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.

Transcription factor E2F3 activates CDC25B to regulate DNA damage and promote mitoxantrone resistance in stomach adenocarcinoma.

BACKGROUND: CDC25B, as a member of the cell cycle regulating protein family, is located in the cytoplasm and is involved in the transition of the cell cycle and mitosis. CDC25B is highly expressed in various tumors and is a newly discovered oncogene. This study aimed to investigate the impact of CDC25B on mitoxantrone resistance in stomach adenocarcinoma (STAD) and its possible mechanisms. METHODS: This study analyzed the expression of CDC25B and its potential transcription factor E2F3 in STAD, as well as the IC50 values of tumor tissues by bioinformatics analysis. Expression levels of CDC25B and E2F3 in STAD cells were measured by qRT-PCR. MTT was utilized to evaluate cell viability and IC50 values of STAD cells, and comet assay was utilized to analyze the level of DNA damage in STAD cells. Western blot was used to analyze the expression of DNA damage-related proteins. The targeting relationship between E2F3 and CDC25B was validated by dual-luciferase and ChIP assays. RESULTS: Bioinformatics analysis and molecular experiments showed that CDC25B and E2F3 were highly expressed in STAD, and CDC25B was enriched in the mismatch repair and nucleotide excision repair pathways. The IC50 values of tumor tissues with high expression of CDC25B were relatively high. Dual-luciferase and ChIP assays confirmed that CDC25B could be transcriptionally activated by E2F3. Cell experiments revealed that CDC25B promoted mitoxantrone resistance in STAD cells by regulating DNA damage. Further research found that low expression of E2F3 inhibited mitoxantrone resistance in STAD cells by DNA damage, but overexpression of CDC25B reversed the impact of E2F3 knockdown on mitoxantrone resistance in STAD cells. CONCLUSION: This study confirmed a novel mechanism by which E2F3/CDC25B mediated DNA damage to promote mitoxantrone resistance in STAD cells, providing a new therapeutic target for STAD treatment.

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.

E2F3 renders an immunosuppressive tumor microenvironment in nasopharyngeal carcinoma: Involvements of the transcription activation of PRC1 and BIRC5.

BACKGROUND: E2F transcription factors are well-recognized oncogenic molecules, and their correlation with immune cell infiltration has recently been reported. This work studies the impacts and mechanism of E2F transcription factor 3 (E2F3) in the growth and tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC). METHODS: Aberrantly expressed transcription factors in NPC were screened by abundant bioinformatics analyses. Gene expression in NPC cells was analyzed by reverse transcription-quantitative polymerase chain reaction and Western blot analyses. Malignant behaviors of NPC cells were analyzed by cell counting kit-8, 5-ethynyl-2'-deoxyuridine labeling, Transwell assays, and xenograft tumor models. TPA-induced THP-1 cells (macrophages) were cultured in the conditioned medium of NPC cells to mimic tumor-associated macrophages (TAMs) in vivo, and these TAMs were cocultured with CD8+ T cells. Regulation of E2F3 on protein regulator of cytokinesis 1 (PRC1) and baculoviral IAP repeat containing 5 (BIRC5) was validated by chromatin immunoprecipitation and luciferase reporter assays. RESULTS: E2F3 was highly expressed in NPC cells, and its knockdown suppressed malignant behavior and tumorigenic ability of the cells. The E2F3 knockdown condition downregulated M2 cytokines CD163 and interleukin-10 in TAMs, which further enhanced proliferation and activation of the cocultured CD8+ T cells. E2F3 promoted transcription of PRC1 and BRIC5. Furthermore, PRC1 or BRIC5 upregulation in NPC cells restored the malignant properties of NPC cells, reprogrammed the TAMs to M2 phenotype, and suppressed the CD8+ T cell proliferation and activation. CONCLUSION: This work suggests that E2F3 renders an immunosuppressive TME in NPC by activating PRC1 and BIRC5. Suppression of any member involved might favor tumor elimination.

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.

Circ_001422 aggravates osteosarcoma progression through targeting miR-497-5p/E2F3 axis.

Circular RNAs exert vital functions in the pathogenesis of osteosarcoma (OS). Circ_001422 has been confirmed to be involved in regulating OS progression, but its specific mechanism has not been clearly studied. This work aimed to analyze circ_001422's role in OS cell biological behaviors and the possible molecular mechanisms. This work carried out reverse transcription-quantitative polymerase chain reaction for detecting circ_001422, E2F3 and miR-497-5p levels, whereas Cell counting kit-8 together with Transwell assays for measuring cell growth, migration as well as invasion abilities. Relation of miR-497-5p with E2F3, as well as circ_001422 with miR-497-5p was analyzed through dual-luciferase reporter gene assay. Protein level was identified by western blot. According to our results, circ_001422 expression within OS tissue significantly increased compared with corresponding healthy samples. Inhibition of circ_001422 significantly decreased OS cell growth, invasion and migration. From mechanism research, miR-497-5p was proved as circ_001422's target, and E2F3 was miR-497-5p's target. Besides, miR-497-5p downregulation or E2F3 overexpression abolished circ_001422 inhibition-mediated inhibition on OS cell proliferation, invasion and migration. Collectively, this study has first suggested circ_001422's role in enhancing OS proliferation, migration as well as invasion via miR-497-5p/E2F3 axis. Our results will offer new ideas and new anti-OS targets.

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.

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.

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.

Loss of exosomal miR-200b-3p from hypoxia cancer-associated fibroblasts promotes tumorigenesis and reduces sensitivity to 5-Flourouracil in colorectal cancer via upregulation of ZEB1 and E2F3.

Hypoxia-mediated tumor progression is a major clinical challenge in human cancers including colorectal cancer (CRC). In addition, exosome-mediated transfer of miRNAs from cancer-associated fibroblasts (CAFs) to cancer cells could promote tumor progression. However, the mechanisms by which hypoxia CAFs promotes CRC progression remain largely unknown. CAFs and normal fibroblasts (NFs) were isolated from CRC tissues and adjacent normal tissues. Next, exosomes were isolated from the supernatant of CAFs that cultured under normoxia (CAFs-N-Exo) and hypoxia (CAFs-H-Exo). RNA-sequencing was then performed to identify differentially expressed miRNAs (DEMs) between CAFs-N-Exo and CAFs-H-Exo. Compared with exosomes derived from normoxia CAFs, exosomes derived from hypoxic CAFs were able to promote CRC cell proliferation, migration, invasion, stemness and reduce the sensitivity of CRC cells to 5-fluorouracil (5-FU). In addition, miR-200b-3p levels were dramatically decreased in exosomes derived from hypoxic CAFs. Remarkably, increasing exosomal miR-200b-3p in hypoxic CAFs reversed the promoting effects of hypoxic CAFs on CRC cell growth in vitro and in vivo. Furthermore, miR-200b-3p agomir could inhibit CRC cell migration, invasion, stemness and increase the sensitivity of SW480 cells to 5-FU via downregulating ZEB1 and E2F3. Collectively, loss of exosomal miR-200b-3p in hypoxia CAFs could contribute to CRC progression via upregulation of ZEB1 and E2F3. Thus, increasing exosomal miR-200b-3p might serve as an alternative approach for the treatment of CRC.

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 SPINT1-AS1/miR-433-3p/E2F3 positive feedback loop promotes the KRAS-mutant colorectal cancer cell proliferation, migration and invasion.

Colorectal cancer (CRC) features high prevalence and mortality. Long non-coding RNAs (lncRNAs) exert nonnegligible roles in human cancer development. Nevertheless, the functions of most lncRNAs still remain unexplored. We currently focused on detecting the influence of SPINT1 antisense RNA 1 (SPINT1-AS1) on CRC development and investigating into the potential regulatory mechanism. RT-qPCR analysis first confirmed that SPINT1-AS1 exhibited high expression in KRAS-mutant (KRASMUT) CRC cells. Through series of functional experiments, we observed that knockdown of SPINT1-AS1 weakened KRASMUT CRC cell proliferation, migration and invasion. Afterwards, the implementation of mechanism assays help to verify that SPINT1-AS1 sequestered microRNA-433-3p (miR-433-3p) to regulate the expression of E2F transcription factor 3 (E2F3). Besides, E2F3 was validated to activate the transcription of SPINT1-AS1 in turn. Rescue experiments confirmed the functional influence of SPINT1-AS1/miR-433-3p/E2F3 on CRC cells. In summary, the molecular axis of SPINT1-AS1/miR-433-3p/E2F3 forms a positive loop which might become a potential biomarker in CRC.

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.

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.

miR-15a-5p Regulates Liver Cancer Cell Migration, Apoptosis and Cell Cycle Progression by Targeting Transcription Factor E2F3.

Liver cancer is a prevalent tumor with high incidence and mortality. MicroRNAs participate in cancer pathogenesis and miR-15a-5p may influence tumor suppression in many cancers. Herein, we analyzed the effect of miR-15a-5p in liver cancer cell migration, apoptosis, and the cell cycle. RT-PCR was performed to measure miR-15a-5p expression levels, transwell assays were applied to investigate the effect of miR-15a-5p on cell migration, and flow cytometry was performed to explore the impact of miR-15a-5p on apoptosis and the cell cycle in Hep3B and HepG2 cells. Luciferase reporter assays and Western blotting were employed to determinate the relationship between E2F3 and miR-15a-5p in liver cancer cells. Expression of E2F3 was detected by bioinformatics analysis and RT-PCR in liver cancer. Small interfering RNA (si-RNA) was used to silence E2F3 expression and assess the effect on migration, apoptosis, and the cell cycle in Hep3B/HepG2 cells. The results demonstrated that miR-15a-5p was downregulated in human liver cancer tissue, and enhancing the expression of miR-15a-5p suppressed migration in liver cancer cells, induced apoptosis, and caused G1 phase arrest. In vivo assays were further performed and miR-15a-5p inhibited the growth of liver cancer. miR-15a-5p appeared to target E2F3, and RT-PCR and bioinformatic analyses indicated that E2F3 expression was higher in liver cancer than control tissues. Silencing E2F3 expression decreased cell migration, induced apoptosis, and caused G1 phase arrest in Hep3B/HepG2 cells. These findings indicate that miR-15a-5p regulates liver cancer cell migration, apoptosis, and growth by targeting E2F3. Thus, miR-15a-5p may act as a suppressor role in liver cancer.

A novel circ_0000654/miR-375/E2F3 ceRNA network in esophageal squamous cell carcinoma.

BACKGROUND: The competing endogenous RNA (ceRNA) activity of circular RNAs (circRNAs) has been implicated in the pathogenesis of cancers, including esophageal squamous cell carcinoma (ESCC). Here, we identified the ceRNA mechanism of circ_0000654 regulation in ESCC. METHODS: The levels of circ_0000654, E2F transcription factor 3 (E2F3), and microRNA (miR)-375 were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell apoptosis was detected by flow cytometry. Cell colony formation was tested by colony formation assay. Dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to confirm the direct relationship between miR-375 and circ_0000654 or E2F3. Xenograft model assays were used to evaluate the effect of circ_0000654 in vivo. RESULTS: Circ_0000654 and E2F3 were upregulated in ESCC. Circ_0000654 depletion enhanced cell apoptosis and hindered cell proliferation and glycolysis in vitro, as well as weakened tumor growth in vivo. Increased expression of E2F3 counteracted the effects of circ_0000654 depletion. Mechanistically, E2F3 was a target of miR-375, and circ_0000654 modulated E2F3 expression through sequestering miR-375. Furthermore, miR-375 upregulation phenocopied circ_0000654 knockdown in inhibiting ESCC progression. CONCLUSION: Our findings identify a new circ_0000654/miR-375/E2F3 ceRNA crosstalk for the oncogenic role of circ_0000654 in ESCC and establish a notion that targeting circ_0000654 and its pathways may have the potential to improve ESCC outcome.

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.