Unveiling the role of UPF3B in hepatocellular carcinoma: Potential therapeutic target.

RNA-binding proteins can regulate nucleotide metabolism and gene expression. UPF3B regulator of nonsense mediated mRNA decay (UPF3B) exhibits dysfunction in cancers. However, its role in the progression of hepatocellular carcinoma (HCC) is still insufficiently understood. Here, we found that UPF3B was markedly upregulated in HCC samples and associated with adverse prognosis in patients. UPF3B dramatically promoted HCC growth both in vivo and in vitro. Mechanistically, UPF3B was found to bind to PPP2R2C, a regulatory subunit of PP2A, boosting its mRNA degradation and activating the PI3K/AKT/mTOR pathway. E2F transcription factor 6 (E2F6) directly binds to the UPF3B promoter to facilitate its transcription. Together, the E2F6/UPF3B/PPP2R2C axis promotes HCC growth through the PI3K/AKT/mTOR pathway. Hence, it could be a promising therapeutic target for treating HCC.

A hsa_circ_001726 axis regulated by E2F6 contributes to metastasis of hepatocellular carcinoma.

BACKGROUND: CircRNAs participate in the development of hepatocellular carcinoma (HCC). This work aims to explore the key tumor promoting circRNA as a gene therapy target. METHODS: The differentially expressed gene circRNAs in HCC tumor tissues was identified by mining GSE121714 dataset. EdU staining, wound healing, transwell invasion assay, TUNEL staining and western blotting examined proliferation, migration, invasion, apoptosis and epithelial mesenchymal transition (EMT). Xenograft mouse model and orthotopic transplantation tumor mouse model were constructed to verify the role of hsa_circ_001726 in growth and metastasis of HCC. The relationship among CCT2, E2F6, hsa_circ_001726, miR-671-5p and PRMT9 was identified by RNA-fluorescence in situ hybridization, luciferase reporter assay and RNA Immunoprecipitation. RESULTS: Eleven differentially expressed circRNAs were found in HCC tumors. Among them, hsa_circ_001726 was highly expressed in HCC tumors and cells, which was transcribed from CCT2. As a transcription factor of CCT2, E2F6 knockdown inactivated CCT2 promoter and reduced hsa_circ_001726 expression. Moreover, hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway. Additionally, hsa_circ_001726 deficiency repressed malignant phenotypes of HCC cells, including proliferation, migration, invasion, EMT and apoptosis. In vivo, hsa_circ_001726 deficiency reduced tumor growth and lung metastasis of HCC in xenograft mouse models and orthotopic transplantation tumor mouse models. CONCLUSION: Hsa_circ_001726 functioned as an oncogene in HCC, which was derived from CCT2 and regulated by E2F6. Hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway, thereby accelerating malignant phenotypes of HCC. Therefore, targeting hsa_circ_001726 may be a new avenue for HCC treatment.

Circ_0004676 exacerbates triple-negative breast cancer progression through regulation of the miR-377-3p/E2F6/PNO1 axis.

BACKGROUND: The significant roles of circular RNAs (circRNAs) in different cancers and diseases have been reported. We now focused on the possible role of a newly recognized circRNA, circ_0004674 in triple-negative breast cancer (TNBC), and the related downstream mechanism. METHODS: The expression of circ_0004674 in TNBC tissues and cells was determined followed by analysis of the correlation between circ_0004674 and TNBC patients' prognosis. The interaction between circ_0004674, miR-377-3p, E2F6, and PNO1 was then identified using bioinformatics analysis combined with FISH, RIP, RNA pull-down, RT-qPCR, and Western blot analysis. Using gain-of-function and loss-of-function methods, we analyzed the effect of circ_0004674, miR-377-3p, E2F6, and PNO1 on TNBC in vivo and in vitro. RESULTS: Increased circ_0004674 and E2F6 but decreased miR-377-3p were observed in TNBC tissues and MDA-MB-231 TNBC cells, all of which findings were associated with poor prognosis in patients with TNBC. Silencing of circ_0004676 remarkably suppressed the proliferation, cell cycle progression, and migration of TNBC cells in vitro, as well as inhibiting tumorigenesis and metastasis in vivo. Additionally, circ_0004676 served as a sponge of miR-377-3p which bound to the transcription factor E2F6. In the presence of overexpression of circ_0004676, E2F6 expression and its target PNO1 expression were elevated, while miR-377-3p expression was decreased. Interestingly, overexpression of E2F6 could reverse the inhibitory effect on tumor growth caused by downregulation of circ_0004676. CONCLUSION: Our study highlighted the carcinogenic effect of circ_0004676 on TNBC through regulation of the miR-377-3p/E2F6/PNO1 axis. 1. Circ_0004674 is highly expressed in TNBC tissues and cells. 2. Circ_0004674 upregulates the expression of E2F6 by sponging miR-377-3p. 3. E2F6 upregulates PNO1 by binding to the PNO1 promoter. 4. Circ_0004674 favors TNBC progression by regulating the miR-377-3p/E2F6/PNO1 axis. 5. This study provides a new target for the treatment of TNBC.

circ_0089153 exacerbates breast cancer cells proliferation and metastasis via sponging miR-2467-3p/E2F6.

The role of circ_0089153 in breast cancer (BCa) malignancy development was explored. circ_0089153 expression in BCa was analyzed by Gene Expression Omnibus database. Clinical tissues were obtained from 90 BCa patients. Cell counting kit-8 assay, 5-ethnyl-2 deoxyuridine assay and colony formation experiment were applied for proliferation detection. Wound healing assay and Transwell experiment were used for migration and invasion detection. Dual luciferase reporter gene assay, RNA immunoprecipitation assay and RNA pull-down assay were conducted. In vivo growth and metastasis of BCa cells were performed. Quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry were applied for RNAs and proteins expression. The up-modulated circ_0089153 indicated an unfavorable survival of BCa patients. circ_0089153 knockdown attenuated BCa cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) (P < .01). circ_0089153 was miR-2467-3p sponge. Low miR-2467-3p expression indicated a worse survival of BCa patients. miR-2467-3p overexpression reduced BCa cells proliferation, migration, invasion and EMT (P < .05). circ_0089153 enhanced BCa cells proliferation, migration, invasion and EMT by sponging miR-2467-3p (P < .05). E2F6 was directly suppressed by miR-2467-3p. E2F6 high expression in BCa patients associated with worse survival. circ_0089153 knockdown suppressed in vivo BCa cells growth and lung metastasis (P < .01). circ_0089153 was an oncogene in breast cancer, which enhanced proliferation and metastasis through sponging miR-2467-3p/E2F6. circ_0089153 was suggested to be a potential target for BCa target treatment.

LncRNA TMPO-AS1 promotes LCN2 transcriptional activity and exerts oncogenic functions in ovarian cancer.

Ovarian cancer remains the sixth most frequently occurring cancer in women worldwide. Long noncoding RNAs (lncRNAs) are capable of regulating gene expression, and thus, participating in a wide range of biological functions and disease processes including cancer development. Our work suggests that lncRNA TMPO antisense RNA 1 (TMPO-AS1) represents an oncogenic lncRNA in ovarian cancer and presents a novel mechanism involving transcription factor E2F transcription factor 6 (E2F6) and lipocalin-2 (LCN2). We identified upregulated lncRNA TMPO-AS1 in ovarian cancer tissues and cells. siRNA-mediated silencing of lncRNA TMPO-AS1 restrained the aggressiveness of ovarian cancer cells and their pro-angiogenic ability, and reduced the expression of LCN2. LncRNA TMPO-AS1 was found to interact with E2F6, a transcriptional repressor that could bind to the promoter region of LCN2 gene. In addition, silencing of E2F6 or overexpression of LCN2 restored the aggressiveness of ovarian cancer cells and their pro-angiogenic ability following siRNA-mediated silencing of lncRNA TMPO-AS1. Taken together, we demonstrated lncRNA TMPO-AS1 could potentially promote LCN2 transcriptional activity by binding to transcription factor E2F6, and thus, stimulated the progression of ovarian cancer. These findings underscore a possible alternative therapeutic strategy for ovarian cancer treatment.

miR-454 suppresses the proliferation and invasion of ovarian cancer by targeting E2F6.

BACKGROUND: The aberrant expression of microRNA-454 (miR-454) has been confirmed to be involved in the development of cancers. However, the functional role of miR-454 in the progression of ovarian cancer remains unclear. METHODS: The expression of miR-454 in ovarian cancer cells and serum of ovarian cancer patients was detected by RT-PCR. CCK8, colony formation, transwell, and flow cytometry assays were conducted to assess the effects of miR-454 on ovarian cancer cell proliferation, migration, invasion, and apoptosis, respectively. Dual-luciferase reporter assay was used to confirm the targeting relationship between miR-454 and E2F6. The expression pattern of E2F6 in ovarian cancer tissues was detected using immunohistochemistry (IHC) assay. The relative expression of related proteins was examined using western blot analysis. RESULTS: miR-454 was markedly down-regulated by hypoxia in ovarian cancer cells. Compared with normal samples, the expression of miR-454 was up-regulated in the serum of ovarian cancer patients, and correlated with the clinicopathological stages of ovarian cancer. Next, we found that miR-454 overexpression inhibited the proliferation, migration and invasion of OVCAR3 and SKOV3 cells, as well as promoted apoptosis. In addition, the Akt/mTOR and Wnt/ß-catenin signaling pathway were inhibited by miR-454 in ovarian cancer cells. Mechanically, bioinformatic analysis and dual-luciferase reporter assay confirmed that E2F6 was a direct target of miR-454 and negatively regulated by miR-454 in ovarian cancer cells. Moreover, IHC analysis showed that E2F6 was highly expressed in ovarian cancer tissues. Finally, we found that the increasing cell proliferation and migration triggered by E2F6 overexpression were abolished by miR-454 overexpression. CONCLUSION: Taken together, these results highlight the role of miR-454 as a tumor suppressor in ovarian cancer cells by targeting E2F6, indicating that miR-454 may be a potential diagnostic biomarker and therapeutic target for ovarian cancer.

E2F6 functions as a competing endogenous RNA, and transcriptional repressor, to promote ovarian cancer stemness.

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long-term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen-mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c-Kit, by epigenetic silencing the tumor suppressor miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c-KIT, but downregulated miR-193a. Luciferase assays further confirmed that microRNA-193a targets both E2F6 and c-Kit. Interestingly, ChIP-PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR-193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR-193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR-193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen-mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA-193a activity, promoting ovarian cancer stemness and tumorigenesis.

Nuclear receptor TLX regulates islet beta cell proliferation via E2F6.

Both type 1 and type 2 diabetes are associated with loss of functional beta cell mass, and strategies to restore beta cells are urgently needed. We reported previously that overexpression of the nuclear receptor TLX induces beta cell proliferation, but the underlying molecular mechanism has not been defined. Here, we identified direct targets of TLX in beta cells at the genome-wide level by ChIP-Seq. These targets include a cadre of regulators that are known to be critical for proliferation. Among these ChIP targets, E2F6 was tightly associated with the cell cycle modules, and thus, we further analyzed E2F6 expression and function in beta cells. We showed that E2F6 is strongly downregulated by TLX, and its expression inhibits beta cell proliferation. Moreover, coexpression of E2F6 with TLX partially abrogated the proliferative effects of TLX. These results strongly suggest that TLX acts through E2F6 to regulate beta cell proliferation. Together, the results of this study reveal a direct interaction between TLX and E2F6 and suggest new targets for the expansion of functional beta cell mass.

Predictive role of E2F6 in cancer prognosis and responses of immunotherapy.

BACKGROUND: E2F6 is a member of the E2F transcription factor family. Numerous studies have demonstrated that E2F6 is critical to cancer development and progression, but its role in cancer immunotherapy remains unclear. METHODS: Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases were used to obtain RNA-seq data for cancer and normal tissues, and we utilized the cBioPortal to analyze E2F6 genomic alterations in pan-cancer. The protein localization of E2F6 was obtained using the Human Protein Atlas (HPA), and the upregulation of E2F6 expression in clinical glioblastoma multiforme (GBM) tissues was detected by Western blot analysis. The ComPPI website was used to analyze the protein interaction information of E2F6. To evaluate the role of E2F6 in pan-cancer prognosis, we used univariate Cox regression and Kaplan-Meier methods, and gene set enrichment analysis (GSEA) was utilized to identify markers associated with E2F6 expression in tumors. TIMER 2.0 was used to study E2F6-related immune cell infiltration in tumor tissues, and the correlation of E2F6 with immunotherapy biomarkers was investigated using Spearman correlation analysis. The role of E2F6 in the cell cycle was analyzed by flow cytometry, and the Cell Counting Kit-8 (CCK-8) and colony formation assays were utilized to determine the proliferative ability of cells. RESULTS: In most tumor types, E2F6 was highly expressed and was a good predictor of prognosis. E2F6 was significantly related to markers of immune activation, tumor immune cell infiltration, and immune regulators. Furthermore, E2F6 knockdown significantly attenuated the proliferative ability of glioma cells. Finally, E2F6 effectively predicted anti-programmed cell death 1 (PD1) treatment response. CONCLUSION: E2F6 is an effective biomarker that predicts the prognosis of cancer patients treated with anti-immune checkpoint therapy.

The osteoporosis susceptibility SNP rs188303909 at 2q14.2 regulates EN1 expression by modulating DNA methylation and E2F6 binding.

EN1 encodes a homeodomain-containing transcription factor and is a determinant of bone density and fracture. Previous powerful genome-wide association studies (GWASs) have identified multiple single-nucleotide polymorphisms (SNPs) near EN1 at 2q14.2 locus for osteoporosis, but the causal SNPs and functional mechanisms underlying these associations are poorly understood. The target genes regulated by the transcription factor EN1 are also unclear. In this study, we identified rs188303909, a functional CpG-SNP, as a causal SNP for osteoporosis at 2q14.2 through the integration of functional and epigenomic analyses. Functional experiments demonstrated that unmethylated rs188303909 acted as a strong allele-specific distal enhancer to regulate EN1 expression by modifying the binding of transcription factor E2F6, but rs188303909 methylation attenuated the active effect of E2F6 on EN1 expression. Importantly, transcription factor EN1 could differentially bind osteoporosis GWAS lead SNPs rs4869739-T and rs4355801-G to upregulate CCDC170 and COLEC10 expression, thus promoting bone formation. Our study provided a mechanistic insight into expression regulation of the osteoporosis susceptibility gene EN1, which could be a potential therapeutic target for osteoporosis precision medicine. KEY MESSAGES: CpG-SNP rs188303909 is a causal SNP at the osteoporosis susceptibility locus 2q14.2. Rs188303909 distally regulates EN1 expression by modulating DNA methylation and E2F6 binding. EN1 upregulates CCDC170 and COLEC10 expression through osteoporosis GWAS lead SNPs rs4869739 and rs4355801.

Relationship Between Changes in the Expression Levels of miR-134 and E2F6 in Mediating Control of Apoptosis in NMDA-Induced Glaucomatous Mice.

OBJECTIVE: This investigation was to determine the relationship between changes in the expression levels of miR-134 and the E2F transcription factor 6 (E2F6) in mediating control of apoptosis in N-methyl-D-aspartate (NMDA)-induced glaucomatous mice. METHODS: Morphological and structural changes were quantitatively analyzed along with apoptosis in the retinal ganglion cell (RGC) layer, internal plexiform layer and RGCs. Glaucomatous RGCs were transfected, and cell viability and apoptosis were examined. The targeting relationship between miR-134 and E2F6 was analyzed, as well as their expression pattern. RESULTS: Intravitreal injection of NMDA induced a significant reduction in the number of RGCs and thinning of IPL thickness. miR-134 was highly expressed and E2F6 was lowly expressed in glaucoma mice. Suppression of miR-134 or E2F6 overexpression inhibited apoptosis in the glaucomatous RGCs and instead their proliferative activity. MiR-134 targeted inhibition of E2F6 expression. Suppressing rises in E2F6 expression reduced the interfering effect of miR-134 on glaucomatous RGC development. CONCLUSION: Depleting miR134 expression increases, in turn, E2F6 expression levels and in turn reduces glaucomatous RGC apoptosis expression.

AB045. E2F6 promotes temozolomide resistance in glioblastoma by enrichment in CARD6 and POSTN promoter region.

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor. Temozolomide (TMZ) is the most used first-line chemotherapeutic agent for GBM after surgery, but acquired resistance to TMZ frequently leads to treatment failure and is a major challenge in the clinical treatment of GBM. Increasing evidence suggests that E2F transcription factor 6 (E2F6) is associated with a variety of tumor malignant biological behaviors and drug resistance, but its biological function and underlying molecular mechanisms in GBM are unknown. METHODS: The study investigated the levels of E2F6 in both TMZ-sensitive and TMZ-resistant GBM cells and tissues using Western blotting and immunofluorescence assays. In vitro experiments were conducted to explore the impact of E2F6 on TMZ resistance and glioma stem cell stemness. These experiments included Western blotting, colony formation assay, flow cytometry assay, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Bioinformatic analyses were conducted to investigate the mechanism behind the high expression of E2F6 in TMZ-resistant cells and its correlation with caspase recruitment domain 6 (CARD6) and disulfide-linked cell adhesion protein (POSTN). The study employed bioinformatic analyses, messenger RNA (mRNA) sequencing, chromatin immunoprecipitation sequencing assay, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. To examine the function of E2F6, an intracranial xenograft tumor mouse model was used for in vivo experiments. RESULTS: It was found that CARD6 and POSTN were significantly associated with TMZ resistance and survival of GBM patients. E2F6 was up-regulated in TMZ-resistant cells and tissues. Knockdown of E2F6 down-regulated the expression of CARD6, promoted TMZ-induced apoptosis, and enhanced chemo-sensitivity, whereas its overexpression significantly increased TMZ resistance in vitro and in vivo. In addition, E2F6 can promote TMZ resistance through stem-like properties acquisition. We identified a signaling pathway related to E2F6 and POSTN, which maintains the self-renewal of GBM stem cells (GSCs). E2F6 concentrates in the promoter region of POSTN, thereby regulating the expression of GSCs-related genes cluster of differentiation 133 (CD133), Nestin, and sex-determining region Y-box 2 (SOX2), which may be involved in tumor metabolism and drug resistance processes. Down-regulation of E2F6 down-regulated the expression of POSTN and inhibited tumor growth in nude mice. CONCLUSIONS: These results suggest that the E2F6-CARD6/POSTN signaling axis regulates the malignant biological behaviors of GBM and TMZ resistance. These findings are expected to provide promising therapeutic targets for CARD6 overcoming GBM TMZ resistance.

lncRNA ENST00000585827 Contributes to the Progression of Endometrial Carcinoma via Regulating miR-424/E2F6/E2F7 Axis.

Endometrial cancer (EC) ranks fourth among the most common gynecologic malignancies. Despite advances in medical technology, the pathogenesis is still unclear. Numerous reports have identified the involvement of lncRNA in the malignant progression of endometrial cancer. The aim of the study was to investigate the expression level of lncRNA ENST00000585827 (lncRNA E27) in endometrial cancer and the molecular mechanism that regulates the development of endometrial cancer. Combined with the results of the previous study, PCR analysis confirmed that lncRNA E27 was significantly upregulated in endometrial cancer cell lines. The results of CCK-8, wound healing assay, and transwell experiments showed that lncRNA E27 could significantly inhibit cell proliferation, migration, and invasion. Flow cytometry results confirmed that lncRNA E27 could promote apoptosis. Furthermore, based on bioinformatics predictions, dual-luciferase assay and RT-qPCR analysis confirmed that miR-424, as its downstream molecule, competitively regulates the expression of E2F6/E2F7. Rescue experiments further supported that lncRNA E27 inhibited proliferation, migration, invasion, and promoted apoptosis of endometrial cancer through miR-424/E2F6/E2F7 signaling axis. Conclusively, our findings revealed the role of lncRNA E27 in regulating the miR-424/E2F6/E2F7 signaling axis during EC progression, opening up new strategies for the treatment of endometrial cancer.

Tumor-associated macrophage-derived exosomes LINC01592 induce the immune escape of esophageal cancer by decreasing MHC-I surface expression.

BACKGROUND: TAMs (tumor-associated macrophages) infiltration promotes the progression of esophageal cancer (EC). However, the underlying mechanisms remain unclear. METHODS: Abnormal expression of LINC01592 from EC microarrays of the TCGA database was analyzed. LINC01592 expression level was validated in both EC cell lines and tissues. Stable LINC01592 knockdown and overexpression of EC cell lines were established. In vitro and in vivo trials were conducted to test the impact of LINC01592 knockdown and overexpression on EC cells. RNA binding protein immunoprecipitation (RIP), RNA pulldown assays, and Immunofluorescence (IF) were used to verify the combination of E2F6 and LINC01592. The combination of E2F6 and NBR1 was verified through the utilization of ChIP and dual luciferase reporter assays. RESULTS: LINC01592 is carried and transferred by exosomes secreted by M2-TAMs to tumor cells. The molecular mechanism underlying the promotion of NBR1 transcription involves the direct binding of LINC01592 to E2F6, which facilitates the nuclear entry of E2F6. The collaborative action of LINC01592 and E2F6 results in improved NBR1 transcription. The elevation of NBR1 binding to the ubiquitinated protein MHC-I via the ubiquitin domain caused a higher degradation of MHC-I in autophagolysosomes and a reduction in MHC-I expression on the exterior of cancerous cell. Consequently, this caused cancerous cells to escape from CD8+ CTL immune attack. The tumor-promoting impacts of LINC01592, as well as the growth of M2-type macrophage-driven tumors, were significantly suppressed by the interruption of E2F6/NBR1/MHC-I signaling through the effect of siRNA or the corresponding antibody blockade. Significantly, the suppression of LINC01592 resulted in an upregulation of MHC-I expression on the tumor cell membrane, thereby enhancing the efficacy of CD8+ T cell reinfusion therapy. CONCLUSIONS: The investigation conducted has revealed a significant molecular interaction between TAMs and EC via the LINC01592/E2F6/NBR1/MHC-I axis, which facilitates the progression of malignant tumors. This suggests that a therapeutic intervention targeting this axis may hold promise for the treatment of the disease.

A positive feedback loop of CENPU/E2F6/E2F1 facilitates proliferation and metastasis via ubiquitination of E2F6 in hepatocellular carcinoma.

Centromere protein U (CENPU), a centromere-binding protein required for cellular mitosis, has been reported to be closely associated with carcinogenesis in multiple malignancies; however, the role of CENPU in hepatocellular carcinoma (HCC) is still unclear. Herein, we investigated its biological role and molecular mechanism in the development of HCC. High CENPU expression in HCC tissue was observed and correlated positively with a poor prognosis in HCC patients. CENPU knockdown inhibited the proliferation, metastasis, and G1/S transition of HCC cells in vivo and in vitro, while ectopic expression of CENPU exerted the opposite effects. Mechanistically, CENPU physically interacted with E2F6 and promoted its ubiquitin-mediated degradation, thus affecting the transcription level of E2F1 and further accelerating the G1/S transition to promote HCC cell proliferation. E2F1 directly binds to the CENPU promoter and increases the transcription of CENPU, thereby forming a positive regulatory loop. Collectively, our findings indicate a crucial role for CENPU in E2F1-mediated signalling for cell cycle progression and reveal a role for CENPU as a predictive biomarker and therapeutic target for HCC patients.

Deubiquitination of the repressor E2F6 by USP22 facilitates AKT activation and tumor growth in hepatocellular carcinoma.

Dysregulated ubiquitination of tumor-related proteins plays a critical role in tumor development and progression. The deubiquitinase USP22 is aberrantly expressed in certain types of cancer and contributes to aggressive tumor progression. However, the precise mechanism underlying the pro-tumorigenic function of USP22 in hepatocellular carcinoma (HCC) remains unclear. Here, we report that E2F6, a pocket protein-independent transcription repressor, is essential for HCC cell growth, and that its activities are controlled by USP22-mediated deubiquitination. USP22 interacts with and stabilizes E2F6, resulting in the transcriptional repression of phosphatase DUSP1. Moreover, the process involving DUSP1 repression by E2F6 strengthens AKT activation in HCC cells. Therefore, these findings provide mechanistic insights into the USP22-mediated control of oncogenic AKT signaling, emphasizing the importance of USP22-E2F6 regulation in HCC development.

Circular RNA ZNF609 functions as a competing endogenous RNA in regulating E2F transcription factor 6 through competitively binding to microRNA-197-3p to promote the progression of cervical cancer progression.

Countless studies have demonstrated that Circular RNAs (circRNAs) exert vital effects in regulating tumorigenesis of various cancers. CircRNA ZNF609 (circ-ZNF609) has been reported as an oncogene in various human cancers. Nevertheless, its regulating effect in cervical cancer (CC) remains to be further explored. RT-qPCR was adopted to measure circ-ZNF609, miR-197-3p and E2F6 levels. CC cell proliferation, migration and invasion were analyzed via CCK-8 and transwell assays. Dual-luciferase reporter assay was adopted to confirm the interaction between miR-197-3p and circ-ZNF609 or E2F6. In the present study, it was found that circ-ZNF609 was elevated in CC tissues and cell lines, and circ-ZNF609 deletion repressed cell viability, migration and invasion in CC. Moreover, circ-ZNF609 was identified to negatively regulate miR-197-3p expression in CC cells. The inhibition of miR-197-3p abrogated the inhibitory effect on CC cell proliferation, migration and invasion induced by circ-ZNF609 knockdown. Additionally, we further demonstrated that circ-ZNF609 upregulated E2F6 by interacting with miR-197-3p. Finally, rescue assays indicated that E2F6 overexpression upended the suppression of CC progression induced by circ-ZNF609 deletion. In conclusion, circ-ZNF609 promoted CC progression through modulating the miR-197-3p/E2F6 axis as an oncogene. This finding offers a unique insight into CC molecular mechanism and suggests a potential target for CC therapy.

Anticancer Potential of Temozolomide-Loaded Eudragit-Chitosan Coated Selenium Nanoparticles: In Vitro Evaluation of Cytotoxicity, Apoptosis and Gene Regulation.

Resistance to temozolomide (TMZ) is the main cause of death in glioblastoma multiforme (GBM). The use of nanocarriers for drug delivery applications is one of the known approaches to overcome drug resistance. This study aimed to investigate the possible effect of selenium-chitosan nanoparticles loaded with TMZ on the efficacy of TMZ on the expression of MGMT, E2F6, and RELA genes and the rate of apoptosis in the C6 cell line. Selenium nanoparticles (SNPs) were loaded with TMZ and then they were coated by Eudragit® RS100 (Eud) and chitosan (CS) to prepare Se@TMZ/Eud-Cs. Physicochemical properties were determined by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDAX), thermal gravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS) methods. Se@TMZ/Eud-Cs was evaluated for loading and release of TMZ by spectrophotometric method. Subsequently, SNPs loaded with curcumin (as a fluorophore) were analyzed for in vitro uptake by C6 cells. Cytotoxicity and apoptosis assay were measured by MTT assay and Annexin-PI methods. Finally, real-time PCR was utilized to determine the expression of MGMT, E2F6, and RELA genes. Se@TMZ/Eud-Cs was prepared with an average size of 200 nm as confirmed by the DLS and microscopical methods. Se@TMZ/Eud-Cs presented 82.77 ± 5.30 loading efficiency with slow and pH-sensitive release kinetics. SNPs loaded with curcumin showed a better uptake performance by C6 cells compared with free curcumin (p-value < 0.01). Coated nanoparticles loaded with TMZ showed higher cytotoxicity, apoptosis (p-value < 0.0001), and down-regulation of MGMT, E2F6, and RELA and lower IC50 value (p-value < 0.0001) than free TMZ and control (p-value < 0.0001) groups. Using Cs as a targeting agent in Se@TMZ/Eud-Cs system improved the possibility for targeted drug delivery to C6 cells. This drug delivery system enhanced the apoptosis rate and decreased the expression of genes related to TMZ resistance. In conclusion, Se@TMZ/Eud-Cs may be an option for the enhancement of TMZ efficiency in GBM treatment.

The COX10-AS1/miR-641/E2F6 Feedback Loop Is Involved in the Progression of Glioma.

Glioma is the most common primary tumour of the central nervous system and is considered one of the greatest challenges for neurosurgery. Mounting evidence has shown that lncRNAs participate in various biological processes of tumours, including glioma. This study aimed to reveal the role and relevant mechanism of COX10-AS1 in glioma. The expression of COX10-AS1, miR-641 and E2F6 was measured by qRT-PCR and/or western blot. Clone formation assays, EdU assays, Transwell assays and tumour xenograft experiments were performed to evaluate the effects of COX10-AS1, miR-641 and E2F6 on glioma proliferation, migration and invasion. Luciferase reporter assays, RNA pull-down assays and ChIP assays were conducted to analyse the relationship among COX10-AS1, miR-641 and E2F6. We demonstrated that COX10-AS1 was upregulated in glioma tissues and cell lines, which was related to the grade of glioma and patient survival. Next, through functional assays, we found that COX10-AS1 influenced the proliferation, migration and invasion of glioma cell lines. Then, with the help of bioinformatics analysis, we confirmed that COX10-AS1 regulated glioma progress by acting as a sponge of miR-641 to regulate E2F6. Moreover, further study indicated that E2F6 could promote COX10-AS1 expression by binding to its promoter region. Taken together, the data indicated that COX10-AS1 acts as an oncogene in combination with COX10-AS1/miR-641/E2F6 in glioma, which may be beneficial to the diagnosis and treatment of glioma.

CircRNA DUSP16 Knockdown Suppresses Colorectal Cancer Progression by Regulating the miR-432-5p/E2F6 Axis.

BACKGROUND: Circular RNAs (circRNAs) play critical roles in various types of cancer. The aim of the present study was to investigate the function and underlying mechanism of CircRNA DUSP16 (circDUSP16) in colorectal cancer (CRC) development. MATERIALS AND METHODS: The expression levels of circDUSP16, microRNA-432-5p (miR-432-5p), and E2F transcription factor 6 (E2F6) were measured by RT-qPCR. Cell proliferation, migration, invasion, and apoptosis were investigated by CCK-8, Transwell, and flow cytometry assays. Western blot analysis was used to evaluate the levels of the pro-apoptotic protein (Bax and cleaved-caspase 3) and the anti-apoptotic protein (Bcl-2). Luciferase reporter assay and RIP assay were used to analyze the association between miR-432-5p and circDUSP16 or E2F6. A xenograft tumor model was employed to explore the effect of circDUSP16 on CRC tumor growth in vivo. RESULTS: CircDUSP16 expression was upregulated in CRC tissues and cell lines. High circDUSP16 expression was correlated with low survival rate. Furthermore, circDUSP16 knockdown repressed cell proliferation, migration, and invasion and induced apoptosis in CRC. CircDUSP16 caused a negative regulation in miR-432-5p expression. In addition, E2F6 expression was elevated in CRC tissues. Inhibition of miR-432-5p promoted the proliferative and metastatic activity of CRC cells and inhibited the induction of apoptosis. Inhibition of E2F6 expression partially abolished the effects caused by miR-432-5p depletion. Moreover, circDUSP16 upregulated E2F6 expression by reducing miR-432-5p expression. Furthermore, circDUSP16 silencing repressed CRC tumor growth in vivo. CONCLUSION: The results supported the hypothesis that circDUSP16 knockdown suppressed CRC progression by regulating the miR-432-5p/E2F6 axis, suggesting that the circDUSP16/miR-432-5p/E2F6 network may be a potential therapeutic target for CRC.