mice with cardiac hypertrophy were established through transverse aortic constriction (TAC). Cardiac hypertrophy in cardiomyocytes was induced by angiotensin II (angII). The expression of AK045171 and its target gene SP1 was examined in cardiomyocytes transfected with miRNA. The AK045171 expression level was downregulated in mice after TAC surgery. Overexpression of AK045171 attenuated cardiac hypertrophy both in vitro and in vivo. The mechanism study indicated that AK045171 binds with SP1, which promotes transcription activation of MEG3. It is suggested that overexpression of AK045171 might have clinical potential to suppress cardiac hypertrophy and heart failure.

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP3) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology., It is not yet clear how ubiquitously-expressed proteins can cause the selective degeneration of particular populations of neurons, such as in spinocerebellar ataxia type 17, SCA17, which results from a CAG trinucleotide repeat expansion in the ubiquitously expressed transcription factor TBP. Here, the authors show that mutant TBP suppresses the cerebellum-enriched transcription of Inpp5a and link altered levels of INPP5A to the selective degeneration of cerebellar neurons.

Background This study is conducted to investigate the protective role of elevated microRNA-375 (miR-375) in dopaminergic neurons in Parkinson's disease through down-regulating transcription factor specificity protein 1 (SP1). Results The successfully modeled rats with Parkinson's disease showed aggregated neurobehavioral change, increased neuroinflammatory response and oxidative stress, and lowered dopamine content. Parkinson's disease rats treated with overexpressed miR-375 displayed improved neurobehavioral change, ameliorated neuroinflammatory response and oxidative stress, heightened dopamine content and abated neuronal apoptosis by down-regulating SP1. Up-regulation of SP1 reversed the protective effect of upregulated miR-375 on Parkinson's disease. Conclusion Up-regulation of miR-375 ameliorated the damage of dopaminergic neurons, reduced oxidative stress and inflammation in Parkinson's disease by inhibiting SP1. Methods Parkinson's disease rat model was established by targeted injection of 6-hydroxydopamine to damage the substantia nigra striatum. The successfully modeled Parkinson's disease rats were intracerebroventricularly injected with miR-375 mimics or pcDNA3.1-SP1. The functions of miR-375 and SP1 in neurobehavioral change, neuroinflammatory response, oxidative stress, dopamine content and expression of apoptosis-related proteins in the substantia nigra of Parkinson's disease rats were evaluated. The target relation of miR-375 and SP1 was confirmed by bioinformatics analysis and dual luciferase reporter gene assay.

Background Survivin has an anti-apoptotic effect against anthracycline-induced cardiotoxicity. Clinically, statin use is associated with a lower risk for heart failure in breast cancer patients with anthracycline chemotherapy. So, the purpose of our study was to investigate whether survivin mediates the protective effect of statin against anthracycline-induced cardiotoxicity. Methods Mice were treated once a week with 5 mg/kg doxorubicin for 4 weeks with or without atorvastatin 20 mg/kg every day then heart tissues were analyzed. Molecular and cellular biology analyses were performed with H9c2 cell lysates. Results Doxorubicin suppressed survivin expression via activation of FOXO1 in H9c2 cardiomyocytes. Whereas, atorvastatin inhibited FOXO1 by increasing phosphorylation and inhibiting nuclear localization. Doxorubicin induced FOXO1 binding to STAT3 and prevented STAT3 from interacting with Sp1. However, atorvastatin inhibited these interactions and stabilized STAT3/Sp1 transcription complex. Chromatin immunoprecipitation analysis demonstrated that doxorubicin decreased STAT3/Sp1 complex binding to survivin promoter, whereas atorvastatin stabilized this binding. In mouse model, atorvastatin rescued doxorubicin-induced reduction of survivin expression and of heart function measured by cardiac magnetic resonance imaging. Conclusions Our study suggested a new pathophysiologic mechanism that survivin mediated protective effect of atorvastatin against doxorubicin-induced cardiotoxicity via FOXO1/STAT3/Sp1 transcriptional network.

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction, and eventual organ failure. Therefore, the development of effective antifibrotic drugs is urgently required. IMB-S7 is novel biphenyl compound derived from bifendate (biphenyldicarboxylate) that is used for the treatment of chronic hepatitis in China. In the current study we investigated the potential of IMB-S7 as an antihepatic fibrosis agent. In bile duct ligation (BDL) rat model, oral administration of IMB-S7 (400 mg· kg−1· d−1, for 14 days) significantly ameliorated BDL-induced liver necrosis, bile duct proliferation, and collagen accumulation. We then showed that IMB-S7 treatment markedly suppressed the TGF-β/Smad pathway in human hepatic stellate cell line LX2 and mouse primary HSCs, as well as in liver samples of BDL rats, thus inhibiting the transcription of most fibrogenesis-associated genes, including TGF-β1, COL1A1, and ACTA2. Furthermore, IMB-S7 treatment significantly suppressed the expression of integrin αv at the mRNA and protein levels in TGF-β-treated LX2 cells and liver samples of BDL rats. Using integrin αv overexpression and silencing, we demonstrated that integrin αv activity correlated positively with the activation of TGF-β/Smad pathway. Based on dual luciferase assay and DNA affinity precipitation assay, we revealed that IMB-S7 inactivated integrin αv through competitively inhibiting the binding of Sp1, a transcription factor, to the integrin αv (ITGAV) promoter (−173/−163 bp). These results suggest that IMB-S7 inhibits HSCs activation and liver fibrosis through Sp1-integrin αv signaling, and IMB-S7 may be a promising candidate to combat hepatic fibrosis in the future.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to SCARB1 promoter. Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at least partly accelerated Hcy-mediated atherosclerosis through promoting lipid accumulation in foam cells, which was attributed to the decreased binding of SP1 to SCARB1 promoter. In our point, these findings will provide novel insight into an epigenetic mechanism for atherosclerosis.

Background: JWA gene is known to down-regulate SP1 and reduces the expression level of Integrin αvβ3. Here, we identified a functional polypeptide (JP1) based on the active fragment of the JWA protein to suppress melanoma growth and metastasis by inhibiting the Integrin αvβ3. Methods: We conducted a series of melanoma growth and metastasis mouse models to evaluate anti-melanoma effect of JP1 peptide. 18F-labeled JP1 (18F-NFP-JP1) was detected by Micro-PET assay to demonstrate drug biodistribution. Toxicity test in cynomolgus monkeys and pharmacokinetic studies in rats were done to assess the druggability. The expression of MEK1/2, NEDD4L, SP1 and Integrin αvβ3 were detected in vitro and vivo models. Results: The peptide JP1 with the best anticancer effect was obtained. Micro-PET assay showed that JP1 specifically targeting to melanoma cells in vivo. JP1 inhibited melanoma growth, metastasis, and prolonged the survival of mouse. JP1 reduced the dosage and toxicity in combination with DTIC in melanoma xenograft and allograft mouse models. Cynomolgus monkey toxicity test showed no observed adverse effect level (NOAEL) of JP1 was 150 mg/kg. Mechanistically, JP1 was shown to activate p-MEK1/2 and triggered SP1 ubiquitination in melanoma cells. NEDD4L, an E3 ubiquitin ligase, was activated by p-MEK1/2 and to ubiquitinate SP1 at K685 site, resulting in subsequent degradation. Conclusions: JP1 was developed as a novel peptide that indicated therapeutic roles on proliferation and metastasis of melanoma through the NEDD4L-SP1-Integrin αvβ3 signaling.

Background & Aims Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. Methods Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. Results Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966–0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. Conclusions LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR–extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681)., Graphical abstract

Long noncoding RNAs (lncRNAs) have been identified to be critical regulator in the osteosarcoma (OS) tumorigenesis. However, the role of lncRNA MIR17HG in the OS proliferation and chemotherapy resistance is still unclear. Here, this research aims to investigate the function of lncRNA MIR17HG in the OS proliferation and cisplatin resistance. Clinically, results revealed that higher MIR17HG expression was associated with shorter overall survival. Functional investigations indicated that MIR17HG promoted the proliferation, invasion and cisplatin resistance of OS cells in vitro, and the MIR17HG knockdown inhibited the growth in vivo. Mechanistically, MIR17HG targeted the miR-130a-3p/SP1 axis, moreover, transcription factor SP1 bind with the MIR17HG promoter region to promote its expression. Taken together, MIR17HG displays the tumor-promotive role in the progression of OS through SP1/MIR17HG/miR-130a-3p/SP1 feedback loop. Our findings might help us to offer novel therapeutic strategies for OS.

Although increasing evidence has suggested crosstalk between Parkinson's disease (PD) and type 2 diabetes mellitus (T2DM), the common mechanisms between the two diseases remain unclear. The aim of our study was to characterize the interconnection between T2DM and PD by exploring their shared biological pathways and convergent molecules. The intersections among the differentially expressed genes (DEGs) in the T2DM dataset GSE95849 and PD dataset GSE6613 from the Gene Expression Omnibus (GEO) database were identified as the communal DEGs between the two diseases. Then, an enrichment analysis, protein-protein interaction (PPI) network analysis, correlation analysis, and transcription factor-target regulatory network analysis were performed for the communal DEGs. As a result, 113 communal DEGs were found between PD and T2DM. They were enriched in lipid metabolism, including protein modifications that regulate metabolism, lipid synthesis and decomposition, and the biological effects of lipid products. All these pathways and their biological processes play important roles in both diseases. Fifteen hub genes identified from the PPI network could be core molecules. Their function annotations also focused on lipid metabolism. According to the correlation analysis and the regulatory network analysis based on the 15 hub genes, Sp1 transcription factor (SP1) could be a key molecule since it affected other hub genes that participate in the common mechanisms between PD and T2DM. In conclusion, our analyses reveal that changes in lipid metabolism could be a key intersection between PD and T2DM, and that SP1 could be a key molecule regulating these processes. Our findings provide novel points for the association between PD and T2DM.

Gastric cancer (GC) is a highly prevalent type of metastatic tumor. The mechanisms underlying GC metastasis are poorly understood. Some long noncoding RNAs (lncRNAs) reportedly play key roles in regulating metastasis of GC. However, the biological roles of five natural antisense lncRNAs (AC093818.1, CTD-2541M15.1, BC047644, RP11-597M12.1, and RP11-40A13.1) in GC metastasis remain unclear. In this study, the expression of these lncRNAs was measured by quantitative reverse transcription-polymerase chain reaction. Migration and invasion were evaluated by wound-healing and the Transwell assay, respectively. Stable cells were injected into the tail veins of nude mice. Sections of collected lung and liver tissues were stained using hematoxylin and eosin. Protein expression was analyzed by western blot. RNA immunoprecipitation (RIP) assay was used to verify whether the STAT3 and SP1 transcription factors bound to AC093818.1 in GC cells. Expression levels of the five lncRNAs, especially AC093818.1, were significantly upregulated in metastatic GC tissues relative to those in nonmetastatic GC tissues. AC093818.1 expression was correlated with invasion, lymphatic metastasis, distal metastasis, and tumor-node-metastasis stage. AC093818.1 expression was highly sensitive and specific in the diagnosis of metastatic or nonmetastatic GC. AC093818.1 overexpression promoted GC migration and invasion in vitro and in vivo. AC093818.1 overexpression increased PDK1, p-AKT1, and p-mTOR expression levels. AC093818.1 silencing decreased these expressions. AC093818.1 bound to transcription factors STAT3 and SP1, and SP1 or STAT3 silencing could alleviated the effect of AC093818.1 overexpression. The data demonstrate that lncRNA AC093818.1 accelerates gastric cancer metastasis by epigenetically promoting PDK1 expression. LncRNA AC093818.1 may be a potential therapeutic target for metastatic GC.

NAMI-A is highly reactive to Sp1, a tumor metastasis related protein, resulting in the perturbation of the protein structure and disruption of the DNA recognition of Sp1. Interestingly, Sp1 is more susceptible than other zinc finger proteins to NAMI-A, suggesting that Sp1 could be the anti-metastasis target of NAMI-A.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3β inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFβ-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFβ, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFβ, and α-SMA increased in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFβ treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFβ treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.