FXR/Menin-mediated epigenetic regulation of E2F3 expression controls ß-cell proliferation and is increased in islets from diabetic GK rats after RYGB.

Farnesoid X receptor (FXR) improves the function of islets, especially in the setting of Roux-en-Y gastric bypass (RYGB). Here we investigated how FXR activation regulates ß-cell proliferation and explored the potential link between FXR signaling and the menin pathway in controlling E2F3 expression, a key transcription factor for controlling adult ß-cell proliferation. Stimulation with the FXR agonist GW4064 or chenodeoxycholic acid (CDCA) increased E2F3 expression and ß-cell proliferation. Consistently, E2F3 knockdown abolished GW4064-induced proliferation. Treatment with GW4064 increased E2F3 expression in ß-cells via enhancing Steroid receptor coactivator-1 (SRC1) recruitment, increasing the pro-transcriptional acetylation of histone H3 at the E2f3 promoter. GW4064 treatment also decreased the association between FXR and menin, leading to the induction of FXR-mediated SRC1 recruitment. Mimicking the impact of FXR agonists, RYGB also increased E2F3 expression and ß-cell proliferation in GK rats and SD rats. These findings unravel the crucial role of the FXR/menin signaling in epigenetically controlling E2F3 expression and ß-cell proliferation, a mechanism possibly underlying RYGB-induced ß-cell proliferation.

FTO promotes the progression of retinoblastoma through YTHDF2-dependent N6-methyladenosine modification in E2F3.

Early treatment of retinoblastoma (RB) has significantly improved clinical outcomes. N6-methyladenosine (m6A) methylation is crucial for cancer progression. Thus, we investigated the role of FTO-dependent demethylation in RB and its underlying mechanisms. The biological behavior of RB cells was analyzed using cell counting kit-8, colony formation analysis, transwell assay, flow cytometry, and western blot analysis. m6A modification was evaluated using methylated RNA immunoprecipitation and dual-luciferase reporter assays, and E2F3 stability was assessed using Actinomycin D. The roles of FTO and E2F3 were also elucidated in vivo. These results indicated that FTO was highly expressed in RB cells with low m6A levels. FTO knockdown inhibited RB cell growth, migration, invasion, and epithelial-mesenchymal transition and arrested the cell cycle at the G0/G1 phase. Mechanistically, FTO interference promoted m6A methylation of E2F3, which was recognized by YTHDF2, thereby reducing mRNA stability. E2F3 overexpression partially rescued the effects of FTO knockdown on biological behavior. Moreover, FTO knockdown reduced tumor weight, tumor volume, ki67 expression, and tumor cell infiltration by mediating E2F3. Taken together, FTO silencing inhibited the malignant processes of RB by suppressing E2F3 in an m6A-YTHD2-dependent manner. These findings suggest that FTO is a novel therapeutic target for RB.

E2F3/5/8 serve as potential prognostic biomarkers and new therapeutic direction for human bladder cancer.

OBJECTS: Human bladder cancer (BC) is the most common urogenital system malignancy. E2F transcription factors (E2Fs) have been reported to be involved in the growth of various cancers. However, the expression patterns, prognostic value and immune infiltration in the tumor microenvironment of the 8 E2Fs in BC have yet fully to be explored. METHODS AND STRATEGY: We investigated the differential expression of E2Fs in BC patients, the prognostic value and correlation with immune infiltration by analyzing a range of databases. RESULTS: We found that the mRNA expression levels of E2F1/2/3/4/5/7/8 were significantly higher in BC patients than that of control tissues. And the increased mRNA expression levels of all E2Fs were associated with tumor stage of BC. The survival analysis revealed that the elevated mRNA expression levels of E2F3/5/8 were significantly correlated with the overall survival (OS) of BC patients. And the genetic changes of E2Fs in BC patients were associated with shorter overall survival (OS) and progression-free survival (PFS). In addition, we revealed that the E2F3/5/8 expressions were closely correlated with tumor-infiltrating lymphocytes (TILs). CONCLUSIONS: E2F3/5/8 might serve as promising prognostic biomarkers and new therapeutic direction for BC patients.

E2F3/CDCA2 reduces radiosensitivity in gastric adenocarcinoma by activating PI3K/AKT pathway.

OBJECTIVES: Gastric adenocarcinoma is primarily responsible for tumor-associated deaths and its incidence is increasing global. CDCA2 is a nuclear protein binding to protein phosphatase one γ (PP1γ) and plays a pro-oncogenic role in tumors. This study aimed to elucidate the biological function of CDCA2 in gastric adenocarcinoma progression and radiosensitivity, as well as its potential mechanisms. METHODS: Differentially expressed mRNAs in gastric adenocarcinoma were obtained by bioinformatics and upstream regulatory factors were predicted. The correlation between their expressions was analyzed. The expressions of E2F3 and CDCA2 in cells were assayed by qRT-PCR and their regulatory relationship was validated by molecular experiments. Cell viability was tested via CCK-8. Cell proliferation and survival after radiotherapy were determined by colony formation assay. The expressions of PI3K/AKT pathway-related proteins were assessed through western blot. RESULTS: CDCA2 was significantly upregulated in gastric adenocarcinoma tissues and cells, promoted cell proliferation, and reduced radiosensitivity. The impact of CDCA2 on cell proliferation and radiosensitivity was reversed by the PI3K/AKT inhibitor. Furthermore, the upstream transcription factor of CDCA2 was found to be E2F3, which was highly expressed in gastric adenocarcinoma. The binding relationship between the two was validated by dual luciferase and ChIP experiments. The rescue experiment showed that E2F3 activated CDCA2 to drive cell proliferation and reduce radiosensitivity through PI3K/AKT pathway in gastric adenocarcinoma. CONCLUSION: In summary, this study found that E2F3 activated CDCA2 to drive cell proliferation and reduce radiosensitivity in gastric adenocarcinoma through the PI3K/AKT pathway, suggesting that E2F3/CDCA2 axis is a new therapeutic target for gastric adenocarcinoma. ADVANCES IN KNOWLEDGE: 1. CDCA2 reduced the radiosensitivity of gastric adenocarcinoma cells;2. CDCA2 reduced the radiosensitivity of gastric adenocarcinoma cells through the PI3K/AKT pathway;3. E2F3 activated CDCA2 to reduce the radiosensitivity of gastric adenocarcinoma cells through the PI3K/AKT pathway.

CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism.

Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle-specific deletion of Cdk4's target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber-specification that is of relevance to metabolic and muscular diseases.

E2F3 induces DNA damage repair, stem-like properties and therapy resistance in breast cancer.

Therapy resistance is a major hurdle to the treatment of human malignant tumors. Both DNA damage repair and stem-like properties contribute to chemoresistance and radioresistance. E2F transcription factor 3 (E2F3) is overexpressed in breast cancer tissues, and promotes proliferation of breast cancer cells. Higher E2F3 level is associated with shorter survival of breast cancer patients. Functional studies further showed that E2F3 promotes S-phage entry, DNA replication, DNA damage repair and stem-like properties. Accordingly, E2F3 knockdown sensitizes breast cancer cells to DNA-damaging agents Adriamycin, Cisplatin, Olaparib and X-ray. Forkhead box M1 (FOXM1) is a downstream molecule of E2F3 signaling, mediating the effects of E2F3 on breast cancer cells. In an m6A methyltransferase METTL14-dependent manner, YTH RNA binding protein F2 (YTHDF2) increase E2F3 mRNA stability and expression, promotes DNA damage repair and induces therapy resistance. These data demonstrate that YTHDF2-E2F3 pathway is a novel target to overcome chemoresistance and radioresistance in breast cancer.

Brevilin A shows an anti-tumor role in prostate cancer via the lncRNA H19/miR-194/E2F3 signaling pathway.

Brevilin A, a natural sesquiterpene lactone extracted from Centipeda minima, has been found with antitumor properties. Our study probed the functions of Brevilin A in prostate cancer cells and the mechanisms among Brevilin A, lncRNA H19, miR-194, and E2F3 on the biological behaviors of the cells. CCK8, Transwell, and TUNEL staining assays examined the impact of Brevilin A on prostate cancer cell proliferation, migration, invasion, and apoptosis, respectively. qRT-PCR and western blot determined lncRNA H19, miR-194, and E2F3 profiles. The influence of Brevilin A on the profiles of lncRNA H19, miR-194, and E2F3 was measured. A xenograft model of prostate cancer nude mice was taken to confirm the impact of Brevilin A and lncRNA H19 on cancer cell growth. Consequently, Brevilin A dampened prostate cancer cell proliferation, migration, and invasion, suppressed the expressions of lncRNA H19 and E2F3, and enhanced miR-194 level. LncRNA H19 and E2F3 were uplifted, whereas miR-194 was abated in prostate cancer cells and tissues. LncRNA H19 targeted miR-194 to positively modulate E2F3 expression, boosted DU145 cell proliferation, invasion, and migration, and curbed apoptosis. In the xenograft model, Brevilin A repressed tumor growth, whereas lncRNA H19 fostered tumor growth. Brevilin A suppressed the promotive effect of lncRNA H19 in PC cell growth in vivo. To conclude, Brevilin A modulates the biological behaviors of prostate cancer cells via the lncRNA H19/miR-194/E2F3 axis. Brevilin A exerts an anti-tumor function in prostate cancer.

The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F.

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Long noncoding RNA POU6F2-AS2 contributes to the aggressiveness of nonsmall-cell lung cancer via microRNA-125b-5p-mediated E2F3 upregulation.

The role of the majority of long noncoding RNAs (lncRNAs) in the progression of nonsmall-cell lung cancer (NSCLC) remains elusive, despite their potential value, thus warranting in-depth studies. For example, detailed functions of the lncRNA POU6F2 antisense RNA 2 (POU6F2-AS2) in NSCLC are unknown. Herein, we investigated the expression status of POU6F2-AS2 in NSCLC. Furthermore, we systematically delineated the biological roles of POU6F2-AS2 in NSCLC alongside its downstream molecular events. We measured the expression levels of POU6F2-AS2 using quantitative real-time polymerase chain reaction and performed a series of functional experiments to address its regulatory effects in NSCLC cells. Using bioinformatic platforms, RNA immunoprecipitation, luciferase reporter assays, and rescue experiments, we investigated the potential mechanisms of POU6F2-AS2 in NSCLC. Subsequently, we confirmed the remarkable overexpression of POU6F2-AS2 in NSCLC using The Cancer Genome Atlas database and our own cohort. Functionally, inhibiting POU6F2-AS2 decreased NSCLC cell proliferation, colony formation, and motility, whereas POU6F2-AS2 overexpression exhibited contrasting effects. Mechanistically, POU6F2-AS2 acts as an endogenous decoy for microRNA-125b-5p (miR-125b-5p) in NSCLC that causes the overexpression of the E2F transcription factor 3 (E2F3). Moreover, suppressing miR-125b-5p or increasing E2F3 expression levels sufficiently recovered the anticarcinostatic activities in NSCLC induced by POU6F2-AS2 silencing. Thus, POU6F2-AS2 aggravates the oncogenicity of NSCLC by targeting the miR-125b-5p/E2F3 axis. Our findings suggest that POU6F2-AS2 is a novel therapeutic target for NSCLC.

CircKRT14 upregulates E2F3 by interacting with miR-1256 to act as an oncogenic factor in esophageal cancer.

BACKGROUND: A growing number of studies have focused on the regulatory role of circular RNAs (circRNAs) in a variety of cancers. The purpose of this study was to investigate the effect of circRNA Keratin 14 (circKRT14) on the progression of esophageal cancer (EC). METHODS: The levels of circKRT14, miR-1256 and E2F transcription factor 3 (E2F3) were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot. The circular structure of circKRT14 was confirmed by RNase R digestion assay. Cell apoptosis, migration and invasion were detected by flow cytometry and transwell assay. The protein levels of related factors were determined by western blot. The relationship between miR-1256 and circKRT14 or E2F3 was verified by dual-luciferase reporter assay. The in vivo function of circKRT14 was studied by xenograft tumor assay. RESULTS: CircKRT14 was significantly increased in EC tissues and cells. CircKRT14 silencing inhibited EC cell proliferation, migration, and invasion, but promoted EC cell apoptosis in vitro. CircKRT1 acted as a sponge for miR-1256 in EC, and in-miR-1256 abolished the inhibitory effect of circKRT14 suppression on EC cell progression. E2F3 was a target of miR-1256 and functioned as an oncogene in EC cells. MiR-1256 curbed EC progression by downregulating E2F3. CircKRT14 could affect E2F3 expression by targeting miR-1256. CircKRT14 regulated EC progression in vivo through miR-1256/E2F3 axis. CONCLUSIONS: These results uncovered that circKRT14 up-regulated the expression of E2F3 and promoted the malignant development of EC through sponging miR-1256.

LncRNA NEAT1 regulates the growth, migration, and invasion of the human esophageal cancer cells via the miR-377/E2F3 axis.

Esophageal cancer is a highly aggressive and lethal human malignancy. The current study was established with the aim of studying the role of the lncRNA NEAT1 in regulating the growth and progression of esophageal cancer. Esophageal cancer tissues and cell lines showed significantly (P<0.05) upregulated transcript levels of lncRNA NEAT1. The expression of NEAT1 was also upregulated in metastatic tissues compared to nonmetastatic. The elimination of lncRNA NEAT1 led to a significant decrease (P<0.05) decrease in the viabilities of cancer cells due to the induction of apoptosis. Cancer cell migration and invasion were also significantly reduced (P<0.05) upon lncRNA NEAT1. In silico analysis indicated that miR-377 targets lncRNA NEAT1 at the post-transcriptional level, whose overexpression in cancer cells was found to mimic the tumor-suppressive regulatory effects of lncRNA-NEAT1. At the molecular level, the regulatory effects of lncRNA NEAT1 were shown to be modulated by the miR-377/E2F3 signaling axis. The results suggest that the molecular targeting of the lncRNA NEAT1 and miR-377/E2F3 axis could prove beneficial in the management of esophageal cancer.

Identifying the E2F3-MEX3A-KLF4 signaling axis that sustains cancer cells in undifferentiated and proliferative state.

Rationale: Dysregulation of signaling that governs self-renewal and differentiation of intestinal stem cells (ISCs) is a major cause of colorectal cancer (CRC) initiation and progression. Methods: qRT-PCR, western blotting, in situ hybridization, immunohistochemistry and immunofluorescence assays were used to detect the expression levels of MEX3A, KLF4 and E2F3 in CRC tissues. The biological functions of MEX3A were studied using Mex3a knockout (KO) and intestinal epithelium specific conditional knockout (cKO) mice, AOM-DSS mouse colorectal tumor model, Apc floxed mouse tumor model and intestinal and tumor organoids. Transcriptomic RNA sequencing (RNA-seq), RNA crosslinking immunoprecipitation (CLIP) and luciferase reporter assays were performed to explore the molecular mechanisms of MEX3A. Results: RNA-binding protein MEX3A, a specific ISC marker gene, becomes ectopically upregulated upon CRC and its levels negatively correlate with patient survival prognosis. MEX3A functions as an oncoprotein that retains cancer cells in undifferentiated and proliferative status and it enhances their radioresistance to DNA damage. Mechanistically, a rate limiting factor of cellular proliferation E2F3 induces MEX3A, which in turn activates WNT pathway by directly suppressing expression of its pro-differentiation transcription factor KLF4. Knockdown of MEX3A with siRNA or addition of KLF4 agonist significantly suppressed tumor growth both by increasing differentiation status of cancer cells and by suppressing their proliferation. Conclusions: It identifies E2F3-MEX3A-KLF4 axis as an essential coordinator of cancer stem cell self-renewal and differentiation, representing a potent new druggable target for cancer differentiation therapy.

Hsa_hsa_circ_0081069 promotes the progression of colorectal cancer through sponging miR-665 and regulating E2F3 expression.

BACKGROUND: Circular RNAs (circRNAs) have been implicated in the initiation and development of various cancers. This study explored the potential contribution of hsa_hsa_circ_0081069 in the progression of colorectal cancer (CRC). METHODS: The gene expression was analyzed by qRT-PCR. Functional roles of hsa_circ_0081069 were examined by shRNA-mediated silencing using CCK-8 proliferation assay, Transwell migration and invasion assay, tube formation assay. The tumorigenesis and metastasis of CRC cells were assess in a xenograft mouse model. RESULTS: Hsa_circ_0081069 was significantly upregulated in CRC tissues and cells. Hsa_circ_0081069 knockdown suppressed the proliferation, migration and invasion in CRC cells, as well as the angiogenesis. Silencing hsa_circ_0081069 also impaired the tumorigenesis of CRC cells in a xenograft mouse model. Furthermore, miR-665 was identified as an interacting partner of hsa_circ_0081069, which was negatively regulated by hsa_circ_0081069. miR-665 targeted the mRNA of E2F3 to suppress its expression. We further demonsatred that miR-665/E2F3 axis mediated the functional role of hsa_circ_0081069 in regulating the malignant phenotype of CRC cells. CONCLUSIONS: Collectively, our study suggests that hsa_circ_0081069 could serve as a prognostic marker in progression of CRC. Targeting hsa_circ_0081069 and miR-665/E2F3 axis could serve as potential therapeutic strategies for CRC treatment.

Retinoblastoma-E2F Transcription Factor Interplay Is Essential for Testicular Development and Male Fertility.

The retinoblastoma (RB) protein family members (pRB, p107 and p130) are key regulators of cell cycle progression, but also play crucial roles in apoptosis, and stem cell self-renewal and differentiation. RB proteins exert their effects through binding to E2F transcription factors, which are essential developmental and physiological regulators of tissue and organ homeostasis. According to the canonical view, phosphorylation of RB results in release of E2Fs and induction of genes needed for progress of the cell cycle. However, there are eight members in the E2F transcription factor family with both activator (E2F1-3a) and repressor (E2F3b-E2F8) roles, highlighting the functional diversity of RB-E2F pathway. In this review article we summarize the data showing that RB-E2F interaction is a key cell-autonomous mechanism responsible for establishment and maintenance of lifelong male fertility. We also review the expression pattern of RB proteins and E2F transcription factors in the testis and male germ cells. The available evidence supports that RB and E2F family members are widely and dynamically expressed in the testis, and they are known to have versatile roles during spermatogenesis. Knowledge of the function and significance of RB-E2F interplay for testicular development and spermatogenesis comes primarily from gene knock-out (KO) studies. Several studies conducted in Sertoli cell-specific pRB-KO mice have demonstrated that pRB-mediated inhibition of E2F3 is essential for Sertoli cell functional maturation and cell cycle exit, highlighting that RB-E2F interaction in Sertoli cells is paramount to male fertility. Similarly, ablation of either pRB or E2F1 in the germline results in progressive testicular atrophy due to germline stem cell (GSC) depletion, emphasizing the importance of proper RB-E2F interplay for germline maintenance and lifelong sperm production. In summary, while balanced RB-E2F interplay is essential for cell-autonomous maintenance of GSCs and, the pRB-E2F3 system in Sertoli cells is critical for providing GSC niche thus laying the basis for spermatogenesis.

E2F3 gene expression is a potential negative prognostic marker for localised and MYCN not-amplified neuroblastoma: Results of in silico analysis of 786 samples.

BACKGROUND: Neuroblastoma (NB) is an enigmatic childhood malignancy characterised by a wide range of clinical behaviour. Many potential oncogenes for NB have recently been identified. Among them, E2 transcription factor 3 (E2F3) expression was associated with a poor survival in 134 stage 4S patients, but evidence for other stage groups remains poorly investigated. METHODS: We have analysed the expression of E2F3 gene from a database of 786 NB samples. Overall and event-free survivals (EFS) were assessed by the Kaplan-Meier method, splitting the data on the median and tertile expression values. The Cox model was applied to control for the confounding by stage, age and MYCN amplification. Validation was performed by an in silico analysis of an independent cohort of 283 NB patients. Furthermore, an immunofluorescence analysis on 48 formalin-fixed, paraffin-embedded NB specimens was also performed. RESULTS: E2F3 overexpression was associated with a poor survival (EFS = 84%, 95% CI: 79%-95%, for low expression levels; EFS = 62%, 95% CI: 56%-68% for middle levels; EFS = 30%, 95% CI: 24%-36%, for high levels, p < .001). This association was confirmed in multivariable analysis and was more evident in patients with MYCN not-amplified and localised stages. Immunofluorescence results and the validation on an independent cohort of NB primary samples confirmed these findings. CONCLUSIONS: E2F3 is a new potential prognostic marker in NB with favourable characteristics at diagnosis. Further studies are needed to elucidate the potential role of E2F3 in NB oncogenesis and progression, in order to identify new targets for therapeutic interventions.

MicroRNA­141 inhibits the differentiation of bone marrow­derived mesenchymal stem cells in steroid­induced osteonecrosis via E2F3.

Osteonecrosis of the femoral head (ONFH) affects the life of patients. MicroRNA­141 (miR­141) has been found associated with proliferation of bone marrow­derived mesenchymal stem cells (BMSCs). E2F transcription factor 3 (E2F3) has been identified as the target of miR­141 to regulate cell proliferation. The aim of the present study was to investigate whether miR­141 and E2F3 were involved in the osteogenic differentiation of BMSCs during ONFH. BMSCs from 4­week­old Sprague­Dawley rats were transduced with miR­141 mimic or inhibitor lentiviruses. Alkaline phosphatase staining was performed to confirm osteogenic differentiation. Reverse transcription­quantitative PCR, luciferase reporter assays and western blot analysis were also used to examine the interaction between E2F3 and miR­141 in BMSCs from the control and ONFH rats. The lentiviral transductions were carried out successfully. The mRNA expression levels of miR­141 in ONFH were upregulated, while those of E2F3 were downregulated compared with the control rat. The luciferase reporter assays indicated that miR­141 could target E2F3. miR­141 knockdown upregulated the mRNA expression levels of E2F3. In addition, osteogenic differentiation of BMSCs was inhibited following miR­141 overexpression, but increased following miR­141 knockdown, as evidenced by the results of the alkaline phosphatase staining and western blot analysis. In conclusion, miR­141 inhibits the osteogenic differentiation of BMSCs in ONFH by targeting E2F3. These two molecules may represent novel candidates to examine in order to investigate the mechanism underlying ONFH.

Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC­derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a­regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

CircPAG1 interacts with miR-211-5p to promote the E2F3 expression and inhibit the high glucose-induced cell apoptosis and oxidative stress in diabetic cataract.

Circular RNAs (circRNAs) are regulatory endogenous RNAs in human diseases by sponging microRNAs (miRNAs) to affect the gene expression. However, little research focused on the circRNA/miRNA/mRNA axis in diabetic cataract. This study was performed for the exploration of circRNA phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (circPAG1) in diabetic cataract. Human lens epithelial cells were treated with high glucose. The quantitative real-time polymerase chain reaction was used for the expression detection of circPAG1, microRNA-211-5p (miR-211-5p), and E2F transcription factor 3 (E2F3). Cell viability and proliferation were detected using Cell Counting Kit-8 assay and EdU assay. Cell apoptosis was analyzed by flow cytometry. The protein levels were measured by Western blot. Oxidative stress was assessed by malondialdehyde, reactive oxygen species, and superoxide dismutase via the corresponding detection kits. The target interaction was validated using the dual-luciferase reporter assay and RNA immunoprecipitation assay. The expression of circPAG1 was downregulated in diabetic cataract patients. The upregulation of circPAG1 could attenuate the high glucose-induced inhibition of cell viability and proliferation but promotion of cell apoptosis and oxidative stress. CircPAG1 served as a miR-211-5p sponge, and the protective role of circPAG1 was partly achieved by sponging miR-211-5p. MiR-211-5p targeted E2F3 and circPAG1 upregulated the E2F3 level by absorbing miR-211-5p. Inhibition of miR-211-5p repressed the high glucose-mediated cell dysfunction by increasing the expression of E2F3. This study clarified that circPAG1 protected human lens epithelial cells from the high glucose-induced cell damages by the mediation of miR-211-5p/E2F3 axis.

Smad3 deficiency improves islet-based therapy for diabetes and diabetic kidney injury by promoting ß cell proliferation via the E2F3-dependent mechanism.

Rationale: Poor ß cell proliferation is one of the detrimental factors hindering islet cell replacement therapy for patients with diabetes. Smad3 is an important transcriptional factor of TGF-ß signaling and has been shown to promote diabetes by inhibiting ß cell proliferation. Therefore, we hypothesize that Smad3-deficient islets may be a novel cell replacement therapy for diabetes. Methods: We examined this hypothesis in streptozocin-induced type-1 diabetic mice and type-2 diabetic db/db mice by transplanting Smad3 knockout (KO) and wild type (WT) islets under the renal capsule, respectively. The effects of Smad3KO versus WT islet replacement therapy on diabetes and diabetic kidney injury were examined. In addition, RNA-seq was applied to identify the downstream target gene underlying Smad3-regulated ß cell proliferation in Smad3KO-db/db versus Smad3WT-db/db mouse islets. Results: Compared to Smad3WT islet therapy, treatment with Smad3KO islets produced a much better therapeutic effect on both type-1 and type-2 diabetes by significantly lowering serum levels of blood glucose and HbA1c and protected against diabetic kidney injuries by preventing an increase in serum creatinine and the development of proteinuria, mesangial matrix expansion, and fibrosis. These were associated with a significant increase in grafted ß cell proliferation and blood insulin levels, resulting in improved glucose intolerance. Mechanistically, RNA-seq revealed that compared with Smad3WT-db/db mouse islets, deletion of Smad3 from db/db mouse islets markedly upregulated E2F3, a pivotal regulator of cell cycle G1/S entry. Further studies found that Smad3 could bind to the promoter of E2F3, and thus inhibit ß cell proliferation via an E2F3-dependent mechanism as silencing E2F3 abrogated the proliferative effect on Smad3KO ß cells. Conclusion: Smad3-deficient islet replacement therapy can significantly improve both type-1 and type-2 diabetes and protect against diabetic kidney injury, which is mediated by a novel mechanism of E2F3-dependent ß cell proliferation.

eIF4A3-induced circWAC promotes breast cancer progression through mediating miR-599/E2F3 axis.

Circular RNAs (circRNAs) are implicated in the regulation of tumor progression via the "competitive endogenous RNAs (ceRNAs)" mechanism. We intended to explore the molecular mechanism of circRNA WW domain containing adaptor with coiled-coil (circWAC) in breast cancer (BC) progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were applied to analyze RNA and protein expression. Cell proliferation, migration, invasion, apoptosis, glycolysis, and tumorigenesis in nude mice were assessed to analyze the role of circWAC/microRNA-599 (miR-599)/E2F transcription factor 3 (E2F3) axis in BC. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA-pull down assay were performed to verify intermolecular interactions. CircWAC was up-regulated in BC tissues and cell lines. CircWAC knockdown restrained the proliferation, migration, invasion, and glycolysis and promoted the apoptosis of BC cells. CircWAC acted as miR-599 sponge, and miR-599 interference largely reversed circWAC silencing-induced effects in BC cells. MiR-599 interacted with the 3' untranslated region (3'UTR) of E2F3, and miR-599 overexpression-induced suppressive effect on cellular malignant potential was overturned by the accumulation of E2F3 in BC cells. Eukaryotic initiation factor 4A3 (eIF4A3) induced the expression of circWAC in BC cells. CircWAC knockdown suppressed xenograft tumor growth in vivo. Our results demonstrated that eIF4A3-induced circWAC promoted the proliferation, migration, invasion, and glycolysis and suppressed the apoptosis of BC cells through mediating miR-599/E2F3 axis, which provided novel potential targets for BC therapy.