Complement factor H attenuates TNF-α-induced inflammation by upregulating EIF3C in rheumatoid arthritis.

OBJECTIVE: To explore the role and underlying mechanism of Complement Factor H (CFH) in the peripheral and joint inflammation of RA patients. METHODS: The levels of CFH in the serum and synovial fluid were determined by ELISA. The pyroptosis of monocytes was determined by western blotting and flow cytometry. The inflammation cytokine release was tested by ELISA. The cell migration and invasion ability of fibroblast-like synoviocytes (FLS) were tested by Wound healing Assay and transwell assay, respectively. The potential target of CFH was identified by RNA sequencing. RESULTS: CFH levels were significantly elevated in the serum and synovial fluid from RA and associated with high sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate (ESR), and disease activity score 28 (DAS28). TNF-α could inhibit CFH expression, and CFH combined with TNF-α significantly decreased cell death, cleaved-caspase 3, gasdermin E N-terminal (GSDME-N), and inflammatory cytokines release (IL-1ß and IL-6) of RA-derived monocytes. Stimulated with TNF-α increased CFH levels in RA FLS and CFH inhibits the migration, invasion, and TNF-α-induced production of inflammatory mediators, including proinflammatory cytokines (IL-6, IL-8) as well as matrix metalloproteinases (MMPs, MMP1 and MMP3) of RA FLSs. The RNA-seq results showed that CFH treatment induced upregulation of eukaryotic translation initiation factor 3 (EIF3C) in both RA monocytes and FLS. The migration of RA FLSs was promoted and the expressions of IL-6, IL-8, and MMP-3 were enhanced upon EIF3C knockdown under the stimulation of CFH combined with TNF-α. CONCLUSION: In conclusion, we have unfolded the anti-inflammatory roles of CFH in the peripheral and joints of RA, which might provide a potential therapeutic target for RA patients.

Eukaryotic translation initiation factor 3 subunit B promotes head and neck cancer via CEBPB translation.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide. Deregulation of mRNA translation is a frequent feature of cancer. Eukaryotic translation initiation factor 3 subunit B (EIF3B) has been reported as an oncogene; however, its role in HNSCC has yet to be fully elucidated. METHODS: In this study, the clinical significance of EIF3B expression was analyzed based on TCGA datasets. Then, EIF3B expression was knocked down and its role in HNSCC was revealed. To explore the molecular mechanisms of EIF3B, we applied RNA sequencing and proteomics and acquired deregulated pathways. RNA immunoprecipitation (RIP) sequencing was conducted to reveal the target mRNAs of EIF3B, and TCGA datasets were used to validate potential targets of EIF3B. RESULTS: Elevated expression of EIF3B was observed in the HNSCC cancer samples. The expression of EIF3B was significantly correlated with the patient's sex, age, HPV infection status, T stage, N stage, perineural invasion status and survival status. EIF3B serves as a marker of an unfavorable HNSCC prognosis. EIF3B-silenced Fadu and Cal27 cells exhibited reduced cell numbers, and EIF3B knockdown induced apoptosis in both cell lines. The EIF3B-silenced cells demonstrated decreased invasion and migration capabilities, and the EIF3B knockdown group mice showed significantly decreased tumor volumes. The results show that EIF3B promotes CEBPB translation and activates the MAPK pathway and revealed that IL6R and CCNG2 are targets of EIF3B-regulated CEBPB translation. CONCLUSION: In summary, the results indicated that EIF3B is a novel oncogene in HNSCC that promotes CEBPB translation and IL6R expression, and these findings provide a link between the molecular basis and pathogenesis of HNSCC.

Correcting the F508del-CFTR variant by modulating eukaryotic translation initiation factor 3-mediated translation initiation.

Inherited and somatic rare diseases result from >200,000 genetic variants leading to loss- or gain-of-toxic function, often caused by protein misfolding. Many of these misfolded variants fail to properly interact with other proteins. Understanding the link between factors mediating the transcription, translation, and protein folding of these disease-associated variants remains a major challenge in cell biology. Herein, we utilized the cystic fibrosis transmembrane conductance regulator (CFTR) protein as a model and performed a proteomics-based high-throughput screen (HTS) to identify pathways and components affecting the folding and function of the most common cystic fibrosis-associated mutation, the F508del variant of CFTR. Using a shortest-path algorithm we developed, we mapped HTS hits to the CFTR interactome to provide functional context to the targets and identified the eukaryotic translation initiation factor 3a (eIF3a) as a central hub for the biogenesis of CFTR. Of note, siRNA-mediated silencing of eIF3a reduced the polysome-to-monosome ratio in F508del-expressing cells, which, in turn, decreased the translation of CFTR variants, leading to increased CFTR stability, trafficking, and function at the cell surface. This finding suggested that eIF3a is involved in mediating the impact of genetic variations in CFTR on the folding of this protein. We posit that the number of ribosomes on a CFTR mRNA transcript is inversely correlated with the stability of the translated polypeptide. Polysome-based translation challenges the capacity of the proteostasis environment to balance message fidelity with protein folding, leading to disease. We suggest that this deficit can be corrected through control of translation initiation.

Transcriptomic analyses of RNA-binding proteins reveal eIF3c promotes cell proliferation in hepatocellular carcinoma.

RNA-binding proteins (RBPs) play fundamental roles in the RNA life cycle. The aberrant expression of RBPs is often observed in human disease, including cancer. In this study, we screened for the expression levels of 1542 human RBPs in The Cancer Genome Atlas liver hepatocellular carcinoma samples and found 92 consistently upregulated RBP genes in HCC compared with normal samples. Additionally, we undertook a Kaplan-Meier analysis and found that high expression of 15 RBP genes was associated with poor prognosis in patients with HCC. Furthermore, we found that eIF3c promotes HCC cell proliferation in vitro as well as tumorigenicity in vivo. Gene Set Enrichment Analysis showed that high eIF3c expression is positively associated with KRAS, vascular endothelial growth factor, and Hedgehog signaling pathways, all of which are closely associated with specific cancer-related gene sets. Our study provides the basis for further investigation of the molecular mechanism by which eIF3c promotes the development and progression of HCC.

Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis.

We have found that eIF3a plays an important role in bleomycin-induced pulmonary fibrosis, and up-regulation of eIF3a induced by TGF-ß1 is mediated via the ERK1/2 pathway. Whether ERK1/2 - eIF3a signal pathway is involved in calcitonin gene-related peptide (CGRP)-mediated pathogenesis of bleomycin-induced pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. Sensory CGRP depletion by capsaicin exacerbated bleomycin-induced pulmonary fibrosis in rats, as shown by a significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, accompanied with increased expression of TGF-ß1, eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. Exogenous application of CGRP significantly inhibited TGF-ß1-induced proliferation and differentiation of pulmonary fibroblasts concomitantly with decreased expression of eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. These effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the ERK1/2 - eIF3a signaling pathway.

Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody.

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

Synthesis, pharmacophores, and mechanism study of pyridin-2(1H)-one derivatives as regulators of translation initiation factor 3A.

Twenty-seven 1,5-disubstituted-pyridin-2(1H)-one derivatives were synthesized and evaluated for their anti-cancer and anti-fibrosis activity by A549 and NIH3T3 cell viability assays, respectively. To study the selectivity between the cancer and fibrosis cell lines, pharmacophore models (F1-F4) were built in advance for compounds with pyridin-2(1H)-one scaffold, which revealed the relationship between the occupation of the aromatic sub-site F4 and potent anti-cancer activity. The relationship between structure and anti-cancer activity for all target compounds is also reported herein: 1-Phenyl-5-((m-tolylamino)methyl)pyridine-2(1H)-one (22) displayed both potency and selectivity (IC50=0.13 mM) toward the A549 cell line through the inhibition of translation initiation, especially by eIF3a suppression, and can be treated as a lead for the design of novel eIF3a regulators and anti-lung cancer agents.

Plasmodium falciparum Eukaryotic Translation Initiation Factor 3 is Stabilized by Quinazoline-Quinoline Bisubstrate Inhibitors.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum. This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

LAMR1 restricts Zika virus infection by attenuating the envelope protein ubiquitination.

Zika virus (ZIKV) infection can cause severe neurological disorders, including Guillain-Barre syndrome and meningoencephalitis in adults and microcephaly in fetuses. Here, we reveal that laminin receptor 1 (LAMR1) is a novel host resistance factor against ZIKV infection. Mechanistically, we found that LAMR1 binds to ZIKV envelope (E) protein via its intracellular region and attenuates E protein ubiquitination through recruiting the deubiquitinase eukaryotic translation initiation factor 3 subunit 5 (EIF3S5). We further found that the conserved G282 residue of E protein is essential for its interaction with LAMR1. Moreover, a G282A substitution abolished the binding of E protein to LAMR1 and inhibited LAMR1-mediated E protein deubiquitination. Together, our results indicated that LAMR1 represses ZIKV infection through binding to E protein and attenuating its ubiquitination.

EIF3B Associates with Exacerbated Clinical Features, Poor Treatment Response and Survival in Adult Philadelphia Chromosome Negative Acute Lymphoblastic Leukemia Patients.

Objective: This study was undertaken to investigate eukaryotic translation initiation factor 3 subunit B (EIF3B) expression and its clinical value for indicating disease progression and prognosis in adult Philadelphia chromosome negative acute lymphoblastic leukemia (Ph- ALL) patients. Methods: Totally, 76 adult Ph- ALL patients and 30 healthy donors (HDs) were included. Bone marrow (BM) samples before therapy (baseline), after 4-week therapy of Ph- ALL patients and the BM samples of HDs were collected. Then, EIF3B expression in BM was detected by reverse transcription quantitative polymerase chain reaction. Results: EIF3B expression was increased in Ph- ALL patients compared with HDs, which distinguished Ph- ALL patients from HDs (area under the curve [AUC]: 0.928; 95% confidence interval [CI]: 0.882-0.974) by receiver operating characteristic curve. Furthermore, higher baseline EIF3B expression was associated with elevated white blood cell and bone marrow blasts, while it was associated with lower complete remission (CR) within 4 weeks and less allogeneic hematopoietic stem cell transplant achievements in Ph- ALL patients. Additionally, higher baseline EIF3B expression was associated with decreased disease-free survival but not overall survival. However, it was associated with raised 1-year mortality and 3-year mortality in Ph- ALL patients. After 4-week therapy, EIF3B expression was reduced in total Ph- ALL patients. Notably, the reduction of EIF3B expression was more obvious in Ph- ALL patients who achieved CR within 4 weeks compared with Ph- ALL patients who did not achieve CR within 4 weeks. Conclusion: EIF3B overexpression is related to worsened clinical features, poor treatment response and survival in adult Ph- ALL patients.

Expression of eukaryotic translation initiation factor 3 subunit B in liver cancer and its prognostic significance.

Liver cancer is one of the major malignancies with the worst prognosis among all solid tumor types. It is therefore ponderable to explore prognostic biomarkers and therapeutic targets for liver cancer. Eukaryotic translation initiation factor 3 subunit B (EIF3B) is closely linked to the transcription initiation of cancer-associated genes. In the present study, EIF3B was indicated to be a potential prognostic biomarker of liver cancer. The mRNA expression level of EIF3B in liver cancer was assessed by analyzing the Cancer Genome Atlas dataset. χ2 and Fisher's exact tests were used to assess the association of EIF3B expression with clinical parameters. Receiver-operating characteristic curve analysis was used for evaluating the diagnostic value of EIF3B. Overall and relapse-free survival were assessed using Kaplan-Meier curves to determine the association between EIF3B expression and survival. Univariate and multivariate Cox regression analysis were performed to identify the factors affecting overall/relapse-free survival. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with EIF3B in liver cancer. It was revealed that EIF3B was highly expressed in liver cancer tissues and it had a promising diagnostic ability. Furthermore, the survival analysis indicated that patients with high EIF3B expression generally had shorter overall as well as relapse-free survival. Univariate and multivariate Cox analysis suggested that high EIF3B mRNA expression may serve as an independent biomarker for the prognostication of patients with liver cancer. GSEA suggested that MYC-V1 (HALLMARK_MYC_TARGETS_V1 geneset; P=0.009), MYC-V2 (HALLMARK_MYC_TARGETS_V2 geneset; P=0.004) and DNA repair pathways (HALLMARK_DNA_REPAIR geneset; P<0.001) were differentially enriched in high EIF3B expression and low EIF3B expression groups. In conclusion, high EIF3B expression was indicated to be an independent prognostic biomarker for patients with liver cancer.

MicroRNA­875­5p inhibits tumor growth and metastasis of hepatocellular carcinoma by targeting eukaryotic translation initiation factor 3 subunit a.

Accumulating evidence has demonstrated that aberrant microRNA (miRNA) expression is involved in hepatocellular carcinoma (HCC) progression. Previous findings suggested that miRNA (miR)­875­5p participates in the development of various types of cancer. However, the expression and function of miR­875­5p in HCC remains largely unclear. The analysis of clinical samples in the present study demonstrated that miR­875­5p expression was downregulated in HCC tissues compared to adjacent non­tumor tissues, which was associated with a large tumor size, venous infiltration, advanced tumor­node­metastasis stage and unfavorable overall survival. In vitro experiments revealed that ectopic expression of miR­875­5p suppressed, whereas inhibition of miR­875­5p promoted HCC cell proliferation, migration, invasion and epithelial­to­mesenchymal transition (EMT) progression. Overexpression of miR­875­5p restrained HCC tumor growth and metastasis in vivo. Mechanistically, eukaryotic translation initiation factor 3 subunit a (eIF3a) was identified as the downstream target of miR­875­5p in HCC. Further experiments demonstrated that the expression of eIF3a was upregulated and negatively correlated with that of miR­875­5p in HCC tissues. In addition, miR­875­5p negatively regulated the luciferase activity of wild­type, but not mutant 3'­untranslated region (3'UTR) of eIF3a mRNA. miR­875­5p suppressed eIF3a expression at the mRNA and protein level in HCC cells. Additionally, eIF3a exerted an oncogenic role, and knockdown of eIF3a inhibited the proliferation, motility and EMT of HCC cells. In addition, eIF3a overexpression abolished the inhibitory effects of miR­875­5p on the proliferation, motility and EMT in HCC cells. In conclusion, miR­875­5p, which was downregulated in HCC, may inhibit tumor growth and metastasis by eIF3a downregulation via targeting its 3'UTR and may be a promising prognostic and therapeutic strategy in HCC.

Eukaryotic translation initiation factor 3B is overexpressed and correlates with deteriorated tumor features and unfavorable survival profiles in cervical cancer patients.

OBJECTIVE: This study aimed to detect the expression of eukaryotic translation initiation factor 3B (EIF3B) and investigate its correlation with tumor features and survival in cervical cancer patients. METHODS: This study retrospectively reviewed 187 cervical cancer (squamous cell carcinoma) patients underwent tumor resection. Immunohistochemistry was performed to determine the expression of EIF3B in tissue samples. Besides, disease free survival (DFS) and overall survival (OS) were calculated. The median follow-up duration was 69 months, and the last follow-up date was 2017/12/31. RESULTS: EIF3B expression was higher in tumor tissue compared to paired adjacent tissue (45.5% vs. 32.1%, P= 0.015). Besides, EIF3B high expression was associated with higher Federation of Gynecology and Obstetrics (FIGO) Stage (P= 0.001) and presence of lymph node metastasis (P= 0.002). As to survival profiles, Kaplan-Meier curves disclosed that DFS (P< 0.001) and OS (P< 0.001) were both shorter in EIF3B high expression group compared to EIF3B low expression group. Multivariate Cox's regression analysis disclosed that EIF3B high expression, pathological grade III (vs I/II) and FIGO Stage III/IV (vs I/II) were independent predictive factors for unfavorable DFS as well as OS in cervical cancer patients (all P value < 0.05). CONCLUSION: EIF3B is overexpressed, and its high expression correlates with higher FIGO Stage, lymph node metastasis and unfavorable survival profiles in cervical cancer patients.

The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Eukaryotic translation initiation factor 3B downregulation inhibits cell proliferation and promotes cell apoptosis through negatively regulating tumor necrosis factor receptor superfamily member 21 in gastric cancer.

BACKGROUND: This study aimed to detect eukaryotic translation initiation factor 3B (EIF3B) expression in gastric cancer (GC) cell lines, and further explore the effect of EIF3B downregulation on GC cell proliferation and apoptosis. METHODS: EIF3B mRNA expression and protein expression in human GC cell lines (NCI-N87, AGS, HGC-27, BGC-823 and MGC80-3) and human gastric mucosal epithelial cell line (GES-1) were detected. Control siRNA (Si-NC group) and EIF3B siRNA (Si-EIF3B group) were transfected into NCI-N87 cells. Rescue experiment was performed by transfection of EIF3B siRNA (Si-EIF3B group) and EIF3B siRNA plus tumor necrosis factor receptor superfamily member 21 (TNFRSF21) siRNA (Si-EIF3B & Si-TNFRSF21 group) into NCI-N87 cells. Besides, cell proliferation, apoptosis, TNFRSF21 expression and TRAF1 expression were assessed. RESULTS: EIF3B mRNA expression and protein expression were elevated in NCI-N87, AGS, HGC-27 and BGC-823 cell lines compared to GES-1 cell line. In NCI-N87 cells, proliferation was reduced in Si-EIF3B group compared to Si-NC group. For cell apoptosis, its rate and apoptotic marker C-Caspase 3 expression were increased but anti-apoptosis marker Bcl-2 expression was reduced in Si-EIF3B group compared to Si-NC group. Moreover, mRNA expression and protein expression of TNFRSF21 were increased in Si-EIF3B group compared to Si-NC group, while mRNA expression and protein expression of TRAF1 were reduced in Si-EIF3B group compared to Si-NC group. In rescue experiment, cell proliferation was increased but apoptosis was decreased in Si-EIF3B & Si-TNFRSF21 group compared to Si-EIF3B group. CONCLUSIONS: EIF3B is overexpressed in GC cell lines, and its downregulation inhibits cell proliferation while promotes apoptosis through negatively regulating TNFRSF21 in GC.

The function and clinical significance of eIF3 in cancer.

Abnormal regulation of gene expression is essential for tumorigenesis. Several studies indicate that regulation of oncogene expression and neoplastic transformation are controlled by subunits of eukaryotic translation initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest (800 kDa) and the most complex mammalian initiation factor. It is composed of 13 non-identical polypeptides designated as eIF3a-m and plays a pivotal role in protein synthesis that bridges the 43S pre-initiation complex and eIF4F-bound mRNA. However, the functional roles of individual subunits are not yet very clear. This review presents on several of aberrant expressed eIF3 subunits which are detected in various human cancers and the associated mechanisms have been acknowledged or are still not sure. Finally, identifying novel targets and biomarkers for caner is of great importance in early diagnosis and treatment of cancer. eIF3 may be a novel target molecule in drug development for cancer treatment and prevention.

Knockdown of elF3a inhibits TGF­ß1­induced extracellular matrix protein expression in keloid fibroblasts.

Keloid formation is characterized by hyperproliferation of secretory and responsive keloid fibroblasts (KFs) and overproduction of extracellular matrix (ECM). Eukaryotic translation initiation factor 3 subunit A (eIF3a) one of the core subunits of the translation initiation complex, eIF3, has previously been reported to possess an anti­fibrogenic effect. However, the role of eIF3a in keloid formation has not yet been investigated. Therefore, the present study examined the effect of eIF3a on transforming growth factor­ß1 (TGF­ß1)­mediated ECM expression in KFs. The expression levels of eIF3a in human keloid tissues was evaluated using reverse transcription­quantitative polymerase chain reaction and western blotting. KFs were incubated with siRNA­eIF3a or siRNA­mock for 48 h. The cells were then treated with TGF­ß1 (10 ng/ml) for 72 h. Cell proliferation was evaluated using the CCK­8 assay. The expression levels of α­SMA, collagen type I, TGF­ß receptor I (RI), TGF­ß RII, phosphorylated (p)­mothers against decapentaplegic homolog (Smad2), Smad2, p­Smad3 and Smad3 were detected western blotting. The present study identified significant upregulation of eIF3a mRNA and protein and in human keloid tissues compared with in normal tissues. Knockdown of eIF3a inhibited KF proliferation induced by TGF­ß1. In addition, eIF3a silencing significantly suppressed the TGF­ß1­induced expression of α­smooth muscle actin, collagen I, TGF­ß RI and TGF­ß RII in KFs. Furthermore, eIF3a silencing inhibited the phosphorylation levels of Smad2 and Smad3 in TGF­ß1­induced KFs. To the best of our knowledge, the current study is the first to demonstrate that siRNA­eIF3a inhibits the expression ECM proteins via the TGF­ß1/Smad signaling pathway in KFs. Therefore, eIF3a may be a potential, novel target for treatment of keloids.

Eukaryotic translation initiation factor 3 (eIF3) subunit e is essential for embryonic development and cell proliferation.

Mammalian eukaryotic translation initiation factor 3 (eIF3) is the largest complex of the translation initiation factors. The eIF3 complex is comprised of thirteen subunits, which are named eIF3a to eIF3 m in most multicellular organisms. The eIF3e gene locus is one of the most frequent integration sites of mouse mammary tumor virus (MMTV), which induces mammary tumors in mice. MMTV-integration events result in the expression of C-terminal-truncated eIF3e proteins, leading to mammary tumor formation. We have shown that tumor formation can be partly caused by activation of hypoxia-inducible factor 2α. To investigate the function of eIF3e in mammals, we generated eIF3e-deficient mice. These eIF3e-/- mice are embryonically lethal, while eIF3e+/- mice are much smaller than wild-type mice. In addition, eIF3e+/- mouse embryonic fibroblasts (MEFs) contained reduced levels of eIF3a and eIF3c subunits and exhibited reduced cellular proliferation. These results suggest that eIF3e is essential for embryonic development in mice and plays a role in maintaining eIF3 integrity.

Eukaryotic translation initiation factor 3 subunit C is associated with acquired resistance to erlotinib in non-small cell lung cancer.

The acquisition of resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) is one of the major problems in the pharmacotherapy against non-small cell lung cancers; however, molecular mechanisms remain to be fully elucidated. Here, using a newly-established erlotinib-resistant cell line, PC9/ER, from PC9 lung cancer cells, we demonstrated that the expression of translation-related molecules, including eukaryotic translation initiation factor 3 subunit C (eIF3c), was upregulated in PC9/ER cells by proteome analyses. Immunoblot analyses confirmed that eIF3c protein increased in PC9/ER cells, compared with PC9 cells. Importantly, the knockdown of eIF3c with its siRNAs enhanced the drug sensitivity in PC9/ER cells. Mechanistically, we found that LC3B-II was upregulated in PC9/ER cells, while downregulated by the knockdown of eIF3c. Consistently, the overexpression of eIF3c increased the number of autophagosomes, proposing the causality between eIF3c expression and autophagy. Moreover, chloroquine, an autophagy inhibitor, restored the sensitivity to erlotinib. Finally, immunohistochemical analyses of biopsy samples showed that the frequency of eIF3c-positive cases was higher in the patients with EGFR-TKI resistance than those prior to EGFR-TKI treatment. Moreover, the eIF3c-positive cases exhibited poor prognosis in EGFR-TKI treatment. Collectively, the upregulation of eIF3c could impair the sensitivity to EGFR-TKI as a novel mechanism of the drug resistance.

Fluorofenidone attenuates bleomycin-induced pulmonary fibrosis by inhibiting eukaryotic translation initiation factor 3a (eIF3a) in rats.

Fluorofenidone is a novel derivative of l-mimosine. It has remarkable anti-fibrotic properties. In this study, we established that fluorofenidone ameliorates pulmonary fibrosis (PF) both in vivo and in vitro by specifically inhibiting the expression of eukaryotic translation initiation factor 3a (eIF3a). eIF3a plays an important role in the development and progression of PF. An animal model of PF was induced by intratracheal instillation of bleomycin (5mg/kg) in rats. Rats were orally administered with fluorofenidone (250, 500 mg/kg/d·[i.g.]) and pirfenidone (500 mg/kg/d·[i.g.]) for 28 days. Primary pulmonary fibroblasts were cultured to determine the effect of fluorofenidone on TGF-ß1-induced (5 ng/ml) proliferation and differentiation of fibroblasts. The expression/level of eIF3a, TGF-ß1, α-SMA, collagen I, and collagen III were analyzed by ELISA, real-time PCR, and western blot. The cell proliferation rate was determined by MTS assay. The results indicate that fluorofenidone significantly improves the pathological changes in lung tissues and reduces the deposition of collagen by inhibiting eIF3a in rats with bleomycin-induced PF. Moreover, in a culture of pulmonary fibroblasts, fluorofenidone decreased the up-regulation of TGF-ß1-induced eIF3a by inhibiting the proliferation of cells and reducing the expression of α-SMA, collagen I, and collagen III. These findings suggest that eIF3a is a new and special target of fluorofenidone, which could be potentially used in the development of a drug that treats PF.