Dexamethasone-tamoxifen combination exerts synergistic therapeutic effects in tamoxifen-resistance breast cancer cells.

Tamoxifen (TAM) is a key player in estrogen receptor-positive (ER+) breast cancer (BC); however, ∼30% of patients experience relapse and a lower survival rate due to TAM resistance. TAM resistance was related to the over expression of SOX-2 gene, which is regulated by the E2F3 transcription factor in the Wnt signaling pathway. It was suggested that SOX-2 overexpression was suppressed by dexamethasone (DEX), a glucocorticoid commonly prescribed to BC patients. The aim of the present study is to explore the effect of combining DEX and TAM on the inhibition of TAM-resistant LCC-2 cells (TAMR-1) through modulating the E2F3/SOX-2-mediated Wnt signaling pathway. The effect of the combination therapy on MCF-7 and TAMR-1 cell viability was assessed. Drug interactions were analyzed using CompuSyn and SynergyFinder softwares. Cell cycle distribution, apoptotic protein expression, gene expression levels of SOX-2 and E2F3, and cell migration were also assessed. Combining DEX with TAM led to synergistic inhibition of TAMR-1 cell proliferation and migration, induced apoptosis, reduced SOX-2 and E2F3 expression and was also associated with S and G2-M phase arrest. Therefore, combining DEX with TAM may present an effective therapeutic option to overcome TAM resistance, by targeting the E2F3/SOX-2/Wnt signaling pathway, in addition to its anti-inflammatory effect.

PA28γ coordinates the cross-talk between cancer-associated fibroblasts and tumor cells to promote OSCC progression via HDAC1/E2F3/IGF2 signaling.

PA28γ overexpression is aberrant and accompanied by poor patient prognosis in various cancers, the precise regulatory mechanism of this crucial gene in the tumor microenvironment remains incompletely understood. In this study, using oral squamous cell carcinoma as a model, we demonstrated that PA28γ exhibits high expression in cancer-associated fibroblasts (CAFs), and its expression significantly correlates with the severity of clinical indicators of malignancy. Remarkably, we found that elevated levels of secreted IGF2 from PA28γ+ CAFs can enhance stemness maintenance and promote tumor cell aggressiveness through the activation of the MAPK/AKT pathway in a paracrine manner. Mechanistically, PA28γ upregulates IGF2 expression by stabilizing the E2F3 protein, a transcription factor of IGF2. Further mechanistic insights reveal that HDAC1 predominantly mediates the deacetylation and subsequent ubiquitination and degradation of E2F3. Notably, PA28γ interacts with HDAC1 and accelerates its degradation via a 20S proteasome-dependent pathway. Additionally, PA28γ+ CAFs exert an impact on the tumor immune microenvironment by secreting IGF2. Excitingly, our study suggests that targeting PA28γ+ CAFs or secreted IGF2 could increase the efficacy of PD-L1 therapy. Thus, our findings reveal the pivotal role of PA28γ in cell interactions in the tumor microenvironment and propose novel strategies for augmenting the effectiveness of immune checkpoint blockade in oral squamous cell carcinoma.

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.

Circ_0001495 influences the development of endometriosis through the miRNA-34c-5p/E2F3 axis.

Endometriosis is a chronic gynecological condition characterized by the presence of endometrial glands and stroma outside the uterine cavity., accounting for 7% of all female malignant tumors and 20%- 30% of malignant tumors of the female reproductive system. Multiple studies have shown that circular RNA (circRNA) has the potential to become a targeted target and marker for EM. However, the roles of circ_0001495 in EM are still unclear. Our research aims to reveal the molecular mechanism of circ_0001495 in EM. In this study, RT-PCR or western blot were conducted to determine mRNA and protein expression. cell viability, proliferation, migration, invasion, and apoptosis were assessed by CCK-8, EdU, wound healing, transwell, and flow cytometry analyses, respectively. Additionally, the targeting relationship between miR-34c-5p and circ_0001495 or E2F3 was confirmed through dual-luciferase reporter gene assay. We found significant overexpression of circ_0001495 in EM tissues and cells. Knockdown of circ_0001495 inhibited the proliferation, migration and invasion of ectopic endometrial stromal cells (EESCs) and increased cell apoptosis. Moreover, we found that circ_0001495 regulated E2F3 levels by interacting with miR-34c-5p in EESC. Furthermore, in vitro, miR-34c-5p inhibition or E2F3 overexpression could attenuate the effect of circ_0001495 silencing on EM progression. In addition, the vivo experiment demonstrated that inhibition of circ_0001495 could repress the development of endometriosis by regulating the miR-34c-5p/E2F3 axis. In conclusion, our study suggested that circ_0001495 promoted EM progression in vitro and in vivo through the miR-34c-5p/E2F3 axis, which might be a potential therapeutic target for EM.

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.

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.

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.

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.

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.

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.