5'-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I / 医学前沿

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNAGln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.

The proliferation promoting effect of eIF2α on fibroblasts of rheumatoid arthritis synovium / 中华风湿病学杂志

Objective:To observe the expression characteristics of eukaryotic translation initiation factor 2α(eIF2α), and analyze its proliferation regulation effect on fibroblasts of rheumatoid arthritis synovium.Methods:The synovial tissues were collected in patients with rheumatoid arthritis(RA)(40 cases) and osteoarthritis(OA)(40 cases). EIF2α and proliferating cell nuclear antigen(PCNA) were detected by immunohistochemistry method. Fibroblast cell line of rheumatoid arthritis synovium(MH7A) were cultured to establish si-eIF2α group(siRNA-eIF2α plasmid transfection), vector transfection group and blank control group in vitro. PCNA was detected by Western blot method, cell proliferation activity was detected by CCK-8 method. χ2 test was performed on count data, two-sample t-test was performed on quantitative data, one-way analysis of variance (ANOVA) was performed to compare the means of more than two groups, regression equation was calculated by correlation regression analysis. Results:The positive rate of eIF2α was significantly higher in RA synovial fibroblasts than that of OA [52.5%(21/4) vs 20.00%(8/20), χ2=9.14, P=0.003]. Positive correlation was found between eIF2α and PCNA in RA ( Y=0.366 X+2.220, P=0.001) . Compared with blank control group and vector transfection group, cell proliferation activity decreased significantly in si-eIF2α group of MH7A cell line at 72 h [(0.65±0.08) vs (0.96±0.12) vs (1.09±0.06), F=4.52, P=0.022] and 96 h [(1.13±0.14) vs (1.42±0.97) vs (1.56±0.12), F=9.87, P=0.001) , PCNA expression decreased significantly [(0.84±0.15) vs (1.32±0.18) vs (1.28±0.14), F=5.22, P=0.012) . Conclusion:High expression of eIF2α can promote the proliferation of fibroblasts of RA synovium.

Triple-negative breast cancer cells respond to T cells severely at the alternative splicing layer

BACKGROUND: Cross talk of tumor­immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor­immune microenvironment. RESULTS: Here, we re-exploited one transcriptomic dataset to gain insight into tumor­immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor­AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. CONCLUSIONS: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway / 中国结合医学杂志

OBJECTIVE@#To test the hypothesis that the inhibition of endoplasmic reticulum (ER) stress-induced apoptosis in oxidized low-density lipoproteins (ox-LDL)-induced human aortic-vascular smooth muscle cells (HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway by Pollen Typhae total flavone (PTF).@*METHODS@#Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an HPTF group (70 μg/mL high ox-LDL+500 μg/mL PTF), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), and a LPTF group (70 μg/mL high ox-LDL+100 μg/mL PTF) in the first part; and a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), a shRNA group (transducted with PERK shRNA lentiviral particles), a scramble shRNA group (transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group (250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group (70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability, and the level of apoptosis was monitored by flow cytometry.@*RESULTS@#The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and shRNA groups. Moreover, the ox-LDL group had increased protein and mRNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, eIF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and shRNA groups.@*CONCLUSIONS@#The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

Aberration in translation initiation and associated diseases:Role of the eukaryotic translation initiation factor 3A / 中南大学学报(医学版)

Translation control in eukaryotes contributes significantly to gene expression regulation during cellular processes,which enables rapid changes of specific proteins to maintain cellular homeostasis.Eukaryotic translation is a multiple-step process that comprised of four phases:initiation,elongation,termination and ribosome recycling.The initiation phase is rate-limiting and orchestrated by a set of eukaryotic translation initiation factors (eIFs).Defects in translation initiation can result in a series of diseases.Among all eIFs,eIF3 is the largest and less-known initiation factor due to its intrinsic complexity.Aberration in eIF3A,the largest subunit of eIF3,is known to contribute to carcinogenesis and protection against evolution into higher-grade malignancy,and the altered expression or mutation of eIF3A affects the responses of cancer patients to platinum-based chemotherapy.Besides its role in cancinogenesis,eIF3A is also implicated in fibrosis,and the agents inhibiting eIF3A delay the progression of this disorder.The dual roles of eIF3A in tumorigenesis are probably due to the regulation of translation of different mRNAs at different stages of tumor progression by eIF3A.In tum the encoded products serve as pro-tumor or anti-tumor proteins at different stages.

HIF-1α pathway: role, regulation and intervention for cancer therapy

Hypoxia-inducible factor-1 (HIF-1) has been recognized as an important cancer drug target. Many recent studies have provided convincing evidences of strong correlation between elevated levels of HIF-1 and tumor metastasis, angiogenesis, poor patient prognosis as well as tumor resistance therapy. It was found that hypoxia (low O2 levels) is a common character in many types of solid tumors. As an adaptive response to hypoxic stress, hypoxic tumor cells activate several survival pathways to carry out their essential biological processes in different ways compared with normal cells. Recent advances in cancer biology at the cellular and molecular levels highlighted the HIF-1α pathway as a crucial survival pathway for which novel strategies of cancer therapy could be developed. However, targeting the HIF-1α pathway has been a challenging but promising progresses have been made in the past twenty years. This review summarizes the role and regulation of the HIF-1α in cancer, and recent therapeutic approaches targeting this important pathway.

Expression of phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated eukaryotic translation initiation factor 4E binding protein 1 in pathologic scar / 中华医学美学美容杂志

Objective To investigate the expression of phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1) in pathologic scar,and comprehend its role and significance in the pathologic scar formation.Methods SP immunohistochemical method was used to detect the expression of p-mTOR and p-4EBP1 in 20 cases of keloid,20 cases of hypertrophic scar,20 cases of non-pathologic scar and 20 cases of normal skin tissue.The positive rates of expression in different tissues were analyzed,and the relationship in pathological scar was explored.Results The positive rates of p-mTOR and p-4EBP1 in keloidand hypertrophic scar were 75.0％ (15/20),60.0％ (12/20) and 60.0％ (12/20),50.0％ (10/20),nom-pathologic scars were 20％ (4/20),10％ (2/20);normal skin tissue were 10％ (2/20),5％ (1/20),which were higher respectively compared with the two control groups (P＜ 0.05);there was a highly positive correlation between p-mTOR and p-4EBP1 expression in pathologic scar (r=0.323,P＜0.05).Conclusions p-mTOR and p-4EBP1 might be involved and cooperated in promoting the progress of pathologic scars.

The effects of EIF4E gene down-regulation induced by siRNA on the proliferation and cell cycle of human breast cancer cell line MDA-MB-231 / 肿瘤

Objective: To study the effect of down-regulation of eukaryotic translation initiation factor 4E (EIF 4E ) induced by the siRNA expression plasmid on the proliferation and cell cycle of human breast cancer cell line MDA-MB-231. Methods: The siRNA expression plasmid pGPU6/GFP/Neo-EIF4E was constructed and then transfected into breast cancer MDA-MB-231 cells by LipofectAMINE2000. The expressions of EIF4E and cyclin D1 mRNAs and proteins were examined by RT-PCR, Western blotting and the immunocytochemistry method, respectively. The MTT method, plate colony formation assay and flow cytometry were performed to detect the proliferation ability, cell colony formation rate and cell cycle distribution of MDA-MB-231 cells transfected with siRNA expression plasmid. Results: The siRNA expression plasmid pGPU6/GFP/Neo-EIF4E was constructed successfully. The expressions of EIF4E and cyclin D1 mRNAs and proteins were all decreased markedly after transfection with pGPU6/GFP/Neo-EIF4E (P <0.05). The growth of MDA-MB-231 cells in the pGPU6/GFP/Neo-EIF4E-tranfected group was inhibited and the cell colony formation rate was statistically decreased (P<0.05) as compared with those in the blank control and the pGPU6/GFP-transfected groups. The result of flow cytometry showed that the percentage of cells in G1 phase in the pGPU6/GFP/Neo-EIF4E-tranfected group was higher than that in the blank control group (71.30%±0.47% vs 53.10%±0.43%); however, the percentage of cells in S phase was signifcantly lower (12.53%±0.13% vs 26.47%±0.38%, P <0.05). Conclusion: EIF 4E siRNA can downregulate EIF 4E gene expression in human breast cancer MDA-MB-231 cells, suppress cell proliferation to some extent, and increase the number of cells in G1 phase with a decreased number of cells in S phase. EIF 4E gene may become one of the important molecular targets to breast cancer. Copyright© 2011 by the Editorial Board of Tumor.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication / 中国病毒学·英文版

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV)usually result in altered hepatitis B surface antigen(HBsAg)secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs)synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

New Solutions of Translation Initiation Site Prediction for Prokaryotic Genomes / 生物化学与生物物理进展

Accurate prediction of the translation initiation site (TIS) is an important issue for prokaryotic genome annotation. However, it is still a challenge for the existing methods to predict the TIS in the genomes over a wide variety of GC content. Besides, the existing methods have not yet undergone a comprehensive evaluation, leaving prediction reliability as a largely open problem. A new algorithm MED-StartPlus, a tool that predicts TIS in prokaryotic genomes with a wide variety of GC content was presented. It makes several efforts to model the nucleotide composition bias, the regulatory motifs upstream of the TIS, the sequence patterns around the TIS, and the operon structure. Tests on hundreds of reliable data sets, with TISs confirmed by experiments or having annotated functions, show that the new method achieves a totally high accuracy of TIS prediction. Compared with existing TIS predictors, the method reports a totally higher performance, especially for genomes that are GC-rich or have complex initiation mechanisms. The potential application of the method to improve the TIS annotation deposited in the public database was also proposed.

The effect of hnRNP K shRNA on K562 cells / 重庆医科大学学报

Objective:To explore the effect of heterogeneous nuclear ribonucleoprotein K(hnRNP K)short hairpin RNA(shRNA)on K562 cells. Methods:hnRNP K shRNA retrovirus was packaged with pSUPER retro hnRNP K shRNA after its identification by enzyme digestion and sequencing analysis,then FCM,Wright staining,MTT assay,RT-PCR were used to analyze its effects on cell cycle and apoptosis,proliferation,hnRNP K and eukaryotic translation initiation factor-4E(eIF4E) mRNA of K562 cells respectively. Results:The sequence of the inserted fragment in pSUPER retro hnRNP K shRNA was correct. Compared with the control K562 cells,hnRNP K shRNA treated group showed significantly more cells at G2/M phase(P

Relationship between Eukaryotic Translation Initiation Factor 4E and Malignant Angiogenesis in Non-Hodgkin Lymphoma / 华中科技大学学报（医学）（英德文版）

The relationship between angiogenesis and eukaryotic translation initiation factor 4E (EIF4E) expression level in non-Hodgkin lymphoma (NHL) was studied. Mean microvessel density (MVD) and EIF4E were detected in 52 lymph node samples paraffin sections of patients with newly diagnosed NHL by the way of immunohistochemistry. Antisense EIF4E cDNA was cloned into plasmid pcDNA3.1 (+) and transfected into Raji cells. A series of angiogenesis related factors,including vascular endothelial growth factor (VEGF), matrix metalloproteinases 9 (MMP-9)and tissue inhibitor of metalloproteinases-2 (TIMP-2) proteins were detected by Western blot. The results showed that: (1) The Expression of EIF4E and MVD was higher in aggressive lymphomas than in indolent lymphomas(P＜0.05)and the expression of EIF4E was positively correlated with MVD in lymph node of NHL(r=0. 695, P＜0.01). (2) Antisense EIF4E eukaryocytic expression vector (pcDNA3.1-EIF4Eas) was constructed successfully. (3) EIF4E, VEGF and MMP-9 were expressed at high levels in Raji cells as compared to normal human peripheral blood monocular cells ( NHPMC), and blockage of EIF4E expression brought down the expression of VEGF and MMP-9.However, TIMP-2 was undetectable in Raji cells, although a moderate level of TIMP-2 was detected in NHPMC. It was concluded that the increased EIF4E expression was associated with aggressive property of NHL.

Translational Regulation: A Novel Target for Breast Cancer Therapy / 한국유방암학회지

Translational initiation is regulated in response to nutrient availabilty and growth stimuli and is coupled with cell cycle progression and cell growth. There is now growing body of evidence which suggests links between translational regulation and the disruption of cell behavior that results in the development and progression of cancer. mRNA translation can be overactivated in breast cancer through eIF4E overexpression or abnormal activation of signal transduction pathways. Among them, rapamycin-sensitive signal transduction pathway (mTOR signaling pathway) is now being studied as a novel target for cancer therapy. In this article, the basic principles of translational control, the alterations encountered in cancer and selected therapy targeting mTOR signaling pathway are reviewed and the preclinical study regarding the determinants of rapamycin sensitivity in breast cancer is presented in order to help elucidate new avenues for breast cancer therapy.

Effect of C2 gene on tumorigenesis of human gastric cancer cells / 中华消化杂志

Objective To investigate the effect of a new cloned full length gene, C2 gene, which encodes human eukaryotic translation initiation factor, on tumorigenesis of human gastric cancer cells in vivo and in vitro. Methods A constructed eukaryotic vector carrying the full length of C2 gene was amplified, purified, and transfected into a gastric cell line SGC7901 cell. The expression of C2 protein was examined with fluorescence activated cell sorting (FACS) and Western Blot. The effect of C2 gene on tumorigenesis of human gastric cancer cells was investigated in vitro and in vivo with methods of clone formation in plate and oncogenesis in nude mouse. Results FACS and Western Blot showed that C2 protein was expressed highly and stably in transfected cells. The average clone formation rate of C2 gene transducted cells is 43.8%, the rate which is lower than that of vector transducted cells (76.9%). In vivo, the time of tumorigenesis of C2 gene transducted cells in nude mouse is prolonged from 1 week to 2 weeks, and the volume of tumor node is smaller than that of vector transducted cells, that is, 1.20 and 5.58 cm 3 respectively ( P

Construction of recombinant interleukin-6 plasmid pBV-IL-6 and its overexpression in E.coli / 中国免疫学杂志

In this paper,by means of PCR amplification,site-specific mutagenesis,DNA recombination in vitro and optimization of translation initiation,we constructed a recombinant plasmid pBV-IL-6 that overproduces biologically active human interleukin-6(rhIL-6)in E.coli.The expressed rhIL-6 lacks the first 25 amino acids of mature hIL-6 and accounts for 71% of the total bacteria proteins.The reasons why a deleted rather than mature IL-6 was chosen for expression in E.coli are as follows:1)The specific bio-activities of the deleted hIL-6 are the same as,even more than that of the mature form according to the published data;2)The deleted part of IL-6 coding region is rich in C-G andeasy to form secondary structure in mRNA,which is not good for expression;3)The re-designed region for initiation site of translation encodes a hydrophilic amino-end which favours highly translation initiation and expression of hIL-6 in E.coli.