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Rb/E2f and DREAM complexes play vital roles in regulating cell cycle progression. To date, how they coordinate their functions to regulate cell cycle-dependent gene expression is not clear. Here, we identified a long noncoding RNA (lncRNA), which we named DREAMer , that bridges the interaction between E2f1 and the dREAM complex to regulate endoreplication specifically in Drosophila salivary gland. We show that E2f1 directly stimulates DREAMer expression, whereas DREAMer mediates the repression of e2f1 transcription by modulating the recruitment of the dREAM complex to the e2f1 promoter via a direct interaction with the dREAM component E2f2. The depletion of DREAMer impairs dREAM binding, leading to derepression of e2f1 transcription, which ultimately increases E2f1 activity and promotes the endoreplication. Furthermore, the transcriptomic analysis revealed profound changes in cell cycle-related gene expression in DREAMer KO salivary glands. Together, our findings reveal an lncRNA-mediated link between the dREAM complex and E2f1, which regulates endoreplication during development.

Recently, RBM15 has emerged as an oncogenic factor in a majority of tumors. However, the mechanism is unclear that accounts for how RBM15-induces colorectal cancer (CRC) progression and it is in need of further study. We determined RBM15 expression through the UALCAN database and RT-qPCR. The role of RBM15 in inducing the malignant and aggressive cancerous phenotype was characterized based on the results of the western blot, RT-qPCR, CCK-8 and transwell assays. The target genes of RBM15 were screened by LinkedOmics. m6A methylation kit was applied to analyze the methylation levels of mRNA. SRAMP website was employed to predict m6A sites of targeted mRNA. RIP, dual luciferase reporter gene and actinomycin D assay were conducted to verify the interactions between RBM15 and its targeted gene, and the presence of m6A modification site of its targeted mRNA, respectively. We confirmed the augmentation of RBM15 expression in CRC, which also has a high clinical diagnostic value for CRC. Functionally, RBM15 silencing clearly restrained malignant cellular processes in CRC cells. Mechanistically, RBM15 bound to E2F2 which increased its m6A binding and stabilized the corresponding E2F2 mRNA formation. Excessive E2F2 largely restored the repression malignant phenotype of tumor cells caused by RBM15 silencing. RBM15 regulated E2F2 in an m6A modification-dependent manner thereby boosting malignant cellular processes in CRC. The RBM15/E2F2 axis may be a novel target for CRC therapy., (© 2024 Wiley Periodicals LLC.)

The E2F family of transcription factors plays a crucial role in the regulation of cell cycle progression and cell proliferation. Accumulative evidence indicates that aberrant expression or activation of E2F2 is a common phenomenon in malignances. E2F2 has emerged as a key player in the development and progression of various types of tumors. A wealth of research has substantiated that E2F2 could contribute to the enhancement of tumor cell proliferation, angiogenesis, and invasiveness. Moreover, E2F2 exerts its influence on a myriad of cellular processes by engaging with a spectrum of auxiliary factors and downstream targets, including apoptosis and DNA repair. The dysregulation of E2F2 in the context of carcinogenesis may be attributable to a multitude of mechanisms, which encompass modifications in upstream regulatory elements or epigenetic alterations. This review explores the function of E2F2 in cancer progression and both established and emerging therapeutic strategies aiming at targeting this oncogenic pathway, while also providing a strong basis for further research on the biological function and clinical applications of E2F2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gao, Qiao, Liu and Zhang.)

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Quercetin is widely distributed in plants as a flavonol compound with multiple biological activities. It has been found that quercetin can regulate bone homeostasis through multiple pathways and targets. This study investigated the role and specific molecular mechanisms of quercetin in regulating osteoblast viability, proliferation, migration and osteogenic differentiation. A mouse model of traumatic fracture was established and then 100 mg/kg quercetin corn oil suspension was gavaged at the same time every day for 28 days. miR-6089 and E2F transcription factor 2 (E2F2) expression levels in mice were measured. Fracture healing in mice was observed. MC3T3-E1 cells were transfected with plasmids targeting miR-6089 and E2F2, and cell viability, proliferation, migration, apoptosis, and osteogenic differentiation were determined. The targeting relationship between miR-6089 and E2F2 was verified. In vivo experiments showed that quercetin significantly increased osteocalcin (OCN) expression (P<0.05) and promoted fracture healing in traumatic fracture (TF) mice. miR-6089 expression was down-regulated (P<0.05) and E2F2 expression was up-regulated (P<0.05) in TF mice. Quercetin promoted miR-6089 expression and inhibited E2F2 expression (both P<0.05). In vitro results showed that quercetin promoted miR-6089 expression and inhibited E2F2 expression in a dose-dependent manner (both P<0.05). Quercetin dose-dependently promoted MC3T3-E1 cell viability, proliferation, migration, and osteogenic differentiation, and inhibited MC3T3-E1 cell apoptosis (all P<0.05). Up-regulating miR-6089 further promoted MC3T3-E1 cell viability, proliferation, migration and osteogenic differentiation, and inhibited MC3T3-E1 cell apoptosis (all P<0.05). miR-6089 targeted and regulated E2F2 expression. Up-regulating E2F2 attenuated the promoting effect of up-regulated miR-6089 on MC3T3-E1 cell viability, proliferation, migration, osteogenic differentiation, and inhibition of apoptosis (all P<0.05). We conclude that quercetin enhances osteoblast viability, proliferation, migration, and osteogenic differentiation by modulating the miR-6089/E2F2 axis, thereby promoting fracture healing.

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most common types of cancer in the upper respiratory tract. It is well-known that it has a high mortality rate and poor prognosis in advanced stages. There are well-known risk factors for LSCC, though new specific and prognostic blood-based markers for LSCC development and prognosis are essential. The current study aimed to evaluate the impact of four different single nucleotide polymorphisms (SNPs), E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028), on LSCC development, morphological features, and patient 5-year survival rate., Methods: A total of 200 LSCC patients and 200 controls were included in this study; both groups were matched by age and sex. In the present study, we analyzed four single nucleotide polymorphisms (SNPs) in the genes E2F1 (rs3213183 and rs3213180) and E2F2 (rs2075993 and rs3820028) and evaluated their associations with the risk of LSCC development, its clinical and morphological manifestation, and patients 5-year survival rate. Genotyping was carried out using RT-PCR., Results: None of the analyzed SNPs showed a direct association with LSCC development. E2F2 rs2075993 G allele carriers (OR = 4.589, 95% CI 1.050-20.051, p = 0.043) and rs3820028 A allele carriers (OR = 4.750, 95% CI 1.088-20.736, p = 0.038) had a statistically significantly higher risk for poor differentiated or undifferentiated LSCC than non-carriers. E2F1 rs3213180 GC heterozygotes were found to have a 3.7-fold increased risk for lymph node involvement (OR = 3.710, 95% CI 1.452-9.479, p = 0.006). There was no statistically significant association between investigated SNPs and patient 5-year survival rate., Conclusions: The present study indicates that E2F2 rs2075993 and rs3820028 impact LSCC differentiation, whereas E2F1 rs3213180 - on lymph node involvement., (© 2024. The Author(s).)

Multiple myeloma (MM) is the second most common haematological malignancy. Despite the development of new drugs and treatments in recent years, the therapeutic outcomes of patients are not satisfactory. It is necessary to further investigate the molecular mechanism underlying MM progression. Herein, we found that high E2F2 expression was correlated with poor overall survival and advanced clinical stages in MM patients. Gain- and loss-of-function studies showed that E2F2 inhibited cell adhesion and consequently activated cell epithelial-to-mesenchymal transition (EMT) and migration. Further experiments revealed that E2F2 interacted with the PECAM1 promoter to suppress its transcriptional activity. The E2F2-knockdown-mediated promotion of cell adhesion was significantly reversed by the repression of PECAM1 expression. Finally, we observed that silencing E2F2 significantly inhibited viability and tumour progression in MM cell models and xenograft mouse models respectively. This study demonstrates that E2F2 plays a vital role as a tumour accelerator by inhibiting PECAM1-dependent cell adhesion and accelerating MM cell proliferation. Therefore, E2F2 may serve as a potential independent prognostic marker and therapeutic target for MM., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

It is known that E2F2 (E2F transcription factor 2) plays an important role as controller in the cell cycle. This study aimed to analyse the expression of the E2F2 gene and E2F2 protein and demonstrate E2F2 target microRNAs (miRNAs) candidates (miR-125b-5p, miR-155-3p, and miR-214-5p) in oral squamous cell carcinoma tumour and margin samples. The study group consisted 50 patients. The E2F2 gene and miRNAs expression levels were assessed by qPCR, while the E2F2 protein was assessed by ELISA. When analysing the effect of miRNAs expression on E2F2 gene expression and E2F2 protein level, we observed no statistically significant correlations. miR-125b-5p was downregulated, while miR-155-3p, and miR-214-5p were upregulated in tumour samples compared to margin. We observed a difference between the miR-125b-5p expression level in smokers and non-smokers in margin samples. Furthermore, HPV-positive individuals had a significantly higher miR-125b-5p and miR-214-5p expression level compared to HPV-negative patients in tumour samples. The study result showed that the E2F2 gene is not the target for analysed miRNAs in OSCC. Moreover, miR-155-3p and miR-125b-5p could play roles in the pathogenesis of OSCC. A differential expression of the analysed miRNAs was observed in response to tobacco smoke and HPV status.

Human embryonic stem cells (hESCs) hold the potential to solve the problem of the shortage of functional hepatocytes in clinical applications and drug development. However, a large number of usable hepatocytes derived from hESCs cannot be effectively obtained due to the limited proliferation capacity. In this study, we found that enhancement of liver transcription factor C/EBPβ during hepatic differentiation could not only significantly promote the expression of hepatic genes, such as albumin, alpha fetoprotein, and alpha-1 antitrypsin, but also dramatically reinforce proliferation-related phenotypes, including increasing the expression of proliferative genes, such as CDC25C, CDC45L, and PCNA, and the activation of cell cycle and DNA replication pathways. In addition, the analysis of CUT&Tag sequencing further revealed that C/EBPβ is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBPβ and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBPβ could significantly inhibit the expression of the aforementioned proliferative genes. RNA transcriptome analysis and GSEA enrichment indicated that the E2F family was enriched, and the expression of E2F2 was changed with the overexpression or knockdown of C/EBPβ. Moreover, the results of CUT&Tag sequencing showed that C/EBPβ also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBPβ and E2F2 proteins, and this interaction between these two proteins was also verified in the LO2 cell line, a hepatic progenitor cell line. Thus, our results demonstrated that C/EBPβ first initiated E2F2 expression and then coupled with E2F2 to regulate the expression of proliferative genes in hepatocytes during the differentiation of hESCs. Therefore, our findings open a new avenue to provide an in vitro efficient approach to generate proliferative hepatocytes to potentially meet the demands for use in cell-based therapeutics as well as for pharmaceutical and toxicological studies.

Endothelial cell senescence has a vital implication for vascular dysfunction, leading to age-related cardiovascular disease, especially hypertension and atherosclerosis. E2F transcription factor 2 (E2F2) plays a critical role in cell proliferation, differentiation, and DNA damage response. Up to date, no study has ever connected E2F2 to vascular endothelial cell senescence. Here, we demonstrate that E2F2 is involved in endothelial cellular senescence. We found that E2F2 expression is decreased during the replicative senescence of human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. The knockdown of E2F2 in young HUVECs induces premature senescence characterized by an increase in senescence-associated β-galactosidase (SA-β-gal) activity, a reduction in phosphorylated endothelial nitric oxide synthase (p-eNOS) and sirtuin 1 (SIRT1), and the upregulation of senescence-associated secretory phenotype (SASP) IL-6 and IL-8. The lack of E2F2 promoted cell cycle arrest, DNA damage, and cell proliferation inhibition. Conversely, E2F2 overexpression reversed the senescence phenotype and enhanced the cellular function in the senescent cells. Furthermore, E2F2 deficiency downregulated downstream target genes including CNNA2, CDK1, and FOXM1, and overexpression restored the expression of these genes. Our findings demonstrate that E2F2 plays an indispensable role in endothelial cell senescence.

Both pro-oncogenic and anti-oncogenic effects of E2F2 have been revealed in different malignancies. However, the precise role of E2F2 in pancreatic cancer, in particular in relation to therapeutic intervention with gemcitabine, remains unclear. In this study, the effect of E2F2 on the proliferation and cell cycle modulation of pancreatic cancer cells, and whether E2F2 plays a role in the treatment of pancreatic cancer cells by gemcitabine, were investigated. The expression of E2F2 in pancreatic cancer was assessed by various methods including bioinformatics prediction, Western blotting, and real-time PCR. The effect of E2F2 on the proliferation and cell cycling of pancreatic cancer cells was analyzed by tissue culture and flow cytometry. In addition, the effect of E2F2 on the intervention of pancreatic cancer by gemcitabine was investigated using both in vitro and in vivo approaches. The expression of E2F2 was found to be significantly increased in pancreatic cancer tissues and cell lines. The pathogenic capacity of E2F2 lied in the fact that this transcription factor promoted the transformation of pancreatic cancer cell cycle from G1-phase to S-phase, thus enhancing the proliferation of pancreatic cancer cells. Furthermore, the expression of E2F2 was increased in pancreatic cancer cells in the presence of gemcitabine, and the augmented expression of E2F2 upregulated the gemcitabine resistance-related gene RRM2 and its downstream signaling molecule deoxycytidine kinase (DCK). The resistance of pancreatic cancer cells to gemcitabine was confirmed using both in vitro and in vivo models. In this study, E2F2 has been demonstrated for the first time to play a pro-oncogenic role in pancreatic cancer by promoting the transition of the cell cycle from G1-phase to S-phase and, therefore, enhancing the proliferation of pancreatic cancer cells. E2F2 has also been demonstrated to enhance the chemotherapy resistance of pancreatic cancer cells to gemcitabine by upregulating the expression of RRM2 and DCK that is downstream of RRM2., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Long noncoding RNAs (lncRNAs) participate in biological processes in multiple types of tumors. However, the regulatory patterns of lncRNAs in prostate cancer remain largely unclear. Here, we evaluated the expression and roles of the lncRNA DLEU2 in prostate cancer. Our results showed that DLEU2 was upregulated in advanced prostate cancer tissues. Patients with prostate cancer displaying high expression of DLEU2 had a poor prognosis. Moreover, we demonstrated that overexpression of DLEU2 facilitated the proliferation, migration, and invasion of prostate cancer in vitro. Mechanistically, DLEU2 promoted serum and glucocorticoid-induced protein kinase 1 (SGK1) expression by acting as an miR-582-5p sponge, and the transcription of DLEU2 was activated by the dysregulation of E2F transcription factor 2 (E2F2) expression in prostate cancer. Furthermore, knockdown of DLEU2 attenuated prostate cancer tumorigenesis in vivo. Notably, these findings suggested that E2F2-activated DLEU2 may function as a competing endogenous RNA to facilitate prostate cancer progression by targeting the miR-582-5p/SGK1 axis., (© 2022. The Author(s).)

The aim of this study was to investigate the effect of circCUL2 on the proliferation, invasion and migration of retinoblastoma cells by regulating the miR-214-5p/E2F2 axis. qRT-PCR and western blot were performed to detect the expressions of circCUL2, miR-214-5p and E2F2 in tumor tissues and adjacent normal tissues from retinoblastoma patients, and in normal human retinal epithelial cells ARPE-19 and human retinoblastoma cells Y79 and SO-Rb50. qRT-PCR and western blot were performed for the detection of RNA levels of circCUL2 and miR-214-5p and the mRNA and protein levels of E2F2, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for cell proliferation ability, Transwell assay for cell invasion ability, and scratch assay for cell migration ability. Luciferase dual reporter assay was used to detect the targeting relationship between circCUL2 and miR-214-5p, and between miR-214-5p and E2F2. CircCUL2 and E2F2 were lowly expressed, while miR-214-5p was highly expressed in retinoblastoma tumor tissues and cells. Transfection with pcDNA3.1-CUL2 or miR-214-5p inhibitor inhibited the proliferation, invasion and migration of Y79 and SO-Rb50 cells compared with the negative control; while transfection with sh-CUL2 or miR-214-5p mimics promoted the proliferation, invasion and migration of Y79 and SO-Rb50 cells. CircCUL2 negatively regulated miR-214-5p, while miR-214-5p negatively regulated E2F2. Overexpression of miR-214-5p or silencing of E2F2 in SO-Rb50 cells partially reversed the inhibitory effect of circCUL2 on the proliferation, invasion and migration of retinoblastoma cells. CircCUL2 inhibited the proliferation, invasion and migration of retinoblastoma cells by regulating the miR-214-5p/E2F2 axis., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: In this study, we aimed to screen methylation signatures associated with the prognosis of patients with clear cell renal cell carcinoma (ccRCC)., Methods: Gene expression and methylation profiles of ccRCC patients were downloaded from publicly available databases, and differentially expressed genes (DEGs)-differentially methylated genes (DMGs) were obtained. Subsequently, gene set enrichment and transcription factor (TF) regulatory network analyses were performed. In addition, a prognostic model was constructed and the relationship between disease progression and immunity was analyzed., Results: A total of 23 common DEGs-DMGs were analyzed, among which 14 DEGs-DMGs were obtained with a cutoff value of PCC < 0 and p < 0.05. The enrichment analysis showed that the 14 DEGs-DMGs were enriched in three GO terms and three KEGG pathways. In addition, a total of six TFs were shown to be associated with the 14 DEGs-DMGs, including RP58, SOX9, NF-κB65, ATF6, OCT, and IK2. A prognostic model using five optimized DEGs-DMGs which efficiently predicted survival was constructed and validated using the GSE105288 dataset. Additionally, four types of immune cells (NK cells, macrophages, neutrophils, and cancer-associated fibroblasts), as well as ESTIMATE, immune, and stromal scores were found to be significantly correlated with ccRCC progression (normal, primary, and metastasis) in addition to the five optimized DEGs-DMGs., Conclusion: A five-gene methylation signature with the predictive ability for ccRCC prognosis was investigated in this study, consisting of CCNB2, CDKN1C, CTSH, E2F2, and ERMP1. In addition, potential targets for methylation-mediated immunotherapy were highlighted., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

B-Myb is an important transcription factor that plays a critical role in gene expression regulation and tumorigenesis. However, its functional implication in colorectal cancer remains elusive. In this study, we found that B-Myb was significantly upregulated at both mRNA and protein levels in colorectal cancer samples compared to non-tumor counterparts. B-Myb overexpression accelerated cell proliferation, cell cycle progression and cell motility in colorectal cancer cells, and promoted tumor growth in orthotopic nude mouse models in vivo. In contrast, B-Myb depletion inhibited these malignant phenotypes. Mechanistic investigations revealed that E2F2 was a novel transcriptional target of B-Myb and is essential to B-Myb-induced malignant phenotypes. Notably, B-Myb and E2F2 exhibited positive expression correlation, and interacted with each other in colorectal cancer cells. In addition to their autoregulatory mechanisms, B-Myb and E2F2 can also directly transactivate each other, thus constituting consolidated reciprocal feed-forward transactivation loops. Moreover, both B-Myb and E2F2 are required for the activation of ERK and AKT signaling pathways in colorectal cancer cells. Taken together, our data clarified a critical role for B-Myb in colorectal cancer and unraveled an exquisite mutual collaboration and reciprocal cross regulation between B-Myb and E2F2 that contribute to the malignant progression of human colorectal cancer., (© 2021. The Author(s).)

Background: Nova Circular RNA (circRNA) of non-coding RNA has gradually become an important regulatory factor, and it has made people attach great concern over the occurrence and development of many diseases, particularly carcinomas. circ&#95;RPPH1 is a newly discovered circRNA. Gene Expression Omnibus (GEO) analysis showed that there are high contents of circ&#95;RPPH1 in breast cancer (BC), but the mechanism of circRNA in BC remains unclear., Methods: Real-time quantitative PCR (qRT-PCR) was applied to test the role of circ&#95;RPPH1 in BC patients, and functional experiments were applied to test the role of circ&#95;RPPH1 on BC tumor. Fluorescence in situ hybridization, double luciferase reporter gene analysis, RNA pull-down and RNA immunoprecipitation experiments were performed to explore the correlation of circ&#95;RPPH1 with miR-146b-3p/E2F2 in BC., Results: circ&#95;RPPH1 was evidently enhanced in BC, and its content was related to the clinical stage and pathological grade. circ&#95;RPPH1 can accelerate the proliferation, migration and invasion, and promote tumorigenesis and metastasis. Mechanism exploration indicated that circ&#95;RPPH1 acted as ceRNA (competing endogenous RNA) of miR-146b-3p, so as to reduce the inhibitory role of miR-146b-3p on its target E2F2., Conclusion: Circ&#95;RPPH1/miR-146b-3p/E2F2 axis can promote the progression of BC, and it might be a latent therapeutic target for clinical BC.

Ovarian cancer (OC) is a gynecological malignancy with a poor prognosis and low survival rate. E2F2 is a transcription activator that plays an indispensable role in cell proliferation and cell cycle progression. The preliminary analysis indicated that the E2F2 gene could produce three circular RNAs (circRNAs). This study aimed to investigate whether these circRNAs would be involved in OC tumorigenesis. The results showed that one of the circRNAs (termed circE2F2) was significantly upregulated in OC tissues and cell lines, and high circE2F2 expression was associated with poor survival in OC patients. The knockdown of circE2F2 in OC cells suppressed cell proliferation, migration, invasion, and cellular glucose metabolism. In circE2F2-deficient cells, the half-life of the E2F2 mRNA was significantly shorter than that in the control group, indicating that sufficient circE2F2 expression could strengthen the stability of the E2F2 mRNA. Further analysis revealed that circE2F2 could bind to RNA-binding protein Hu antigen R (HuR). Moreover, circE2F2 enhanced the stability of the E2F2 mRNA via binding to the HuR protein. Also, E2F2 overexpression significantly enhanced the mobility, invasiveness, and glucose metabolism of OC cells with insufficient circE2F2 expression, suggesting that circE2F2 induced OC cell growth and metastasis by upregulating E2F2. In conclusion, circE2F2 promoted OC cell proliferation, metastasis, and glucose metabolism by stabilizing the E2F2 mRNA via binding to the HuR protein. These findings suggest a novel regulatory mechanism for the oncogenic effects of circE2F2, E2F2, and HuR on ovarian carcinogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Renal cell carcinoma is the second malignant tumors in the urinary system with high mortality and morbidity. Increasing evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in tumor development and progression. In the current study, based on the publicly available data obtained from GEO and TCGA database, we identified five prognosis-related lncRNAs with the ability to predict the prognosis of patients with renal cell carcinoma. Among them, the uncharacterized and upregulated lncRNA RCAT1 (renal cancer-associated transcript 1) was identified as the key lncRNA. Our data further revealed that the expression of lncRNA RCAT1 was significantly upregulated in renal cell carcinoma tissues and cells. Gain-of-function and loss-of-function studies showed that lncRNA RCAT1 promoted cell proliferation, migration, and invasion in vitro and in vivo. Furthermore, we verified that lncRNA RCAT1 could abundantly sponge miR-214-5p, which served as a tumor suppressor in renal cell carcinoma. Significantly, miR-214-5p overexpression could attenuate the promotion of cell proliferation and metastasis induced by lncRNA RCAT1. Moreover, we found that E2F2 was a direct target of miR-214-5p, and lncRNA RCAT1 could protect E2F2 from miR-214-5p-mediated degradation. Taken together, our findings suggested that lncRNA RCAT1 could enhance the malignant phenotype of renal cell carcinoma cells by modulating miR-214-5p/E2F2 axis, and lncRNA RCAT1 might be a novel prognostic biomarker and a potential therapeutic target for renal cell carcinoma., (© 2021. The Author(s).)

Aims: The effects of PFKFB4 on glycolysis during the cancer progression has been investigated, while its role in glioma remains unclear. The present study evaluated the molecular mechanism of PFKFB4 in glycolysis of glioma progression., Materials and Methods: The pan-cancer platform SangerBox was inquired to investigate the E2F2 expression in tumors. The E2F2 expression was studied by qRT-PCR and immunohistochemistry in collected glioma and normal brain tissues and by qRT-PCR and western blot in glioma cells. The relationship between the E2F2 expression in glioma tissues and patients' prognosis was analyzed. The cell malignant phenotype, glycolysis, growth and metastasis were examined by CCK-8, EdU, colony formation, flow cytometry, wound healing, Transwell assays, ELISA kits, and tumorigenesis and metastasis assays. Downstream targets of E2F2 were searched in hTFtarget, followed by pathway enrichment analysis. The expression of these targets and their correlation with E2F2 expression in gliomas were investigated through the GEPIA website. After ChIP and luciferase assays, the effect of the target on glioma was investigated., Key Findings: E2F2 was overexpressed in glioma patients and predicted poor prognoses. E2F2 promoted cell proliferation, colony formation, DNA synthesis, migration, invasion and glycolysis, and inhibited apoptosis. Meanwhile, inhibition of E2F2 suppressed the growth and metastasis of gliomas. E2F2 elevated the PFKFB4 expression transcriptionally by binding to its promoter and activated PI3K/AKT pathway. The promotion of glioma metastasis and glycolysis by E2F2 was mitigated by PFKFB4 knockdown., Significance: E2F2-mediated transcriptional enhancement of PFKFB4 expression regulated the phosphorylation of PI3K/AKT to promote glioma malignancy progression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lipid metabolism rearrangements in nonalcoholic fatty liver disease (NAFLD) contribute to disease progression. NAFLD has emerged as a major risk for hepatocellular carcinoma (HCC), where metabolic reprogramming is a hallmark. Identification of metabolic drivers might reveal therapeutic targets to improve HCC treatment. Here, we investigated the contribution of transcription factors E2F1 and E2F2 to NAFLD-related HCC and their involvement in metabolic rewiring during disease progression. In mice receiving a high-fat diet (HFD) and diethylnitrosamine (DEN) administration, E2f1 and E2f2 expressions were increased in NAFLD-related HCC. In human NAFLD, E2F1 and E2F2 levels were also increased and positively correlated. E2f1 -/- and E2f2 -/- mice were resistant to DEN-HFD-induced hepatocarcinogenesis and associated lipid accumulation. Administration of DEN-HFD in E2f1 -/- and E2f2 -/- mice enhanced fatty acid oxidation (FAO) and increased expression of Cpt2 , an enzyme essential for FAO, whose downregulation is linked to NAFLD-related hepatocarcinogenesis. These results were recapitulated following E2f2 knockdown in liver, and overexpression of E2f2 elicited opposing effects. E2F2 binding to the Cpt2 promoter was enhanced in DEN-HFD-administered mouse livers compared with controls, implying a direct role for E2F2 in transcriptional repression. In human HCC, E2F1 and E2F2 expressions inversely correlated with CPT2 expression. Collectively, these results indicate that activation of the E2F1-E2F2-CPT2 axis provides a lipid-rich environment required for hepatocarcinogenesis. SIGNIFICANCE: These findings identify E2F1 and E2F2 transcription factors as metabolic drivers of hepatocellular carcinoma, where deletion of just one is sufficient to prevent disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2874/F1.large.jpg., (©2021 American Association for Cancer Research.)

Background: E2F2 is a member of the E2F transcription factor family and has important but not fully understood biological functions in cancers. The biological role of E2F2 in gastric cancer (GC) also remains unclear., Methods: We examined the expression levels of E2F2 in GC using publicly available datasets such as TIMER, Oncomine, GEPIA, UALCAN, etc., and in our patient cohort, using quantitative real-time PCR, western blotting, and immunohistochemistry. We further investigated the effects of E2F2 on phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling, autophagy, and the migration and invasion of GC cells by the wound healing assay, Transwell assay and transmission electron microscopy., Results: E2F2 was highly expressed in both GC tissues and cells compared with normal gastric tissues/cells. High E2F2 expression was associated with poor overall survival (OS). In addition, the expression of E2F2 in GC was strongly correlated with a variety of immune markers. E2F2 overexpression promoted the migration and invasiveness of GC cells in vitro through inhibition of PI3K/Akt/mTOR-mediated autophagy., Conclusion: High E2F2 expression was associated with the characteristics of invasive tumors and poor prognosis. E2F2 also had potential modulatory effects on tumor immunity. We discovered a novel function of E2F2 in the regulation of PI3K/Akt/mTOR-mediated autophagy and the downstream processes of cell migration and invasion.

Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are subgroups of head and neck squamous cell carcinoma. E2F Transcription Factor 2 (E2F2) could contribute to cancer development, because it plays a critical role in many cellular processes, including the cell cycle, proliferation, differentiation, DNA damage response, and cell death. In the current study, we assessed the associations of five E2F2 polymorphisms (rs6667575, rs3218121, rs3218211, rs3218148, and rs3218203) with OSCC and OPSCC and influence on the TNM staging and grading. This is the first such survey to concern the European population. The study included 94 primary tumour samples following surgical resection from patients, whereas the control group consisted of 99 healthy individuals. We tried a matching of cases and controls for age and sample size. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination. Our results suggested that the most significant difference between the control group and the cancer group was the A/G heterozygote for rs3218121. Samples containing this genotype were mostly found in the control group. In our samples, rs6667575, rs3218121, rs3218211, and rs3218148 polymorphisms may affect the course of OSCC and OPSCC, while rs3218203 was not associated with OSCC and OPSCC. However, further studies are warranted to confirm our findings., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Karolina Gołąbek et al.)

LBX2-AS1 is a long non-coding RNA that facilitates the development of gastrointestinal cancers and lung cancer, but its participation in ovarian cancer development remained uninvestigated. Clinical data retrieved from TCGA ovarian cancer database and the clinography of 60 ovarian cancer patients who received anti-cancer treatment in our facility were analysed. The overall cell growth, colony formation, migration, invasion, apoptosis and tumour formation on nude mice of ovarian cancer cells were evaluated before and after lentiviral-based LBX2-AS1 knockdown. ENCORI platform was used to explore LBX2-AS1-interacting microRNAs and target genes of the candidate microRNAs. Luciferase reporter gene assay and RNA pulldown assay were used to verify the putative miRNA-RNA interactions. Ovarian cancer tissue specimens showed significant higher LBX2-AS1 expression levels that non-cancerous counterparts. High expression level of LBX2-AS1 was significantly associated with reduced overall survival of patients. LBX2-AS1 knockdown significantly down-regulated the cell growth, colony formation, migration, invasion and tumour formation capacity of ovarian cancer cells and increased their apoptosis in vitro. LBX2-AS1 interacts with and thus inhibits the function of miR-455-5p and miR-491-5p, both of which restrained the expression of E2F2 gene in ovarian cancer cells via mRNA targeting. Transfection of miRNA inhibitors of these two miRNAs or forced expression of E2F2 counteracted the effect of LBX2-AS1 knockdown on ovarian cancer cells. LBX2-AS1 was a novel cancer-promoting lncRNA in ovarian cancer. This lncRNA increased the cell growth, survival, migration, invasion and tumour formation of ovarian cancer cells by inhibiting miR-455-5p and miR-491-5p, thus liberating the expression of E2F2 cancer-promoting gene., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Dioxins are one of the most potent anthropogenic poisons, causing systemic disorders in embryonic development and pathologies in adults. The mechanism of dioxin action requires an aryl hydrocarbon receptor (AhR), but the downstream mechanisms are not yet precisely clear. Here, we performed a meta-analysis of all available transcriptome datasets taken from human cell cultures exposed to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Differentially expressed genes from different experiments overlapped partially, but there were a number of those genes that were systematically affected by TCDD. Some of them have been linked to toxic dioxin effects, but we also identified other attractive targets. Among the genes that were affected by TCDD, there are functionally related gene groups that suggest an interplay between retinoic acid, AhR, and Wnt signaling pathways. Next, we analyzed the upstream regions of differentially expressed genes and identified potential transcription factor (TF) binding sites overrepresented in the genes responding to TCDD. Intriguingly, the dioxin-responsive element (DRE), the binding site of AhR, was not overrepresented as much as other cis-elements were. Bioinformatics analysis of the AhR binding profile unveils potential cooperation of AhR with E2F2, CTCFL, and ZBT14 TFs in the dioxin response. We discuss the potential implication of these predictions for further dioxin studies.

The E2F family, classically known for a central role in cell cycle, has a number of emerging roles in cancer including angiogenesis, metabolic reprogramming, metastasis and DNA repair. E2F1 specifically has been shown to be a critical mediator of DNA repair; however, little is known about DNA repair and other E2F family members. Here we present an integrative bioinformatic and high throughput drug screening study to define the role of E2F2 in maintaining genomic integrity in breast cancer. We utilized in vitro E2F2 ChIP-chip and over expression data to identify transcriptional targets of E2F2. This data was integrated with gene expression from E2F2 knockout tumors in an MMTV-Neu background. Finally, this data was compared to human datasets to identify conserved roles of E2F2 in human breast cancer through the TCGA breast cancer, Cancer Cell Line Encyclopedia, and CancerRx datasets. Through these methods we predict that E2F2 transcriptionally regulates mediators of DNA repair. Our gene expression data supports this hypothesis and low E2F2 activity is associated with a highly unstable tumor. In human breast cancer E2F2, status was also correlated with a patient's response to PARP inhibition therapy. Taken together this manuscript defines a novel role of E2F2 in cancer progression beyond cell cycle and could impact patient treatment.

The cell cycle-related transcription factor E2F1 is a member of the cell cycle-related transcription factor E2F family, mainly involved in various cell processes including cell cycle progression, DNA repair, DNA replication, cell differentiation, proliferation, and apoptosis. E2F1 is highly expressed in a variety of tumor tissues and cells, and it plays a role as a cancer-promoting gene. The up-regulation of E2F1 expression is closely related to the occurrence, development, metastasis and prognosis of tumors. Therefore, E2F1 is expected to become a new target for cancer treatment. This article reviews the latest research progress of E2F1 in current common tumors.
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Background: The pathogenesis of the abnormality of the immune system is still not clear at present. Chemosynthetic drugs, human or animal immune products and microbiological drugs are used as the main drugs in clinics currently, but these drugs have different side effects. So researchers turned to safer natural products in order to find immunomodulatory active substances from natural products and their extracts., Methods: Immunosuppressed mice were induced by cyclophosphamide and administered with Cordyceps militaris polypeptide (CMP) for the study on the effect of CMP on the immune function of mice and its mechanism. Based on the 1748 differential gene sets selected in our previous work, the transcription factors and their corresponding target genes were screened by integrating the TRED (Transcriptional Regulatory Element Database), a transcriptional factor-target gene regulatory network was constructed, then the role of transcription factors in the regulatory network was elucidated by statistically analyzing the key nodes, and finally, the correlation of network genes with diseases was analyzed by using the DAVID database., Results: The results of animal experiments showed that CMP could increase the immune organ indexes, the number of white blood cells, the degree of delayed allergy and the content of hemolysin in the serum of mice. CMP was found to be involved in the regulation of immune function in mice through genes Kdr, Spp1, Ptgs2, Rel, and Smad3, and transcription factors Ets1, E2f2 and E2f1. E2F2 and E2F1 are members of the E2F family, so we speculated that the E2F family might play an important role, and its main regulatory pathways were the PI3K-Akt signaling pathway and TNF signaling pathway., Conclusion: CMP can improve the immunity of mice. CMP can regulate the immune function of mice through multiple genes and transcription factors, and may also play a role in immune-related diseases, such as cancer.

Background: Tumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor)., Methods: Functionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination., Results: Compared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway., Conclusion: Transcriptomic analyses revealed for the first time, that the estrogen-mediated S-phase entry pathway partially contributed to the synergistic effect of the drug combination. More importantly, our studies led to the discoveries of new potential therapeutic targets, such as E2F2, as well as a novel drug-induced targeting of estrogen receptor β (ESR2) approach for triple-negative breast cancer treatment, currently lacking of targeted therapies.

Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Long noncoding RNAs (lncRNAs) display essential roles in cancer progression. FLVCR1-AS1 is a rarely investigated lncRNAs involved in various human cancers, such as hepatocellular carcinoma and lung cancer. However, its function in glioma has not been clarified. In our study, we found that FLVCR1-AS1 was highly expressed in glioma tissues and cell lines. And upregulation of FLVCR1-AS1 predicted poor prognosis in patients with glioma. Moreover, FLVCR1-AS1 knockdown inhibited proliferation, migration and invasion of glioma cells. Through bioinformatics analysis, we identified that FLVCR1-AS1 was a sponge for miR-4731-5p to upregulate E2F2 expression. Moreover, rescue assays indicated that FLVCR1-AS1 modulated E2F2 expression to participate in glioma progression. Altogether, our research demonstrates that the FLVCR1-AS1/miR-4731-5p/E2F2 axis is a novel signaling in glioma and may be a potential target for tumor therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The miR-638 acted as a tumor suppressor and E2F transcription factor 2 (E2F2) was a critical regulator in some cancers, while the role of them on stemness of breast cancer stem cells (BCSCs) was rarely detailed. Hence, we focused on exploring the effects of miR-638 and E2F2 on BCSCs stemness., Methods: The proportion of CD24 -/CD44 + cells of BCSCs was detected by flow cytometry. The target relationship of miR-638 and E2F2 was explored using luciferase assays. The ability of self-renewal, proliferation, and invasion of BCSCs were determined by Mammosphere forming, Cell Counting Kit-8 (CCK-8), colony formation, and transwell assays. Xenograft tumor was established to detect the influence of miR-638 on tumor growth., Results: miR-638 was down-regulated, while E2F2 was elevated in breast cancer. The E2F2 level was negatively correlated with miR-638. The BCSCs represented higher proportion of CD24 -/CD44 + cells and levels of sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4). The miR-638 was down-regulated and E2F2 was increased in BCSCs. MiR-638 could target to E2F2 and decreased the level of E2F2 in BCSCs cells. Overexpression of miR-638 decreased the proportion of CD24 -/CD44 + cells and the levels of SOX2 and OCT4 by inhibiting E2F2. The overexpression of miR-638 also inhibited the abilities of self-renewal, proliferation, and invasion of BCSCs by inhibiting E2F2. The miR-638 overexpression inhibited the breast tumor growth., Conclusion: MiR-638 represses the characteristics and behaviors of BCSCs by targeting E2F2. MiR-638 may be a potential target for breast cancer therapy.

Objective: Lung cancer is the main burden on human health, with high mortality and poor prognosis. The involvement of long non-coding RNAs (lncRNAs) in the development of cancer has attracted wide attention. This study aimed to investigate the role and novel mechanisms of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in the progression of lung cancer., Materials and Methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of NNT-AS1, microRNA-3666 (miR-3666), and E2F transcription factor 2 (E2F2). 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to analyze cell proliferation. Flow cytometry was carried out to investigate cell apoptosis. Transwell assay was conducted to observe cell invasion. The interaction between miR-3666 and NNT-AS1 or E2F2 was predicted by bioinformatics tool starBase v2.0 and verified by Dual-Luciferase reporter assay. The protein level of E2F2 was quantified by Western blot., Results: NNT-AS1 and E2F2 were upregulated, but miR-3666 was downregulated in lung cancer tissues and cells. NNT-AS1 knockdown attenuated proliferation and invasion but enhanced apoptosis of lung cancer cells, while miR-3666 inhibition reversed these effects. It was confirmed that miR-3666 was a target of NNT-AS1 and it directly interacted with E2F2. The inhibitory proliferation and invasion, and acceleratory apoptosis of lung cancer cells, caused by miR-3666 enrichment, were overturned by E2F2 overexpression. Furthermore, E2F2 was regulated by NNT-AS1 through miR-3666., Conclusions: NNT-AS1 participated in the progression of lung cancer through NNT-AS1/miR-3666/E2F2 regulatory axis at least in part. Our study supplied a promising strategy for the treatment of lung cancer.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factor stimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology., (© 2019 Wiley Periodicals, Inc.)

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle., (Copyright © 2019 Elsevier Inc. All rights reserved.)

BACKGROUND Long non-coding RNA differentiation antagonizing nonprotein coding RNA (lncRNA DANCR) has been reported to act as an oncogene in various human cancers. The role of DANCR in development of pancreatic cancer (PC) is unknown. The aim of our research was to investigate the biological role of DANCR in PC. MATERIAL AND METHODS Expressions of DANCR, miR-214-5p, and E2F2 mRNA in PC tissues and cell lines were examined by qRT-PCR. Western blotting was carried out for detection of E2F2 protein expression in PC cells. Transwell assays were used to examine the metastatic ability of PC cells, while CCK-8 and colony formation assay were applied to evaluate cell proliferation. The effects of DANCR on PC cells were assessed by knockdown in vitro and in vivo. The regulatory mechanism of competitive endogenous RNAs were obtained from bioinformatics prediction and luciferase reporter assay. RESULTS DANCR was markedly upregulated in clinical tissues and cell lines of PC. High DANCR expression exhibited a significant correlation with poor prognosis. DANCR knockdown inhibited growth and metastasis of PC cells. Furthermore, DANCR acted as sponge to regulate miR-214-5p, and miR-214-5p inhibitor reversed the effects of DANCR knockdown on PC cells. Moreover, DANCR positively modulated E2F2 expression through miR-214-5p in PC cells. CONCLUSIONS Collectively, our findings demonstrated that lncRNA DANCR/miR-214-5p/E2F2 axis acts as an oncogene in PC development, which might provide a potential target for PC therapy.

MiRNAs regulate the cardiomyocyte (CM) cell cycle at the post-transcriptional level, affect cell proliferation, and intervene in harmed CM repair post-injury. The present study was undertaken to characterize the role of let-7i-5p in the processes of CM cell cycle and proliferation and to reveal the mechanisms thereof. In the present study, we used real-time qPCR (RT-qPCR) to determine the up-regulated let-7i-5p in CMs during the postnatal switch from proliferation to terminal differentiation and further validated the role of let-7i-5p by loss- and gain-of-function of let-7i-5p in CMs in vitro and in vivo We found that the overexpression of let-7i-5p inhibited CM proliferation, whereas the suppression of let-7i-5p significantly facilitated CM proliferation. E2F2 and CCND2 were identified as the targets of let-7i-5p, mediating its effect in regulating the cell cycle of CMs. Supperession of let-7i-5p promoted the recovery of heart function post-myocardial infarction by enhancing E2F2 and CCND2. Collectively, our results revealed that let-7i-5p is involved in the regulation of the CM cell cycle and further impacts proliferation, which may offer a new potential therapeutic strategy for cardiac repair after ischemic injury., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Gastric cancer (GC) is the second most frequent cause of cancer-related mortality in the world, with Eastern Asia having the highest incidence rates. E2F is a family of transcription factor proteins that has a variety of functions, which include control of cell cycle, cell differentiation, DNA damage response and cell death. E2F transcription factors are divided into two subfamilies: transcription activators (E2F transcription factors 1 (E2F1), 2 (E2F2) and 3a (E2F3a)) and repressors (E2F3b, E2F transcription factors 4 (E2F4), 5 (E2F5), 6 (E2F6), 7 (E2F7) and 8 (E2F8)). Studies have demonstrated that E2F had prognostic significance in a number of cancers. However, the entirety of the prognostic roles of E2F mRNA expression in GC has not yet been apparently determined. In the present study, the prognostic value of individual family members of E2F mRNA expression for overall survival (OS) was evaluated by using online Kaplan-Meier Plotter (KM Plotter) database. Our result demonstrated that high expressions of three family members of E2F (E2F1, E2F3, E2F4) mRNA were significantly associated with unfavourable OS in all GC patients. However, increased expressions of E2F2, E2F5, E2F6 and E2F7 were significantly associated with favourable OS, especially for higher clinical stages in GC patients. These results provided a better insight into the prognostic functions of E2F mRNA genes in GC. Although the results should be further verified in clinical trials, our findings may be a favourable prognostic predictor for the development of newer therapeutic drugs in the treatment of GC., (© 2018 The Author(s).)

Traumatic brain injury (TBI) activates multiple neuronal cell death mechanisms, leading to post-traumatic neuronal loss and neurological deficits. TBI-induced cell cycle activation (CCA) in post-mitotic neurons causes regulated cell death involving cyclin-dependent kinase (CDK) activation and initiation of an E2F transcription factor-mediated pro-apoptotic program. Here we examine the mechanisms of CCA-dependent neuronal apoptosis in primary neurons in vitro and in mice exposed to controlled cortical impact (CCI). In contrast to our prior work demonstrating robust neuroprotective effects by CDK inhibitors after TBI, examination of neuronal apoptotic mechanisms in E2F1 -/- /E2F2 -/- or E2F2 -/- transgenic mice following CCI suggests that E2F1 and/or E2F2 likely play only a modest role in neuronal cell loss after brain trauma. To elucidate more critical CCA molecular pathways involved in post-traumatic neuronal cell death, we investigated the neuroprotective effects and mechanisms of the potent CDK inhibitor CR8 in a DNA damage model of cell death in primary cortical neurons. CR8 treatment significantly reduced caspase activation and cleavage of caspase substrates, attenuating neuronal cell death. CR8 neuroprotective effects appeared to reflect inhibition of multiple pathways converging on the mitochondrion, including injury-induced elevation of pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins Puma and Noxa, thereby attenuating mitochondrial permeabilization and release of cytochrome c and AIF, with reduction of both caspase-dependent and -independent apoptosis. CR8 administration also limited injury-induced deficits in mitochondrial respiration. These neuroprotective effects may be explained by CR8-mediated inhibition of key upstream injury responses, including attenuation of c-Jun phosphorylation/activation as well as inhibition of p53 transactivation of BH3-only targets.

We intended to evaluate miR-490-5p expression in hepatocellular carcinoma (HCC) tissues and detect the potential targets of miR-490-5p. In vitro experiments were conducted to further investigate the biological function of miR-490-5p on HCC cell metastasis. We investigated the abnormally expressed miRNAs in HCC tissues, and the miR-490-5p expression level was detected by qRT-PCR. E2F2 and ECT2 were proved to be the potential targets of miR-490-5p by luciferase reporter assay. The expression levels of E2F2 and ECT2 were determined using Western blot. Transwell assay was used to analyse the impact of miR-490-5p on metastasis of HCC cells. Four high-expressed miRNAs, and seven low-expressed miRNAs, including miR-490-5p, were detected in HCC tissues. The expression level of miR-490-5p was connected with the tumor size, tumor node metastasis (TNM) stage, and survival ratio of HCC patients. E2F2 and ECT2 were the targets of miR-490-5p, and miR-490-5p inhibited HCC cell metastasis through down-regulating the expressions of E2F2 and ECT2. The over-expressed miR-490-5p could restrain the metastasis of HCC cells by down-regulating E2F2 and ECT2 expression levels., (© 2018 Wiley Periodicals, Inc.)

Background: Cartilage degradation would result in osteoarthritis (OA). p16INK4awas found in some age-related diseases. In this study, we aimed to determine the role of p16INK4a during OA and to investigate the underlying mechanisms., Methods: Enzyme-linked immunosorbent assay (ELISA) was performed to test the activity of senescence-associated secretory phenotype (SASP). Real-time PCR (RT-PCR) and Western blot were used to determine the expressions of target genes., Results: The increased expressions of p16INK4a and E2F2 were accompanied with cartilage degradation induced by IL-1β. Over-expression of p16INK4a enhanced the secretion of SASP markers (TGFβ, IL-6, IL-8, IL-1α, MMP3 and MMP13), reduced the expression of type II procollagen (COL2A1).Thus, the over-expression of p16INK4a lead to cartilage injury. Moreover, we found that the expression of E2F2 was enhanced in p16INK4a over-expression group, and that cartilage injury caused by p16INK4a was alleviated by depleting E2F2., Conclusions: p16INK4a was up-regulated during the cartilage injury in OA. p16INK4a promoted cartilage injury by increasing the expression of E2F2. Thus, this study extends the molecular regulation network for understanding pathological progression of OA, and provides potential therapeutic target for OA.

Peroxisome proliferator-activated receptor α (PPAR α ) is a member of the nuclear hormone receptor superfamily and functions as a transcription factor. Previous work showed that PPAR α plays multiple roles in lipid metabolism in tissues such as cardiac and skeletal muscle, liver, and adipose tissue. Recent studies have discovered additional roles for PPAR α in cell proliferation and metabolism, as well as tumor progression. PPAR α is aberrantly expressed in various cancers, and activated PPAR α inhibits the proliferation of some tumor cells. However, there have been no studies of PPAR α in human gliomas. Here, we show that PPAR α is expressed at lower levels in anaplastic gliomas and glioblastoma multiforme (GBM) tissue compared with low-grade gliomas tissue, and low expression is associated with poor patient prognosis. PPAR α activates transcription of dynamin-3 opposite strand (DNMO3os), which encodes a cluster of miR-214, miR-199a-3p, and miR-199a-5p microRNAs. Of these, miR-214 is transcribed at particularly high levels. PPAR α -induced miR-214 expression causes downregulation of its target E2F2. Finally, miR-214 overexpression inhibits glioma cell growth in vitro and in vivo by inducing cell cycle arrest in G0/G1. Collectively, these data uncover a novel role for a PPAR α -miR-214-E2F2 pathway in controlling glioma cell proliferation.

The E2F transcription factors control key elements of development, including mammary gland branching morphogenesis, with several E2Fs playing essential roles. Additional prior data has demonstrated that loss of individual E2Fs can be compensated by other E2F family members, but this has not been tested in a mammary gland developmental context. Here we have explored the role of the E2Fs and their ability to functionally compensate for each other during mammary gland development. Using gene expression from terminal end buds and chromatin immunoprecipitation data for E2F1, E2F2 and E2F3, we noted both overlapping and unique mammary development genes regulated by each of the E2Fs. Based on our computational findings and the fact that E2Fs share a common binding motif, we hypothesized that E2F transcription factors would compensate for each other during mammary development and function. To test this hypothesis, we generated RNA from E2F1-/-, E2F2-/- and E2F3+/- mouse mammary glands. QRT-PCR on mammary glands during pregnancy demonstrated increases in E2F2 and E2F3a in the E2F1-/- mice and an increase in E2F2 levels in E2F3+/- mice. During lactation we noted that E2F3b transcript levels were increased in the E2F2-/- mice. Given that E2Fs have previously been noted to have the most striking effects on development during puberty, we hypothesized that loss of individual E2Fs would be compensated for at that time. Double mutant mice were generated and compared with the single knockouts. Loss of both E2F1 and E2F2 revealed a more striking phenotype than either knockout alone, indicating that E2F2 was compensating for E2F1 loss. Interestingly, while E2F2 was not able to functionally compensate for E2F3+/- during mammary outgrowth, increased E2F2 expression was observed in E2F3+/- mammary glands during pregnancy day 14.5 and lactation day 5. Together, these findings illustrate the specificity of E2F family members to compensate during development of the mammary gland.

Retinoblastoma tumor suppressor (Rb) promotes cell cycle exit, survival, differentiation, and tumor suppression in the retina. Here, we show it is also essential for vascularization and lamination. Despite minimal effects on Hif1a target expression, intraretinal vascular plexi did not form in the Rb -/- murine retina. Deleting adenovirus E2 promoter binding factor 3 (E2f3), which rescues starburst amacrine cell differentiation, or E2f2, had no effect, but deleting E2f1, which promotes neuronal cell cycle exit and survival, restored retinal vasculature. We specifically linked cell loss to the defect because removing Bax rescued rod and bipolar neurons and the vasculature, but not cell cycle exit. Despite rescuing Rb -/- neurons, Bax deletion exacerbated a delay in outer retina lamination, and exposed a requirement for Rb in inner retina lamination. The latter resembled Sem5 or FAT atypical cadherin 3 (Fat3) mutants, but expression of Sem5/Fat3 pathway components, or that of Neogenin, which perturbs migration in the Rb -/- cortex, was unchanged. Instead, lamination defects correlated with ectopic division, and were E2f1-dependent, implicating the cell cycle machinery. These in vivo studies expose new developmental roles for Rb, pinpoint aberrant E2f1 and Bax activity in neuronal death and vascular loss, and further implicate E2f1 in defective lamination. Links between Rb, angiogenesis and lamination have implications for the treatment of neovascularization, neurodegeneration and cancer.

As a transcription factor, E2F2 participates in regulation of numerous genes. To investigate the role and mechnism of E2F2 in RA, expression of E2F2 in synovial tissue was detected. Proliferation, invasion, and secretion of inflammatory cytokines were measured after E2F2 was knocked-down in RASFs by siRNA transfection. Induction of TNF-α, IL-6, and LPS on expression and nuclear translocation of E2F2, and signal pathways involved in the process were tested. ChIP was used to investigate direct binding of NF-кB to the promoter of E2F2, and E2F2 to the promoter of IL-6. The correlation between mRNA levels of E2F2 and IL-6 or TNF-α in secreted in supernatant of RASFs were also investigated. As a result, silencing E2F2 could inhibit the proliferation and invasion of RASFs. LPS, IL-6 can stimulate the expression of E2F2 in RASFs both via the NF-кB pathway, while TNF-α via the ERK pathway. TNF-α can facilitate the nuclear translocation of E2F2 and TNF-α can bind to promoter of E2F2, and then E2F2 can bind to the promoter of IL-6 directly. Significant correlations was found between levels of E2F2 and IL-6/TNF-α in synoviocytes of RA patients. Our findings indicate that E2F2 may play an important role in pathogenesis of RA.

Background/aims: High levels of cancer stem cells (CSCs) in patients with triple-negative breast cancer (TNBC) correlate with risk of poor clinical outcome and possibly contribute to chemoresistance and metastasis in patients with highly malignant TNBC. Aberrant microRNA expression is associated with the dysfunction of self-renewal and proliferation in cancer stem cells, while there is little information about the TNBC-specific microRNAs in regulating CSC ability., Methods: Solexa deep sequencing was performed to detect the expression levels of TNBC or non-TNBC stem cells (CSCs) microRNAs. Mammosphere formation assay, qRT-PCR and the xenograft model in nude mice were performed. Bioinformatic analysis and microarray were used to select the target gene, and luciferase reporter assays were used to confirm the binding sites., Results: Solexa sequencing data exhibited differential expression of 193 microRNAs between TNBC and non-TNBC stem cells. The gene ontology analysis and pathways analyses showed that genes were involved in the maintenance of stemness. MiR-4319 could suppress the self-renewal and formation of tumorspheres in TNBC CSCs through E2F2, and also inhibited tumor initiation and metastasis in vivo. Moreover, increased E2F2 could reverse the effect of miR-4319 on the self-renewal in TNBC CSCs., Conclusions: MiR-4319 suppresses the malignancy of TNBC by regulating self-renewal and tumorigenesis of stem cells and might be a remarkable prognostic factor or therapeutic target for patients with TNBC., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Circular RNAs (circRNAs) are key regulators in the development and progression of human cancers, however its role in non-small cell lung cancer (NSCLC) tumorigenesis is not well understood. The aim of this study is to identify the expression level of circPVT1 in NSCLC and further investigated its functional relevance with NSCLC progression both in vitro and in vivo., Methods: Quantative real-time PCR was used for the measurement of circPVT1 in NSCLC specimens and cell lines. Fluorescence in situ hybridization analysis (FISH) assay was used for the identification of sublocation of circPVT1 in NSCLC cells. Bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to verify the binding of c-Fos at circPVT1 promoter region, and the direct interaction between circPVT1 and miR-125b. Gain- or loss-function assays were performed to evaluate the effects of circPVT1 on cell proliferation and invasion. Western blot and immunohistochemistry assays were performed to detect the protein levels involved in E2F2 pathway., Results: We found that circPVT1 was upregulated in NSCLC specimens and cells. The transcription factor c-Fos binded to the promoter region of circPVT1, resulting in the overexpression of circPVT1 in NSCLC. Knockdown of circPVT1 suppressed NSCLC cell proliferation, migration and invasion, and increased apoptosis. In addition, circPVT1 mediated NSCLC progression via the regulation of E2F2 signaling pathway. More importantly, circPVT1 was predominantly abundant in the cytoplasm of NSCLC cells, and circPVT1 could serve as a competing endogenous RNA to regulate E2F2 expression and tumorigenesis in a miR-125b-dependent manner, which is further verified by using an in vivo xenograft model., Conclusion: circPVT1 promotes NSCLC cell growth and invasion, and may serve as a promising therapeutic target for NSCLC patients. Therefore, silence of circPVT1 could be a future direction to develop a novel treatment strategy., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration.

Lung cancer is one of the most aggressive tumours with very low life expectancy. Altered microRNA expression is found in human tumours because it is involved in tumour growth, progression and metastasis. In this study, we analysed microRNA expression in 47 lung cancer biopsies. Among the most downregulated microRNAs we focussed on the miR-99a characterisation. In vitro experiments showed that miR-99a expression decreases the proliferation of H1650, H1975 and H1299 lung cancer cells causing cell cycle arrest and apoptosis. We identified two novel proteins, E2F2 (E2F transcription factor 2) and EMR2 (EGF-like module-containing, mucin-like, hormone receptor-like 2), downregulated by miR-99a by its direct binding to their 3'-UTR. Moreover, miR-99a expression prevented cancer cell epithelial-to-mesenchymal transition (EMT) and repressed the tumourigenic potential of the cancer stem cell (CSC) population in both these cell lines and mice tumours originated from H1975 cells. The expression of E2F2 and EMR2 at protein level was studied in 119 lung cancer biopsies. E2F2 and EMR2 are preferentially expressed in adenocarcinomas subtypes versus other tumour types (squamous and others). Interestingly, the expression of E2F2 correlates with the presence of vimentin and both E2F2 and EMR2 correlate with the presence of β-catenin. Moreover, miR-99a expression correlates inversely with E2F2 and directly with β-catenin expression in lung cancer biopsies. In conclusion, miR-99a reveals two novel targets E2F2 and EMR2 that play a key role in lung tumourigenesis. By inhibiting E2F2 and EMR2, miR-99a represses in vivo the transition of epithelial cells through an EMT process concomitantly with the inhibition of stemness features and consequently decreasing the CSC population.

Normal cell cycle progression and proliferation of palatal mesenchymal cells are important for palatal development. As targets of miR-17-92, E2F transcription factors family has been suggested to induce the transcription of miR-17-92 in several cell types. In the present study, we sought to investigate whether this negative feedback loop exists in mouse PMCs and what the function of this negative feedback loop would be in palatal mesenchymal cells. Using GeneMANIA, we revealed that the most important function of experimentally verified targets of miR-17-92 is cell cycle regulation. E2F1 and E2F3, but not E2F2, were extensively expressed in mouse palate. Over-expression of E2F1 significantly increased the expression of all the members of miR-17-92. After increased by E2F1, miR-17 and miR-20a may negatively target E2F1, and thereby prevent the cells from excessive proliferation. We suggest that the negative feedback loop between E2F1 and miR-17-92 may contribute to palatal development by regulating the proliferation and cell cycle of palatal mesenchymal cells.