LINC01579 promotes cell proliferation by acting as a ceRNA of miR-139-5p to upregulate EIF4G2 expression in glioblastoma.

Glioblastoma (GBM), a malignant and lethal tumor, remains a big threat to human health and life. Increasing explorations have confirmed that long noncoding RNAs are involved in the tumorigenesis and development of multiple cancers. Nevertheless, the regulatory mechanism of (long intergenic nonprotein coding RNA 1579 LINC01579) in GBM remains to be investigated. In this study, the expression of LINC01579 was upregulated in GBM cells and LINC01579 knockdown inhibited cell proliferation as well as promoted cell apoptosis. Additionally, LINC01579 acted as a sponge for miR-139-5p in GBM and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) was found to be a downstream target of miR-139-5p. Furthermore, the positive correlation of LINC01579 and EIF4G2 as well as the converse correlation between miR-139-5p and LINC01579 (or EIF4G2) were revealed by the experiments. Based on rescue assays, EIF4G2 overexpression or miR-139-5p inhibitor partially recovered the function of LINC01579 knockdown on cell proliferation and apoptosis. In summary, the results of this study verified that LINC01579 modulated cell proliferation and cell apoptosis in GBM by competitively binding with miR-139-5p to regulate EIF4G2, which provided a new clue to figure out potential therapy for patients suffered from GBM.

Overexpression of eIF4F components in meningiomas and suppression of meningioma cell growth by inhibiting translation initiation.

Meningiomas frequently display activation of the PI3K/AKT/mTOR pathway, leading to elevated levels of phospho-eukaryotic translation initiation factor 4E binding proteins, which enhances protein synthesis; however, it is not known whether inhibition of protein translation is an effective treatment option for meningiomas. We found that human meningiomas expressed high levels of the three components of the eukaryotic initiation factor 4F (eIF4F) translation initiation complex, eIF4A, eIF4E, and eIF4G. The expression of eIF4A and eIF4E was important in sustaining the growth of NF2-deficient benign meningioma Ben-Men-1 cells, as shRNA-mediated knockdown of these proteins strongly reduced cell proliferation. Among a series of 23 natural compounds evaluated, silvestrol, which inhibits eIF4A, was identified as being the most growth inhibitory in both primary meningioma and Ben-Men-1 cells. Silvestrol treatment of meningioma cells prominently induced G2/M arrest. Consistently, silvestrol significantly decreased the amounts of cyclins D1, E1, A, and B, PCNA, and Aurora A. In addition, total and phosphorylated AKT, ERK, and FAK, which have been shown to be important drivers for meningioma cell proliferation, were markedly lower in silvestrol-treated Ben-Men-1 cells. Our findings suggest that inhibiting protein translation could be a potential treatment for meningiomas.

miR-139-5p controls translation in myeloid leukemia through EIF4G2.

MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34(+)-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27(Kip1). Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML.

Down-regulation of eIF4GII by miR-520c-3p represses diffuse large B cell lymphoma development.

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3.

The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

DNA damage and eIF4G1 in breast cancer cells reprogram translation for survival and DNA repair mRNAs.

The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest, and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1 (eukaryotic initiation factor 4γ1). Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis [Survivin, hypoxia inducible factor 1α (HIF1α), X-linked inhibitor of apoptosis (XIAP)], promoting cell cycle arrest [growth arrest and DNA damage protein 45a (GADD45a), protein 53 (p53), ATR-interacting protein (ATRIP), Check point kinase 1 (Chk1)] and DNA repair [p53 binding protein 1 (53BP1), breast cancer associated proteins 1, 2 (BRCA1/2), Poly-ADP ribose polymerase (PARP), replication factor c2-5 (Rfc2-5), ataxia telangiectasia mutated gene 1 (ATM), meiotic recombination protein 11 (MRE-11), and others]. Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. Although some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest, and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage.

The soy isoflavone equol may increase cancer malignancy via up-regulation of eukaryotic protein synthesis initiation factor eIF4G.

Dietary soy is thought to be cancer-preventive; however, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein or combined soy isoflavones (genistein, daidzein, and glycitein) increased primary mammary tumor growth and metastasis. Cancer-promoting molecules, including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E, were up-regulated in mammary tumors from mice that received dietary daidzein. Herein, we show that increased eIF expression in tumor extracts of mice after daidzein diets is associated with protein expression of mRNAs with internal ribosome entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. Results with metastatic cancer cell lines show that some of the effects of daidzein in vivo can be recapitulated by the daidzein metabolite equol. In vitro, equol, but not daidzein, up-regulated eIF4G without affecting eIF4E or its regulator, 4E-binding protein (4E-BP), levels. Equol also increased metastatic cancer cell viability. Equol specifically increased the protein expression of IRES containing cell survival and proliferation-promoting molecules and up-regulated gene and protein expression of the transcription factor c-Myc. Moreover, equol increased the polysomal association of mRNAs for p 120 catenin and eIF4G. The elevated eIF4G in response to equol was not associated with eIF4E or 4E-binding protein in 5' cap co-capture assays or co-immunoprecipitations. In dual luciferase assays, IRES-dependent protein synthesis was increased by equol. Therefore, up-regulation of eIF4G by equol may result in increased translation of pro-cancer mRNAs with IRESs and, thus, promote cancer malignancy.

Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon.

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f-a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

Increased B cell proliferation and reduced Ig production in DREAM transgenic mice.

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experiments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased protein degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 gene, which encodes a protein related to protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes.

Over-expression of eukaryotic translation initiation factor 4 gamma 1 correlates with tumor progression and poor prognosis in nasopharyngeal carcinoma.

BACKGROUND: The aim of the present study was to analyze the expression of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in nasopharyngeal carcinoma (NPC) and its correlation with clinicopathologic features, including patients' survival time. METHODS: Using real-time PCR, we detected the expression of EIF4G1 in normal nasopharyngeal tissues, immortalized nasopharyngeal epithelial cell lines NP69, NPC tissues and cell lines. EIF4G1 protein expression in NPC tissues was examined using immunohistochemistry. Survival analysis was performed using Kaplan-Meier method. The effect of EIF4G1 on cell invasion and tumorigenesis were investigated. RESULTS: The expression levels of EIF4G1 mRNA were significantly greater in NPC tissues and cell lines than those in the normal nasopharyngeal tissues and NP69 cells (P < 0.001). Immunohistochemical analysis revealed that the expression of EIF4G1 protein was higher in NPC tissues than that in the nasopharyngeal tissues (P < 0.001). In addition, the levels of EIF4G1 protein in tumors were positively correlated with tumor T classification (P = 0.039), lymph node involvement (N classification, P = 0.008), and the clinical stages (P = 0.003) of NPC patients. Patients with higher EIF4G1 expression had shorter overall survival time (P = 0.019). Multivariate analysis showed that EIF4G1 expression was an independent prognostic indicator for the overall survival of NPC patients. Using shRNA to knock down the expression of EIF4G1 not only markedly inhibited cell cycle progression, proliferation, migration, invasion, and colony formation, but also dramatically suppressed in vivo xenograft tumor growth. CONCLUSION: Our data suggest that EIF4G1 can serve as a biomarker for the prognosis of NPC patients.

[Establishment of a stable nasopharyngeal carcinoma cell line with lentivirus-mediated RNA interference for EIF4G1 gene silencing].

OBJECTIVE: To establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA). METHODS: The EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F, 6-10B, C666-1, CNE1, CNE2, HNE1, HONE1, and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells with constant expression of the EIF4G1-siRNA, the efficiency of EIF4G1 mRNA expression interference was determined using QRT-PCR. RESULTS: The 8 NPC cell lines showed differential expression of EIF4G1 mRNA, among which 5-8F cells had the highest EIF4G1 expression. The recombinant lentivirus plasmid pLenti6/BLOCK-iT-DEST/EIF4G1-shRNA was successfully constructed and verified by PCR and sequencing. The EIF4G1 mRNA level of 5-8F cells infected with shRNA-EIF4G1 lentivirus was significantly reduced as compared with the negative control and the blank control cells. CONCLUSION: The recombinant lentivirus vector pLenti6/BLOCK- iT-DEST/EIF4G1-shRNA we constructed results in marked downregulation of EIF4G1 mRNA expression and constant expression of EIF4G1-siRNA after infection of 5-8F cells.

Inhibition of host protein synthesis in B95a cells infected with the HL strain of measles virus.

The shut-off of host protein synthesis in virus-infected cells is one of the important mechanisms for viral replication. In this report, we showed that the HL strain of measles virus (MeV-HL) as well as other field isolates, which were isolated from human blood lymphocytes using B95a cells, induce the shut-off in B95a cells. Since the Edmonston strain of MeV failed to induce the shut-off in B95a cells, the ability to induce the shut-off was considered to be dependent on virus strains. Although, the modification of eukaryotic translation initiation factors (eIF) including eIF4G, eIF4E, and 4E-BP1 was reported for shut-off by various viruses, the involvement of these eIFs was not observed in MeV-HL-infected B95a cells. Instead, the accumulation of phosphorylated eIF2alpha was found to coincide to the decrease of host protein synthesis, suggesting the involvement of phosphorylation of eIF2alpha in inhibition of translation as one of the mechanisms of the shut-off.

Sex-dependent differences in the regulation of myocardial protein synthesis following long-term ethanol consumption.

Chronic heavy alcohol consumption alters cardiac structure and function. Controversies remain as to whether hearts from females respond to the chronic ethanol intake in a manner analogous to males. In particular, sex differences in the myocardial response to chronic alcohol consumption remain unresolved at the molecular level. The purpose of the present set of experiments was to determine whether alterations in cardiac structure and protein metabolism show sexual dimorphism following chronic alcohol consumption for 26 wk. In control animals, hearts from female rats showed lowered heart weights and had thinner ventricular walls compared with males. The smaller heart size was associated with a lower protein content that occurred in part from a reduced rate of protein synthesis. Chronic alcohol consumption in males, but not in females, caused a thinning of the ventricular wall and intraventricular septum, as assessed by echocardiography, correlating with the loss of heart mass. The alterations in cardiac size occurred, in part, through a lowering of the protein content secondary to a diminished rate of protein synthesis. The decreased rate of protein synthesis appeared related to a reduced assembly of active eukaryotic initiation factor (eIF)4G.eIF4E complex secondary to both a diminished phosphorylation of eIF4G and increased formation of inactive 4Ebinding protein (4EBP1).eIF4E complex. The latter effects occurred as a result of decreased phosphorylation of 4EBP1. None of these ethanol-induced alterations in hearts from males were observed in hearts from females. These data suggest that chronic alcohol-induced impairments in myocardial protein synthesis results, in part, from marked decreases in eIF4E.eIF4G complex formation in males. The failure of female rats consuming ethanol to show structural changes appears related to the inability of ethanol to affect the regulation protein synthesis to the same extent as their male counterparts.

[Study on the expression of eIF families after elemene treatment].

OBJECTIVE: To investigate the activity of Caspase-3 and the expression of eukaryotic initiation factor families, bFGF and VEGF after elemene on laryngeal carcinoma HEp-2 cells. METHOD: The HEp-2 cells after elemene treatment were analyzed utilizing Westernblot and reverse transcriptase polymerase chain reaction (RT-PCR). The activity of Caspase-3 was assessed by colorimetric assay. RESULT: The activity of Caspase-3 was enhanced after elemene treatment. The protein expression of eIF4E, eIF4G, bFGF and VEGF were significantly inhibited by elemene; and the mRNA expression of bFGF and VEGF were inhibited either. CONCLUSION: Elemene can effectively inhibit the growth of HEp-2 cells and result in the alteration of activity of Caspase-3. There were significant correlations between the decreased expression of protein eIF4E, eIF4G, bFGF and VEGF. The mechanism of eIF4E and eIF4G decrease the expression of bFGF and VEGF is post-transcriptional.