circE2F1-encoded peptide inhibits circadian machinery essential for nucleotide biosynthesis and tumor progression via repressing SPIB/E2F1 axis.

Circadian clock dominates a variety of biological activities, while its roles and regulatory mechanisms in neuroblastoma (NB), a pediatric extracranial malignancy, still remain largely elusive. Herein, through comprehensive analyses of public datasets, E2F transcription factor 1 (E2F1) and its circular RNA (circE2F1)-encoded 99-amino acid peptide (E2F1-99aa) were identified as vital regulators of circadian machinery essential for purine and pyrimidine biosynthesis during NB progression. Mechanistically, through interaction with Spi-B transcription factor (SPIB), E2F1 was transactivated to up-regulate circadian machinery genes (CRY1 and TIMELESS), resulting in relief of CLOCK/BMAL1-repressed transcription of enzymes (DHODH, PAICS, or PPAT) essential for de novo purine and pyrimidine biosynthesis. The biogenesis of circE2F1 was repressed by eukaryotic translation initiation factor 4A3 (EIF4A3), while E2F1-99aa or its truncated peptide competitively bound to SPIB, leading to decrease in SPIB-E2F1 interaction, circadian machinery and nucleotide biosynthetic gene expression, purine or pyrimidine biosynthesis, tumorigenesis, and aggresiveness of NB cells. In clinical NB cases, high EIF4A3, E2F1 or SPIB expression was correlated with low survival possibility of patients, while lower circE2F1 or E2F1-99aa levels were associated with advanced stages and tumor progression. These results indicate that circE2F1-encoded peptide inhibits circadian machinery essential for nucleotide biosynthesis and tumor progression via repressing SPIB/E2F1 axis.

Up-regulation of NCAPG mediated by E2F1 facilitates the progression of osteosarcoma through the Wnt/ß-catenin signaling pathway.

Background: In recent years, there are few reports on non-SMC condensin I complex subunit G (NCAPG) in osteosarcoma. Our study aims to explore the biological role of NCAPG in osteosarcoma and its underlying molecular mechanism and to further clarify the reasons for the abnormal expression of NCAPG in osteosarcoma. Methods: Here, we mined The Cancer Genome Atlas (TCGA) Program public database through bioinformatics methods, analyzed the differential expression of NCAPG in sarcoma tissue and normal tissue, and explored the relationship between NCAPG expression level and sarcoma tissue differentiation, including tumor recurrence, metastasis, and patient survival. Next, the transcription factors responsible for the abnormal expression of NCAPG in osteosarcoma tumors were predicted by multiple online website tools and verified via cellular experiments. Subsequently, loss of function and cell phenotype experiments were performed to confirm the effect of NCAPG on the malignant biological behavior of osteosarcoma cells. Mechanistically, by reviewing the literature, we found that NCAPG can affect the malignant progression of many solid tumors by regulating the Wnt/ß-catenin signaling pathway. Therefore, we preliminarily investigated the potential effect of NCAPG on this pathway via western blot experiments in osteosarcoma. Results: Increased expression of NCAPG was found in sarcoma compared to normal tissues, which was positively correlated with poor differentiation, metastasis, and poor prognosis. Combining the transcription factor prediction results, correlation analysis, and expression level in the TCGA public database with validation outcomes of in vitro cell assays, we found that E2F transcription factor 1 (E2F1) regulated the increased expression of NCAPG in osteosarcoma. The results of cell phenotype experiments showed that silencing NCAPG could inhibit the proliferation, migration, and invasion of osteosarcoma cells. The preliminary mechanistic investigation suggested that NCAPG may affect osteosarcoma progression through the Wnt/ß-catenin pathway. Conclusions: Our data reveal that E2F1 facilitates NCAPG expression in osteosarcoma by regulating the transcription of the NCAPG gene. Up-regulation of NCAPG promotes osteosarcoma progression via the Wnt/ß-catenin signaling axis.

New link between RNH1 and E2F1: regulates the development of lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a non-small cell carcinoma. Ribonuclease/angiogenin inhibitor 1 (RNH1) exerts multiple roles in virous cancers. E2F1 is a critical transcription factor involved in the LUAD development. Here, we analyze the expression of RNH1 in LUAD patients, investigate the biological function of RNH1 in LUAD, and demonstrate its potential mechanisms through E2F1 in LUAD. METHODS: In the present study, we presented the expression of RNH1 in LUAD based on the database and confirmed it by western blot detection of RNH1 in human LUAD tissues. Lentiviral infection was constructed to silence or overexpress RNH1 in NCI-H1395 and NCI-H1437 cells. We assess the role of RNH1 on proliferation in LUAD cells by MTT assay, colony formation assays, and cell cycle detection. Hoechst staining and flow cytometry were used to evaluate the effects of RNH1 on apoptosis of LUAD cells. The function of RNH1 in invasion and migration was investigated by Transwell assay. Dual luciferase assay, ChIP detection, and pull-down assay were conducted to explore the association of E2F1 in the maintenance of RNH1 expression and function. The regulation of E2F1 on the functions of RNH1 in LUAD cells was explored. Mouse experiments were performed to confirm the in-vivo role of RNH1 in LUAD. mRNA sequencing indicated that RNH1 overexpression altered the expression profile of LUAD cells. RESULTS: RNH1 expression in LUAD tissues of patients was presented in this work. Importantly, RNH1 knockdown improved the proliferation, migration and invasion abilities of cells and RNH1 overexpression produced the opposite effects. Dual luciferase assay proved that E2F1 bound to the RNH1 promoter (-1064 ∼ -1054, -1514 ∼ -1504) to reduce the transcriptional activity of RNH1. ChIP assay indicated that E2F1 DNA was enriched at the RNH1 promoter (-1148 ∼ -943, -1628 ∼ -1423). Pull-down assays also showed the association between E2F1 and RNH1 promoter (-1148 ∼ -943). E2F1 overexpression contributed to the malignant behavior of LUAD cells, while RNH1 overexpression reversed it. High-throughput sequencing showed that RNH1 overexpression induced multiple genes expression changes, thereby modulating LUAD-related processes. CONCLUSION: Our study demonstrates that binding of E2F1 to the RNH1 promoter may lead to inhibition of RNH1 expression and thus promoting the development of LUAD.

Long non­coding RNA DANCR aggravates breast cancer through the miR­34c/E2F1 feedback loop.

Emerging scientific evidence has suggested that the long non­coding (lnc)RNA differentiation antagonizing non­protein coding RNA (DANCR) serves a significant role in human tumorigenesis and cancer progression; however, the precise mechanism of its function in breast cancer remains to be fully understood. Therefore, the objective of the present study was to manipulate DANCR expression in MCF7 and MDA­MB­231 cells using lentiviral vectors to knock down or overexpress DANCR. This manipulation, alongside the analysis of bioinformatics data, was performed to investigate the potential mechanism underlying the role of DANCR in cancer. The mRNA and/or protein expression levels of DANCR, miR­34c­5p and E2F transcription factor 1 (E2F1) were assessed using reverse transcription­quantitative PCR and western blotting, respectively. The interactions between these molecules were validated using chromatin immunoprecipitation and dual­luciferase reporter assays. Additionally, fluorescence in situ hybridization was used to confirm the subcellular localization of DANCR. Cell proliferation, migration and invasion were determined using 5­ethynyl­2'­deoxyuridine, wound healing and Transwell assays, respectively. The results of the present study demonstrated that DANCR had a regulatory role as a competing endogenous RNA and upregulated the expression of E2F1 by sequestering miR­34c­5p in breast cancer cells. Furthermore, E2F1 promoted DANCR transcription by binding to its promoter in breast cancer cells. Notably, the DANCR/miR­34c­5p/E2F1 feedback loop enhanced cell proliferation, migration and invasion in breast cancer cells. Thus, these findings suggested that targeting DANCR may potentially provide a promising future therapeutic strategy for breast cancer treatment.

Blocking the E2F transcription factor 1/high-mobility group box 2 pathway enhances the intervention effects of α-santalol on the malignant behaviors of liver cancer cells.

In view of the tumor-inhibiting effect of α-santalol in various cancers and the role of E2F transcription factor 1 (E2F1) as an important target for anticancer research, this study investigates the relation between α-santalol and E2F1, as well as the effect of α-santalol on liver cancer progression and the corresponding mechanism. Concretely, liver cancer cells were treated with different concentrations of α-santalol. The IC50 value of α-santalol was determined using Probit regression analysis. Then, transcription factors that are targeted by α-santalol and differentially expressed in liver cancer were screened out. The clinicopathological impact of E2F1 and its targets were evaluated and predicted. The expressions of E2F1 and high-mobility group box 2 (HMGB2) and their correlation in the liver cancer tissues were analyzed by bioinformatics. The effects of E2F1 and HMGB2 on the biological characteristics of liver cancer cells were examined through loss/gain-of-function and molecular assays. With the extension of treatment time, the inhibitory effects of 10 µmol/L and 20 µmol/L α-santalol on cancer cell survival rate were enhanced (P < 0.001). E2F1 and HMGB2 were highly expressed and positively correlated in liver cancer tissues (P < 0.05). High E2F1 expression was correlated with large tumors and high TNM stages (P < 0.05). E2F1 knockdown promoted the effects of α-santalol on dose-dependently inhibiting viability, colony formation, invasion and migration (P < 0.05). Moreover, E2F1 knockdown reduced the IC50 value and HMGB2 level, while HMGB2 overexpression produced opposite effects. HMGB2 overexpression and E2F1 knockdown mutually counteracted their effects on the IC50 value and on the viability and apoptosis of α-santalol-treated liver cancer cells (P < 0.01). Collectively, blocking the E2F1/HMGB2 pathway enhances the intervention effects of α-santalol on the proliferation, migration and invasion of liver cancer cells.

Deubiquitylase USP31 Induces Autophagy and Promotes the Progression in Lung Squamous Cell Carcinoma Cells by Stabilizing E2F1 Expression.

BACKGROUND: Autophagy exerts a vital role in the progression of lung squamous cell carcinoma (LUSC). Ubiquitin-specific peptidase 31 (USP31) has recently been found to be involved in the development of a variety of cancers. However, whether USP31 modulates autophagy in LUSC remains unclear. METHODS: This study revealed that high levels of USP31 were discovered in LUSC tissue samples employing the Gene Expression Profiling Interactive Analysis (GEPIA) database, quantitative real- time PCR (qRT-PCR), and Western blot analysis. Cell proliferation was tested via cell counting kit 8 (CCK-8) as well as colony formation, demonstrating that USP31-stable knockdown reduced cell viability. RESULTS: Immunofluorescence analysis illustrated that USP31 knockdown blocked the occurrence of LUSC autophagy. Meanwhile, USP31 has been shown to stabilize the expression of E2F transcription factor 1 (E2F1) through the proteasome pathway. Furthermore, overexpressed E2F1 effectively eliminated the effect of USP31 knockdown on LUSC cell proliferation and autophagy. CONCLUSION: In summary, this investigation proved that USP31 promoted LUSC cell growth and autophagy, at least in part by stabilizing E2F1 expression, which provided a potential therapeutic gene for the treatment of LUSC.

Long non-coding RNA CDKN2B-AS1 promotes hepatocellular carcinoma progression via E2F transcription factor 1/G protein subunit alpha Z axis.

BACKGROUND: A series of long non-coding RNAs (lncRNAs) have been reported to play a crucial role in cancer biology. Some previous studies report that lncRNA CDKN2B-AS1 is involved in some human malignancies. However, its role in hepatocellular carcinoma (HCC) has not been fully deciphered. AIM: To decipher the role of CDKN2B-AS1 in the progression of HCC. METHODS: CDKN2B-AS1 expression in HCC was detected by quantitative real-time polymerase chain reaction. The malignant phenotypes of Li-7 and SNU-182 cells were detected by the CCK-8 method, EdU method, and flow cytometry, respectively. RNA immunoprecipitation was executed to confirm the interaction between CDKN2B-AS1 and E2F transcription factor 1 (E2F1). Luciferase reporter assay and chromatin immunoprecipitation were performed to verify the binding of E2F1 to the promoter of G protein subunit alpha Z (GNAZ). E2F1 and GNAZ were detected by western blot in HCC cells. RESULTS: In HCC tissues, CDKN2B-AS1 was upregulated. Depletion of CDKN2B-AS1 inhibited the proliferation of HCC cells, and the depletion of CDKN2B-AS1 also induced cell cycle arrest and apoptosis. CDKN2B-AS1 could interact with E2F1. Depletion of CDKN2B-AS1 inhibited the binding of E2F1 to the GNAZ promoter region. Overexpression of E2F1 reversed the biological effects of depletion of CDKN2B-AS1 on the malignant behaviors of HCC cells. CONCLUSION: CDKN2B-AS1 recruits E2F1 to facilitate GNAZ transcription to promote HCC progression.

Long noncoding RNA LINC00482 silencing sensitizes non-small cell lung cancer cells to cisplatin by downregulating CLASRP via E2F1.

Long noncoding RNA LINC00482 (LINC00482) is dysregulated in non-small cell lung cancer cells (NSCLC). Herein, this research examined the actions and specific mechanisms of LINC00482 in cisplatin (DDP) resistance in NSCLC. LINC00482 expression was assessed using RT-qPCR in clinical NSCLC tissues and cell lines. Knockdown and ectopic expression assays were conducted in A549 and HCC44 cells, followed by determination of cell proliferation with CCK-8 and clone formation assays, apoptosis with flow cytometry, and DDP sensitivity. The association between LINC00482, E2F1, and CLASRP was evaluated with dual-luciferase reporter, ChIP, and RIP assays. The role of LINC00482 in NSCLC was confirmed in nude mice. NSCLC tissues and cells had upregulated LINC00482 expression. LINC00482 was mainly localized in the cell nucleus, and LINC00482 recruited E2F1 to enhance CLASRP expression in NSCLC cells. LINC00482 knockdown enhanced the DDP sensitivity and apoptosis of NSCLC cells while reducing cell proliferation, which was negated by overexpressing CLASRP. LINC00482 knockdown restricted tumor growth and enhanced DDP sensitivity in NSCLC in vivo. LINC00482 silencing downregulated CLASRP through E2F1 to facilitate the sensitivity to DDP in NSCLC.

Partner of NOB1 homolog transcriptionally activated by E2F transcription factor 1 promotes the malignant progression and inhibits ferroptosis of pancreatic cancer.

Pancreatic cancer (PC) is one of the deadliest malignancies. Partner of NOB1 homolog (PNO1) has been reported to be involved in tumorigenesis. However, the role of PNO1 in PC remains to be elucidated. The purpose of this study was to examine the effects of PNO1 on the progression of PC and the possible mechanism related to E2F transcription factor 1 (E2F1), a transcription factor predicted by the JASPAR database to bind to the PNO1 promoter region and promoted the proliferation of pancreatic ductal adenocarcinoma. First, PNO1 expression in PC tissues and its association with survival rate were analyzed by the Gene Expression Profiling Interactive Analysis database. Western blot and reverse transcription-quantitative polymerase chain reaction were used to evaluate PNO1 expression in several PC cell lines. After PNO1 silencing, cell proliferation, migration, and invasion were measured by colony formation assay, 5-ethynyl-2'-deoxyuridine staining, wound healing, and transwell assays. Then, the lipid reactive oxygen species in PANC-1 cells was estimated by using C11-BODIPY581/591 probe. The levels of glutathione, malondialdehyde, and iron were measured. The binding between PNO1 and E2F1 was confirmed by luciferase and chromatin immunoprecipitation (ChIP) assays. Subsequently, E2F1 was overexpressed in PANC-1 cells with PNO1 knockdown to perform the rescue experiments. Results revealed that PNO1 was highly expressed in PC tissues and PNO1 expression was positively correlated with overall survival rate and disease-free survival rate. Significantly elevated PNO1 expression was also observed in PC cell lines. PNO1 knockdown inhibited the proliferation, migration, and invasion of PANC-1 cells. Moreover, ferroptosis was promoted in PNO1-silenced PANC-1 cells. Results of luciferase and ChIP assays indicated that E2F1 could bind to PNO1 promoter region. Rescue experiments suggested that E2F1 overexpression reversed the impacts of PNO1 depletion on the malignant behaviors and ferroptosis in PANC-1 cells. Summing up, PNO1 transcriptionally activated by E2F1 promotes the malignant progression and inhibits the ferroptosis of PC.

Hydrogen sulfide promoted retinoic acid-related orphan receptor α transcription to alleviate diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is one serious and common complication in diabetes without effective treatments. Hydrogen sulfide (H2S) fights against a variety of cardiovascular diseases including DCM. Retinoic acid-related orphan receptor α (RORα) has protective effects on cardiovascular system. However, whether RORα mediates the protective effect of H2S against DCM remains unknown. The present research was to explore the roles and mechanisms of RORα in H2S against DCM. The study demonstrated that H2S donor sodium hydrosulfide (NaHS) alleviated cell injury but enhanced RORα expression in high glucose (HG)-stimulated cardiomyocytes. However, NaHS no longer had the protective effect on attenuating cell damage and oxidative stress, improving mitochondrial membrane potential, inhibiting necroptosis and enhanced signal transducer and activator of transcription 3 (STAT3) Ser727 phosphorylation in HG-stimulated cardiomyocytes after RORα siRNA transfection. Moreover, NaHS improved cardiac function, attenuated myocardial hypertrophy and fibrosis, alleviated oxidative stress, inhibited necroptosis, but increased STAT3 phosphorylation in wild type (WT) mice but not in RORα knockout mice (a spontaneous staggerer mice, sg/sg mice) with diabetes. Additionally, NaHS increased RORα promoter activity in cardiomyocytes with HG stimulation, which was related to the binding sites of E2F transcription factor 1 (E2F1) in the upstream region of RORα promoter. NaHS enhanced E2F1 expression and increased the binding of E2F1 to RORα promoter in cardiomyocytes with HG stimulation. In sum, H2S promoted RORα transcription via E2F1 to alleviate necroptosis and protect against DCM. It is helpful to propose a novel therapeutic implication for DCM.

Bioinformatics and Experimental Identification of circ_0001535 Associated with Diagnosis and Development of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a type of disease frequently occurs in the elderly population. Diagnosis and treatment methods for this disease are still lacking, and more research is required. In addition, little is known about the function of the circular RNAs (circRNAs) in AD. METHODS: In this research, RNA expression data of AD from the Gene Expression Omnibus (GEO) database were downloaded. The expression levels of circRNAs in cerebrospinal fluid samples of healthy participants and AD patients were measured by reverse transcription­quantitative PCR (RT-qPCR). The diagnosed value of differential expressed circRNAs was analyzed with the Receiver operating characteristic curve (ROC). Pathways related to circ_0001535 were found using gene set enrichment analysis (GSEA) and Metascape. The direct interactions between circ_0001535 and E2F transcription factor 1 (E2F1) or E2F1 and dihydrofolate reductase (DHFR) were verified using Chromatin immunoprecipitation (ChIP) and RNA Binding Protein Immunoprecipitation (RIP) assays. Cell Counting Kit-8 (CCK8) and flow cytometry were used to identify the function of circ_0001535/E2F1/DHFR axis on the proliferation and apoptosis of AD cells. RESULTS: In total, 12 circRNAs have been linked to AD diagnosis. The expression levels of 7 circRNAs differed between AD patients and control groups. Circ_0001535 had the most diagnose value among these circRNAs. Hence, circ_0001535 was regarded as a key circRNA in the present study. E2F1/DHFR axis was predicted to be regulated by circ_0001535. In addition, IP assays experiment results showed that E2F1 could bind to the promoter region of DHFR and be regulated by circ_0001535. In vitro results showed that circ_0001535 overexpression could promote DHFR expression, while E2F1 knock down could inhibit DHFR expression in SH-SY5Y cells. Finally, rescue experiments suggested that circ_0001535 could reduce Aß25-35-induced SH-SY5Y cell proliferation and facilitate apoptosis through E2F1/DHFR axis. CONCLUSIONS: Our research in AD circRNA can offer important information regarding the role of specific circRNAs in the AD environment and point to specific future areas of therapeutic intervention in AD.

E2F1­mediated RAB34 upregulation accelerates the proliferation and inhibits the cell cycle arrest and apoptosis of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a malignant disease that is mainly arisen from myeloid stem/progenitor cells. The pathogenesis of AML is complex. Ras-related protein member RAS oncogene GTPases (RAB) 34 protein has been reported to serve an important role in the development of cancer. However, to the best of our knowledge, the role of RAB34 in AML has not been previously reported. The GEPIA database was used to predict the expression levels of RAB34 in patients with AML. Reverse transcription-quantitative PCR and western blotting were used to detect the expression of RAB34 in AML cell lines. Cell transfection with short hairpin (sh)RNAs targeting RAB34 was used to interfere with RAB34 expression. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining were used to measure cell proliferation. Flow cytometry was used to investigate cell cycle distribution and apoptosis. Western blotting was used to assess the protein expression levels of RAB34 and E2F transcription factor 1 (E2F1), and cell cycle- and apoptosis-associated proteins, including Bcl-2, Bax, CDK4, CDK8 and cyclin D1. The potential binding between E2F1 and RAB34 was then verified by luciferase reporter and chromatin immunoprecipitation assays. Subsequently, cells were co-transfected with RAB34 shRNA and the E2F1 overexpression plasmid before cell proliferation, cell cycle and apoptosis were analyzed further. The expression of RAB34 was found to be significantly increased in AML cell lines. Knocking down RAB34 expression in AML cells was found to significantly inhibit cell proliferation, induce cell cycle arrest and promote apoptosis. E2F1 activated the transcription of RAB34 and E2F1 elevation reversed the impacts of RAB34 silencing on cell proliferation, cell cycle and apoptosis in AML. Therefore, these findings suggest that E2F1-mediated RAB34 upregulation may accelerate the malignant progression of AML.

Pulsed Electromagnetic Fields Combined With Adipose-Derived Stem Cells Protect Ischemic Myocardium by Regulating miR-20a-5p/E2F1/p73 Signaling.

Myocardial infarction (MI) is a serious threat to human health. Although monotherapy with pulsed electromagnetic fields (PEMFs) or adipose-derived stem cells (ADSCs) has been reported to have positive effect on the treatment of MI, a satisfactory outcome has not yet been achieved. In recent years, combination therapy has attracted widespread interest. Herein, we explored the synergistic therapeutic effect of combination therapy with PEMFs and ADSCs on MI and found that the combination of PEMFs and ADSCs effectively reduced infarct size, inhibited cardiomyocyte apoptosis and protected the cardiac function in mice with MI. In addition, bioinformatics analysis and RT-qPCR showed that the combination therapy could affect apoptosis by regulating the expression of miR-20a-5p. A dual-luciferase reporter gene assay also confirmed that the miR-20a-5p could target E2F transcription factor 1 (E2F1) and inhibit cardiomyocyte apoptosis by regulating the E2F1/p73 signaling pathway. Therefore, our study systematically demonstrated the effectiveness of combination therapy on the inhibition of cardiomyocyte apoptosis by regulating the miR-20a-5p/E2F1/p73 signaling pathway in mice with MI. Thus, our study underscored the effectiveness of the combination of PEMFs and ADSCs and identified miR-20a-5p as a promising therapeutic target for the treatment of MI in the future.

CDC25A inhibition suppresses cell proliferation and induces G1/S­phase cell cycle arrest in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a primary malignancy that originates from the nasopharyngeal region. It has been demonstrated that a decrease in the expression level of cell division cycle gene 25A (CDC25A) suppresses cell viability and induces apoptosis in a variety of different types of cancer. However, at present, the role of CDC25A in NPC has yet to be fully elucidated. Therefore, the aim of the present study was to investigate the role of CDC25A in NPC progression and to explore the potential underlying mechanism. Reverse transcription­quantitative PCR was performed to detect the relative mRNA levels of CDC25A and E2F transcription factor 1 (E2F1). Western blot analysis was subsequently used to determine the expression levels of CDC25A, Ki67, proliferating cell nuclear antigen (PCNA) and E2F1. CCK8 assay was employed to measure cell viability and flow cytometric analysis was employed to analyze the cell cycle. The binding sites between the CDC25A promoter and E2F1 were predicted using bioinformatics tools. Finally, luciferase reporter gene and chromatin immunoprecipitation assays were performed to verify the interaction between CDC25A and E2F1. The results obtained suggested that CDC25A is highly expressed in NPC cell lines and CDC25A silencing was found to inhibit cell proliferation, reduce the protein expression levels of Ki67 and PCNA and induce G1 arrest of NPC cells. Furthermore, E2F1 could bind CDC25A and positively regulate its expression at the transcriptional level. In addition, CDC25A silencing abolished the effects of E2F1 overexpression on cell proliferation and the cell cycle in NPC. Taken together, the findings of the present study showed that CDC25A silencing attenuated cell proliferation and induced cell cycle arrest in NPC and CDC25A was regulated by E2F1. Hence, CDC25A may be a promising therapeutic target for treatment of NPC.

Clinical Implication of E2F Transcription Factor 1 in Hepatocellular Carcinoma Tissues.

Background: To date, the clinical management of advanced hepatocellular carcinoma (HCC) patients remains challenging and the mechanisms of E2F transcription factor 1 (E2F1) underlying HCC are obscure. Materials and Methods: Our study integrated datasets mined from several public databases to comprehensively understand the deregulated expression status of E2F1. Tissue microarrays and immunohistochemistry staining was used to validate E2F1 expression level. The prognostic value of E2F1 was assessed. In-depth subgroup analyses were implemented to compare the differentially expressed levels of E2F1 in HCC patients with various tumor stages. Functional enrichments were used to address the predominant targets of E2F1 and shedding light on their potential roles in HCC. Results: We confirmed the elevated expression of E2F1 in HCC. Subgroup analyses indicated that elevated E2F1 level was independent of various stages in HCC. E2F1 possessed moderate discriminatory capability in differentiating HCC patients from non-HCC controls. Elevated E2F1 correlated with Asian race, tumor classification, neoplasm histologic grade, eastern cancer oncology group, and plasma AFP levels. Furthermore, high E2F1 correlated with poor survival condition and pooled HR signified E2F1 as a risk factor for HCC. Enrichment analysis of differentially expressed genes, coexpressed genes, and putative targets of E2F1 emphasized the importance of cell cycle pathway, where CCNE1 and CCNA2 served as hub genes. Conclusions: We confirmed the upregulation of E2F1 and explored the prognostic value of E2F1 in HCC patients. Two putative targeted genes (CCNE1 and CCNA2) of E2F1 were identified for their potential roles in regulating cell cycle and promote antiapoptotic activity in HCC patients.

Effects of over-expression of E2F transcription factor 1 on radiosensitivity glioma U251 cells / 中华行为医学与脑科学杂志

Objective:To investigate the effects of over-expression of E2F transcription factor 1 (E2F1) on proliferation, invasion, apoptosis and radiosensitivity of glioma cell U251.Methods:Real-time quantitative PCR (qRT-PCR) were used to detect the differential expression of E2F1 mRNA in glioma cells LN18, SW1088, U251 and normal brain glial cells. The stable over-expression of E2F1 plasmid was constructed and transfected into U251 cells. qRT-PCR and Western blot test were used to detect the expression of E2F1, pituitary tumor transforming gene 1(PTTG1), C-Myc, B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax) mRNA and protein expression in the control group and E2F1 over-expression group.U251 cells were divided into control group(no X-ray irradiation), irradiation group(6 Gy dose of X-ray), and irradiation + E2F1 over-expression group(transfected with E2F1 first, then irradiated by 6 Gy of X-ray). Cell proliferation ability was detected by cell counting Kit-8(CCK-8) cell viability detection reagent, and cell invasion and migration ability were detected by Transwell chamber. Apoptosis and cell cycle were detected by flow cytometry.GraphPad Prism 8.0 was used for data analysis.The statistical methods were one-way ANOVA and independent sample t-test. Results:qRT-PCR showed that there was statistical difference in the mRNA levels of E2F1( F=201.92, P<0.05) in different cell lines.The expression levels of E2F1 mRNA in LN18(4.04±0.29), SW1088(3.19±0.16)and U251(4.66±0.20) cells were higher than those in HEB(1.02±0.07)cells ( q=27.00, 19.40, 32.52, all P<0.05). After successfully constructing U251 cells with stable over-expression of E2F1 plasmid, qRT-PCR and Western blot detection results showed that: the mRNA and protein levels of E2F1, PTTG1, C-Myc and Bcl-2 in E2F1 over-expression group were higher than those in control group ( t=77.16, 57.88, 4.63, 51.13, 7.50, 70.85, 8.38, 48.81, all P<0.05). Bax mRNA(0.20±0.01) and protein(0.66±0.01) levels were lower than those in control group((1.00±0.02), (0.94±0.01)), and the differences were statistically significant ( t=1.74, 54.65, both P<0.05). After X-ray irradiation (6 Gy), CCK8 detection results showed: the proliferation ability of the three groups at 24, 48, 72 and 96 h were significantly different ( F=95.41, 187.53, 1 158.49, 7 883.78, all P<0.05). The proliferation capacity of the irradiation group were lower than those of the control group at 24, 48, 72 and 96 h ( q=19.51, 27.20, 66.60, 174.9, all P<0.05). The proliferation capacity of irradiation + E2F1 over-expression group at 24, 48, 72 and 96 h were higher than those of irradiation group ( q=10.63, 10.81, 21.11, 60.90, all P<0.05). Transwell assay results showed that there were significant differences in cell invasion and migration ability among the three groups ( F=315.38, 681.10, both P<0.05). The invasion and migration ability of cells in the irradiation group were lower than those in the control group ( q=35.09, 12.76, both P<0.05), and the invasion and migration ability of cells in the irradiation + E2F1 over-expression group were higher than those in the irradiation group ( q=52.06, 22.81, both P<0.05). Flow cytometry showed that there were significant differences in apoptosis rate and percentage of cells in each cycle among the three groups ( F=667.63, 3 213.30, 3 011.26, 861.98, all P<0.05). The percentage of the apoptosis rate, S phase and G2 phase cells in the irradiation group were higher than those in the control group ( q=51.10, 89.39, 51.82, all P<0.05), while the percentage of G1 phase cells in the irradiation group was lower than that in the control group ( q=141.2, P<0.05). The apoptosis rate and percentage of S phase and G2 phase cells in the irradiation + E2F1 over-expression group were lower than those in the irradiation group ( q=18.87, 41.42, 29.31, all P<0.05), while the number of G1 phase cells in the irradiation + E2F1 over-expression group was lower than that in the irradiation group ( q=70.73, P<0.05). Conclusion:Over-expression of E2F1 can reduce the radiosensitivity of glioma U251 cells by regulating the expression of mRNA and protein of genes related to cell cycle and apoptosis, and E2F1 may be involved in the radioresistance of glioma cells.

E2F Transcription Factor 1 Activates FKBP Prolyl Isomerase 4 to Promote Angiogenesis in Cervical Squamous Cell Carcinoma Via the PI3K/AKT Signaling Pathway.

Angiogenesis, namely the formation of blood vessels, is crucial for tumor growth, metastasis and development. E2F transcription factor 1 (E2F1) has been linked to tumorigenesis in several human cancers. This work examines the role of E2F1 and its downstream targets in angiogenesis in cervical squamous cell carcinoma (CSCC). E2F1 was predicted as a candidate oncogene in CSCC using a GSE63514 dataset. Increased E2F1 expression was detected in CSCC tumor samples and cell lines by RT-qPCR, immunohistochemistry, and western blot assays. E2F1 downregulation reduced the angiogenesis activity of HUVECs and the invasiveness of CSCC cells. In vivo, E2F1 knockdown also reduced the xenograft tumor growth and promoted tumor necrosis in mice. FKBP prolyl isomerase 4 (FKBP4) was identified as a target of E2F1. E2F1 bound to FKBP4 promoter for transcriptional activation. Further upregulation of FKBP4 blocked the tumor-suppressive role of E2F1 silencing. FKBP4 was enriched in the PI3K/AKT signaling. In cells and xenograft tumors, the E2F1/FKBP4 axis promoted PI3K and AKT phosphorylation. Activation of the PI3K/AKT signaling restored the angiogenesis activity in cells blocked by E2F1 silencing. In summary, this work demonstrates that E2F1 promotes FKBP4 transcription to activate the PI3K/AKT pathway, which augments the angiogenesis and invasiveness of CSCC.

Health effects and known pathology associated with the use of E-cigarettes.

In recent years, new nicotine delivery methods have emerged, and many users are choosing electronic cigarettes (e-cigarettes) over traditional tobacco cigarettes. E-cigarette use is very popular among adolescents, with more than 3.5 million currently using these products in the US. Despite the increased prevalence of e-cigarette use, there is limited knowledge regarding the health impact of e-cigarettes on the general population. Based on published findings by others, E-cigarette is associated with lung injury outbreak, which increased health and safety concerns related to consuming this product. Different components of e-cigarettes, including food-safe liquid solvents and flavorings, can cause health issues related to pneumonia, pulmonary injury, and bronchiolitis. In addition, e-cigarettes contain alarmingly high levels of carcinogens and toxicants that may have long-lasting effects on other organ systems, including the development of neurological manifestations, lung cancer, cardiovascular disorders, and tooth decay. Despite the well- documented potential for harm, e-cigarettes do not appear to increase susceptibility to SARS-CoV- 2 infection. Furthermore, some studies have found that e-cigarette users experience improvements in lung health and minimal adverse effects. Therefore, more studies are needed to provide a definitive conclusion on the long-term safety of e-cigarettes. The purpose of this review is to inform the readers about the possible health-risks associated with the use of e-cigarettes, especially among the group of young and young-adults, from a molecular biology point of view.

KAT2A/E2F1 Promotes Cell Proliferation and Migration via Upregulating the Expression of UBE2C in Pan-Cancer.

Various studies have shown that lysine acetyltransferase 2A (KAT2A), E2F transcription factor 1 (E2F1), and ubiquitin conjugating enzyme E2 C (UBE2C) genes regulated the proliferation and migration of tumor cells through regulating the cell cycle. However, there is a lack of in-depth and systematic research on their mechanisms of action. This study analyzed The Cancer Genome Atlas (TCGA) to screen potential candidate genes and the regulation network of KAT2A and E2F1 complex in pan-cancer. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB), cell phenotype detection, immunofluorescence co-localization, chromatin immunoprecipitation assay (ChIP), and RNA-Seq techniques were used to explore the functional of a candidate gene, UBE2C. We found that the expression of these three genes was significantly higher in more than 10 tumor types compared to normal tissue. Moreover, UBE2C was mainly expressed in tumor cells, which highlighted the impacts of UBE2C as a specific therapeutic strategy. Moreover, KAT2A and E2F1 could promote cell proliferation and the migration of cancer cells by enhancing the expression of UBE2C. Mechanically, KAT2A was found to cooperate with E2F1 and be recruited by E2F1 to the UBE2C promoter for elevating the expression of UBE2C by increasing the acetylation level of H3K9.

E2F1 induced neuroblastoma cell migration and invasion via activation of CENPE/FOXM1 signaling pathway.

Background: Neuroblastoma (NB) is a common malignancy occurring in infants and young children. Centrosome-associated protein E (CENPE) is a kinetochore-related motor protein highly expressed in NB, with the mechanism largely unknown. This study is committed to investigating the role and mechanism of CENPE in NB.Method: Short hairpin RNAs targeting CENPE and E2F transcription factor 1 (shCENPE and shE2F1) and CENPE overexpression plasmid were transfected into IMR-32 and SK-N-SH cells. The mRNA expressions of CENPE, N-Cadherin, Vimentin, and proliferating cell nuclear antigen (PCNA) in NB cells were detected by qRT-PCR. The viability, migration, and invasion of cells were tested through cell function experiments. Western blot was applied to detect the protein levels of N-Cadherin, Vimentin, PCNA, CENPE and Forkhead box M1 (FOXM1). The relationship between CENPE and E2F1 was verified by dual-luciferase reporter assay, while the interaction between FOXM1 and CENPE in NB cells was analyzed by rescue experiments.Results: CENPE expression was upregulated in NB cells from metastatic sites. Silencing of CENPE suppressed the NB cell viability, migration, and invasion; and decreased N-Cadherin, Vimentin and PCNA expressions, while overexpressed CENPE did oppositely. E2F1 positively targeted CENPE and CENPE partly reversed the effects of shE2F1 on repressing NB cell viability, migration, invasion and the activation of CENPE/FOXM1 signaling pathway. In addition, silenced FOXM1 partly offset the effects of CENPE on promoting NB cell migration and invasion.Conclusion: E2F1 induces NB cell migration and invasion via activating CENPE/FOXM1 pathway.