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.

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.

rtcisE2F promotes the self-renewal and metastasis of liver tumor-initiating cells via N6-methyladenosine-dependent E2F3/E2F6 mRNA stability.

Liver cancer is highly heterogeneous, and the tumor tissue harbors a variety of cell types. Liver tumor initiating cells (TICs) well contribute to tumor heterogeneity and account for tumor initiation and metastasis, but the molecular mechanisms of liver TIC self-renewal are elusive. Here, we identified a functional read-through rt-circRNA, termed rtcisE2F, that is highly expressed in liver cancer and liver TICs. rtcisE2F plays essential roles in the self-renewal and activities of liver TICs. rtcisE2F targets E2F6 and E2F3 mRNAs, attenuates mRNA turnover, and increases E2F6/E2F3 expression. Mechanistically, rtcisE2F functions as a scaffold of N-methyladenosine (m6A) reader IGF2BP2 and E2F6/E2F3 mRNA. rtcisE2F promotes the association of E2F6/E2F3 mRNAs with IGF2BP2, and inhibits their association with another m6A reader, YTHDF2. IGF2BP2 inhibits E2F6/E2F3 mRNA decay, whereas YTHDF2 promotes E2F6/E2F3 mRNA decay. By switching m6A readers, rtcisE2F enhances E2F6/E2F3 mRNA stability. E2F6 and E2F3 are both required for liver TIC self-renewal and Wnt/ß-catenin activation, and inhibition of these pathways is a potential strategy for preventing liver tumorigenesis and metastasis. In conclusion, the rtcisE2F-IGF2BP2/YTHDF2-E2F6/E2F3-Wnt/ß-catenin axis drives liver TIC self-renewal and initiates liver tumorigenesis and metastasis, and may provide a strategy to eliminate liver TICs.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.

Expression patterns for Bcl-2, EMA, ß-catenin, E-cadherin, PAX8, and MIB1 in thymomas.

The expression of immunohistochemical markers has been extensively investigated in thymomas to assist in the differential diagnosis. We have studied six select markers to determine their utility in the evaluation of these tumors. A series of 126 thymomas including 33 type A, 27 type AB, 20 type B1, 22 type B2, and 24 type B3, were examined utilizing a tissue microarray (TMA) technique with antibodies to e-cadherin, ß-catenin, PAX8, bcl-2, EMA, and MIB-1. Keratin AE1/AE3 and p63 were used for quality control. A significant finding was strong and consistent positivity for bcl-2 in type A (90%) and type AB (88.8%) thymoma, while 100% of B1, B2, and B3 were negative. The distribution of e-cadherin and ß-catenin was not useful for differential diagnosis. E-cadherin and ß-catenin were expressed in a high proportion of all the tumors (92-100%), except for B2 thymoma which showed only 45% expression. A significant increase in the expression of the MIB-1 proliferation marker (mean: 12.8% nuclear positivity) was also observed in B3 thymoma compared with the other histologic types. Statistical significance was confirmed using Kruskal's non-parameterized test for distribution. EMA was generally negative except for spindle cells in the fibrous septa in types A and AB thymoma. PAX8 showed less consistent nuclear staining than p63 and was only widely expressed in 55.7% of cases. Bcl-2 may serve as a useful marker to separate spindle cell thymomas (Type A and AB) from the other types, and the MIB1 proliferation index may be of use to differentiate type B2 from type B3 thymoma.

E2F6 initiates stable epigenetic silencing of germline genes during embryonic development.

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse 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.

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.

CREB acts as a common transcription factor for major epigenetic repressors; DNMT3B, EZH2, CUL4B and E2F6.

CREB signaling is known for several decades, but how it regulates both positive and negative regulators of cell proliferation is not well understood. On the other hand functions of major epigenetic repressors such as DNMT3B, EZH2 and CUL4B for their repressive epigenetic modifications on chromatin have also been well studied. However, there is very limited information available on how these repressors are regulated at their transcriptional level. Here, using computational tools and molecular techniques including site directed mutagenesis, promoter reporter assay, chromatin immunoprecipitation (ChIP), we identified that CREB acts as a common transcription factor for DNMT3B, EZH2, CUL4B and E2F6. ChIP assay revealed that pCREB binds to promoters of these repressors at CREs and induce their transcription. As expected, the expression of these repressors and their associated repressive marks particularly H3K27me3 and H2AK119ub are increased and decreased upon CREB overexpression and knock-down conditions respectively in the cancer cells indicating that CREB regulates the functions of these repressors by activating their transcription. Since CREB and these epigenetic repressors are overexpressed in various cancer types, our findings showed the molecular relationship between them and indicate that CREB is an important therapeutic target for cancer therapy.

LncRNA GHET1 promotes cervical cancer progression through regulating AKT/mTOR and Wnt/ß-catenin signaling pathways.

Cervical cancer (CC) is a prevalent gynecological cancer, and the patients with CC usually suffer from dismal prognosis. Long non-coding RNAs (lncRNAs) are demonstrated to serve as promising biological targets in human cancers. Gastric carcinoma proliferation enhancing transcript 1 (GHET1) has been revealed to function as an oncogene in several cancers, but it has never been investigated in CC. We proposed to examine the biological role of GHET1 in CC and the underlying mechanism and validated the up-regulated expression of GHET1 in CC cell lines. Loss-of-function assays demonstrated that down-regulation of GHET1 inhibited cell growth, migration and epithelial-to-mesenchymal transition (EMT) in CC. Furthermore, we validated that GHET1 down-regulation could inactivate AKT/mTOR and Wnt/ß-catenin pathways, and that respective activation of these two pathways abrogated the inhibitive effect of GHET1 knockdown on CC cell growth, migration and EMT. Moreover, we unfolded a preliminary investigation on the modulation of GHET1 on AKT/mTOR and Wnt/ß-catenin pathways. We found that GHET1 stabilized E2F6 mRNA through interacting with IGF2BP2, so as to regulate the activity of AKT/mTOR and Wnt/ß-catenin pathways. Rescue assays also proved that GHET1 regulated these two pathways and CC cell growth, migration and EMT through E2F6. In conclusion, we revealed that down-regulation of GHET1 suppresses cervical cancer progression through regulating AKT/mTOR and Wnt/ß-catenin signaling pathways, indicating GHET1 as a promising molecular biomarker for CC treatment improvement.

Could EMA and cytokeratin 7 be useful in distinguishing tricholemmal carcinoma from clear-cell squamous cell carcinoma? A case series from our department and a brief review of the literature.

Tricholemmal carcinoma is a malignant cutaneous adnexal tumor showing outer root sheath differentiation, thought to be the malignant counterpart of trichilemmoma. Although the real existence of tricholemmal carcinoma continues to be a matter of debate, it has been introduced in the recently published 4th edition of World Health Organization classification of skin tumors. Herein, we evaluated whether immunohistochemistry (EMA, CK7, CK5/14, p63, p16, and Ber-EP4) supports tricholemmal carcinoma as a separate entity and whether it could be useful in this differential diagnosis. A total of 9 cases, 3 tricholemmal carcinomas and 6 clear-cell squamous cell carcinomas were evaluated on the basis of histological criteria suggested by the WHO. In our opinion, although these results need to be validated in larger series, they support tricholemmal carcinoma as a separate entity and suggest an immunohistochemical profile (clear-cell squamous cell carcinomas: EMA diffusely positive, CK7 negative; tricholemmal carcinoma: EMA negative, CK7 patchy or moderately positive) that could be useful for this differential diagnosis.

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.

Hypoxia-sensitive LINC01436 is regulated by E2F6 and acts as an oncogene by targeting miR-30a-3p in non-small cell lung cancer.

Dysregulation of long noncoding RNA (lncRNA) is known to be involved in numerous human diseases, including lung cancer. However, the precise biological functions of most lncRNA remain to be elucidated. Here, we identified a novel up-regulated lncRNA, LINC01436 (RefSeq: NR_110419.1), in non-small cell lung cancer (NSCLC). High expression of LINC01436 was significantly associated with poor overall survival. Notably, LINC01436 expression was transcriptionally repressed by E2F6 under normoxia, and the inhibitory effect was relieved in a hypoxic microenvironment. Gain- and loss-of-function studies revealed that LINC01436 acted as a proto-oncogene by promoting lung cancer cell growth, migration and invasion in vitro. Xenograft tumor assays in nude mice confirmed that LINC01436 promoted tumor growth and metastasis in vivo. Mechanistically, LINC01436 exerted biological functions by acting as a microRNA (miR)-30a-3p sponge to regulate the expression of its target gene EPAS1. Our findings characterize LINC01436 as a new hypoxia-sensitive lncRNA with oncogenic function in NSCLC, suggesting that LINC01436 may be a potential biomarker for prognosis and a potential target for treatment.

microRNA-193a stimulates pancreatic cancer cell repopulation and metastasis through modulating TGF-ß2/TGF-ßRIII signalings.

BACKGROUND: Pancreatic cancer characterizes high recurrence and poor prognosis. In clinical practice, radiotherapy is widely used for pancreatic cancer treatment. However, the outcome remains undesirable due to tumor repopulation and following recurrence and metastasis after radiation. So, it is highly needed to explore the underlying molecular mechanisms and accordingly develop therapeutic strategies. Our previous studies revealed that dying cells from chemoradiation could stimulate repopulation of surviving pancreatic cancer cells. However, we still knew little how dying cells provoke pancreatic cancer cell repopulation. We herein would explore the significance of TGF-ß2 changes and investigate the modulation of microRNA-193a (miR-193a), and identify their contributions to pancreatic cancer repopulation and metastasis. METHODS: In vitro and in vivo repopulation models were established to mimic the biological processes of pancreatic cancer after radiation. Western blot, real-time PCR and dual-luciferase reporter assays were accordingly used to detect miR-193a and TGF-ß2/TGF-ßRIII signalings at the level of molecular, cellular and experimental animal model, respectively. Flow cytometry analysis, wound healing and transwell assay, vascular endothelial cell penetration experiment, and bioluminescence imaging were employed to assessthe biological behaviors of pancreatic cancer after different treatments. Patient-derived tumor xenograft (PDX) mice models were established to evaluate the therapeutic potential of miR-193a antagonist on pancreatic cancer repopulation and metastasis after radiation. RESULTS: miR-193a was highly expressed in the irradiated pancreatic cancer dying cells, accordingly elevated the level of miR-193a in surviving cells, and further promoted pancreatic cancer repopulation and metastasis in vitro and in vivo. miR-193a accelerated pancreatic cancer cell cycle and stimulated cell proliferation and repopulation through inhibiting TGF-ß2/TGF-ßRIII/SMADs/E2F6/c-Myc signaling, and even destroyed normal intercellular junctions and promoted metastasis via repressing TGF-ß2/TGF-ßRIII/ARHGEF15/ABL2 pathway. Knockdown of miR-193a or restoration of TGF-ß2/TGF-ßRIII signaling in pancreatic cancer cells was found to block pancreatic cancer repopulation and metastasis after radiation. In PDX models, the treatment in combination with miR-193a antagonist and radiation was found to dramatically inhibit pancreatic cancer cell repopulation and metastasis, and further improved the survival after radiation. CONCLUSIONS: Our findings demonstrated that miR-193a stimulated pancreatic cancer cell repopulation and metastasis through modulating TGF-ß2/TGF-ßRIII signalings, and miR-193a might be a potential therapeutic target for pancreatic cancer repopulation and metastasis.

Contrasting effects of an Mdm2 functional polymorphism on tumor phenotypes.

MDM2, an E3 ubiquitin ligase, is a potent inhibitor of the p53 tumor suppressor and is elevated in many human cancers that retain wild-type p53. MDM2 SNP309G is a functional polymorphism that results in elevated levels of MDM2 (due to enhanced SP1 binding to the MDM2 promoter) thus decreasing p53 activity. Mdm2SNP309G/G mice are more prone to spontaneous tumor formation than Mdm2SNP309T/T mice, providing direct evidence for the impact of this SNP in tumor development. We asked whether environmental factors impact SNP309G function and show that SNP309G cooperates with ionizing radiation to exacerbate tumor development. Surprisingly, ultraviolet B light or Benzo(a)pyrene exposure of skin shows that SNP309G allele actually protects against squamous cell carcinoma susceptibility. These contrasting differences led us to interrogate the mechanism by which Mdm2 SNP309 regulates tumor susceptibility in a tissue-specific manner. Although basal Mdm2 levels were significantly higher in most tissues in Mdm2SNP309G/G mice compared with Mdm2SNP309T/T mice, they were significantly lower in Mdm2SNP309G/G keratinocytes, the cell-type susceptible to squamous cell carcinoma. The assessment of potential transcriptional regulators in ENCODE ChIP-seq database identified transcriptional repressor E2F6 as a possible negative regulator of MDM2 expression. Our data show that E2F6 suppresses Mdm2 expression in cells harboring the SNP309G allele but not the SNP309T allele. Thus, Mdm2 SNP309G exhibits tissue-specific regulation and differentially impacts cancer risk.

E2F6 Impairs Glycolysis and Activates BDH1 Expression Prior to Dilated Cardiomyopathy.

RATIONALE: The E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme ß-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker. OBJECTIVE: To define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1. METHODS AND RESULTS: We previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the Bdh1 transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone ß-hydroxybutyrate (ß-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to ß-OHB. CONCLUSIONS: The data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium through a mechanism involving cyclin B1. We reveal BDH1 expression as an early biomarker of heart failure and its potential impact, through ketone signaling, on CX-43 levels in E2F6-induced DCM.

[Effects of miR-424 on Proliferation and Migration Abilities in Non-small Cell Lung Cancer A549 Cells and Its Molecular Mechanism].

BACKGROUND: The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC) have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected. METHODS: NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot. RESULTS: After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549. CONCLUSIONS: miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6.
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EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

Interplay between the E2F pathway and ß-adrenergic signaling in the pathological hypertrophic response of myocardium.

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in the myocardium via manipulation of the unique E2F repressor, E2F6, which is believed to repress gene activity independently of Rb. Mice with targeted expression of E2F6 in postnatal myocardium developed dilated cardiomyopathy (DCM) without hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory hypertrophic growth as well as their response to the ß-adrenergic agonist isoproterenol. As early as 2 weeks, E2F6 transgenic (Tg) mice present with dilated thinner left ventricles and significantly reduced ejection fraction and fractional shortening which persists at 6 weeks of age, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with isoproterenol (6.1 mg/kg/day) show double the increase in LVW:BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot analysis revealed the activation of the adrenergic pathway in Tg heart tissue under basal conditions with ~2-fold increase in the level of ß2-adrenergic receptors (p-value: 8.9E-05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-Src tyrosine-protein kinase (p-value: 0.0002), extracellular receptor kinase 2 (ERK2) (p-value: 0.0005), and induction of the anti-apoptotic protein Bcl2 (p-value 0. 0.00001). In contrast, a ~60% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a ~four fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, were evident in the myocardium of E2F6-Tg mice. The expression of E2F3 was down-regulated by E2F6, but was restored by isoproterenol. Further, Rb expression was down-regulated in Tg mice in response to isoproterenol implying a net activation of the E2F pathway. Thus the unique regulation of E2F activity by E2F6 renders the myocardium hypersensitive to adrenergic stimulus resulting in robust hypertrophic growth. These data reveal a novel interplay between the E2F pathway, ß2-adrenergic/PKA/PDE4D, and ERK/c-Src axis in fine tuning the pathological hypertrophic growth response. E2F6 deregulates E2F3 such that pro-hypertrophic growth and survival are enhanced via ß2-adrenergic signaling however this response is outweighed by the induction of anti-hypertrophic signals so that left ventricle dilation proceeds without any increase in muscle mass.

Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells.

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth.