Eukaryotic initiation factor 5A2 and human digestive system neoplasms

Eukaryotic initiation factor 5A2(eIF5A2),as one of the two isoforms in the family,is reported to be a novel oncogenic protein that is involved in multiple aspects of many types of human cancer.Overexpression or gene amplification of EIF5A2 has been demonstrated in many cancers.Accumulated evidence shows that eIF5A2 initiates tumor formation,enhances cancer cell growth,increases cancer cell metastasis,and promotes treatment resistance through multiple means,including inducing epithelial–mesenchymal transition,cytoskeletal rearrangement,angiogenesis,and metabolic reprogramming.Expression of eIF5A2 in cancer correlates with poor survival,advanced disease stage,as well as metastasis,suggesting that eIF5A2 function is crucial for tumor development and maintenance but not for normal tissue homeostasis.All these studies suggest that eIF5A2 is a useful biomarker in the prediction of cancer prognosis and serves as an anticancer molecular target.This review focuses on the expression,subcellular localization,post-translational modifications,and regulatory networks of eIF5A2,as well as its biochemical functions and evolving clinical applications in cancer,especially in human digestive system neoplasms.

Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein

AIM: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67).METHODS: Immunofluorescence staining,35S-Met/Cys metabolic labeling,Western blotting analysis,sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells.Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR).Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins.In vivo 32P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation,eIF2α phosphorylation,and p67 expression in normal and breast cancer cells.RESULTS: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus.In a sucrose gradient,approxi-mately 30% of the overexpressed p67 was bound with ribosomes.p67 interacted with the kinase domain,butnot the dsRNA-binding domains of PKR.Only the glycosylated p67 was associated with the ribosome,and p67 did not compete with PKR for ribosome binding.In breast cancer cells,there was increased autophosphorylation of PKR but no phosphorylation of eIF2α,compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells.CONCLUSION: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.

Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Detection of eukaryotic translation initiation factor 4E and its clinical significance in hepatocellular carcinoma

AIM:To study the expression of eukaryotic translation initiation factor 4E(eIF4E),which is closely correlated with malignant tumors,and its relationship to prognosis in hepatocellular carcinoma. METHODS:Western blotting was performed to quantify the elF4E protein expression in the normal human liver cell line L02 and the hepatoma cell lines Hep3B, HepG2,and Huh7.Forty-six hepatocellular carcinoma samples with complete clinical data were obtained from Changzheng Hospital during the period of December 2008 to July 2009.The expression of eIF4E in the tumor samples and their adjacent tissues were detected by immunohistochemistry.The relationship between the test results and hepatocellular carcinoma(HCC) prognosis was statistically analysed by using a COX proportional hazard model. RESULTS:Western blotting analysis showed that there were distinct eIF4E protein bands in all three of the hepatoma cell lines.In particular,the HepG2 cell line had the highest level of eIF4E protein expression.The L02 cell group had a low eIF4E expression.Immunohistochemical assay showed that there were 32 cases in which the tumour tissue expression was higher than their adjacent tissues,accounting for 69.57%.There were also 14 cases in which the tumour tissue expression was lower or no significant difference was found, accounting for 30.43%.COX proportional hazards model analysis showed that HCC prognosis was related to the depth of invasion,the overexpression of eIF4E and p53, possibly as independent HCC prognostic predictors. CONCLUSION:In summary,eIF4E expression is associated with liver cancer,and patients with high eIF4E expression levels have a higher risk of recurrence.

Relationship between Eukaryotic Translation Initiation Factor 4E and Malignant Angiogenesis in Non-Hodgkin Lymphoma

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 Rail 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.

Eukaryotic Translation Initiation Factors Shape RNA Viruses Resistance in Plants

Viruses are representative of a global threat to agricultural production. Genetic resistance is the preferred strategy for the control of viral infection and against loss of crop yield. Viral protein synthesis requires host cellular factors for translating their viral RNAs, and for regulating their replication and cell to cell systemic movement. Therefore, the viruses are dependent on cellular translation factors. Mutations in the gene encoding eIF4E and eIF4G or their isoforms, eIFiso4 E, eIFiso4 G and eIF2Bβ have been mapped as a source of plant potyvirus while other genus of plant virus recessive resistance genes in many species are originated from these loci. Some of other plant translation factors, such as eIF3,eIF4 A-like helicases, eEF1A and eEF1B, which are required in interacting with viral RNAs and regulating various aspects of the infection cycle,have also been identified. Here, we summarized the mechanisms utilized by RNA viruses of eukaryotic plants and the essential roles of e IFs in virus infection. Moreover, we discussed the potential of e IFs as a target gene in the development of genetic resistance to viruses for crop improvement. This review highlighted newly revealed examples of abnormal translational strategies and provided insights into natural host resistance mechanisms that have been linked to 3 cap-independent translational enhancer activity.

Cloning and Characterization of Eukaryotic Translation Initiation Factor 4E (eIF4E) Gene Family in Ipomoea batatas L. (Lam) for Understanding Hexaploid Sweetpotato-Virus Interactions

Characterization of genes related to sweetpotato viral disease resistance is critical for understanding plant-pathogen interactions, especially with feathery mottle virus infection. For example, genes encoding eukaryotic translation initiation factor (eIF)4E, its isoforms, eIF(iso)4E, and the cap-binding protein (CBP) in plants, have been implicated in viral infections aside from their importance in protein synthesis. Full-length cDNA encoding these putative eIF targets from susceptible/resistant and unknown hexaploid sweetpotato (Ipomoea batatas L. Lam) were amplified based on primers designed from the diploid wild-type relative Ipomoea trifida consensus sequences, and designated IbeIF4E, IbeIF(iso)4E and IbCBP. Comparative analyses following direct-sequencing of PCR-amplified cDNAs versus the cloned cDNA sequences identified multiple homeoalleles: one to four IbeIF4E, two to three IbeIF(iso)4E, and two IbCBP within all cultivars tested. Open reading frames were in the length of 696 bp IbeIF4E, 606 bp IbeIF(iso)4E, and 675 bp IbCBP. The encoded single polypeptide lengths were 232, 202, and 225 amino acids for IbeIF4E, IbeIF(iso)4E, and IbCBP, with a calculated protein molecular mass of 26 kDa, 22.8 kDa, and 25.8 kDa, while their theoretical isoelectric points were 5.1, 5.57, and 6.6, respectively. Although the homeoalleles had similar sequence lengths, single nucleotide polymorphisms and multi-allelic variations were detected within the coding sequences. The multi-sequence alignment performed revealed a 66.9% - 96.7% sequence similarity between the predicted amino acid sequences obtained from the homeoalleles and closely related species. Furthermore, phylogenetic analysis revealed ancestral relationships between the eIF4E homeoalleles and other species. The outcome herein on the eIF4E superfamily and its correlation in sequence variations suggest opportunities to decipher the role of eIF4E in hexaploid sweetpotato feathery mottle virus infection.

Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2 (eEF1A2) on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms.METHODS: eEF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, HepG2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included eEF1A2 silencing in BEL-7402 cells with lentivirus eEF1A2-shRNA (KD group) and eEF1A2 overexpression in SK-HEP-1 cells with eEF1A2 plasmid (OE group). Non-transfected cells (control group) and lentivirus-based empty vector transfected cells (NC group) were considered control groups. Cell proliferation (MTT and colony formation assays), apoptosis (Annexin V-APC assay), cell cycle (DNA ploidy assay), and migration and invasion (Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-κB signaling effectors were evaluated by Western blot.RESULTS: eEF1A2 mRNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower eEF1A2 mRNA levels; HepG2 and BEL-7402 cells showed higher eEF1A2 mRNA levels, with BEL-7402 cells displaying the highest amount. Efficient eEF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-κB signaling pathway was notably inhibited. Inversely, eEF1A2 overexpression resulted in promoted cell proliferation, migration and invasion.CONCLUSION: eEF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-κB signaling.

瘢痕疙瘩miR-557和eIF2a的表达及靶向关系研究

目的检测瘢痕疙瘩中微小RNA-557(miR-557)和真核细胞翻译起始因子2a(eIF2a)在病变组织和细胞株中的表达特征,明确两者的靶向关系。方法选择首都医科大学附属北京朝阳医院诊治患者术后的瘢痕疙瘩(n=98)、增生性瘢痕组织(n=49)和正常皮肤组织(n=49)。应用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-557的表达,应用免疫组化检测eIF2a和Ki-67的表达。分离培养瘢痕疙瘩组织中的成纤维细胞,应用双荧光素酶报告基因实验观察miR-57与eIF2a的靶向关系。结果瘢痕疙瘩中miR-557的表达量明显低于增生性瘢痕组织和正常皮肤组织(P<0.05),瘢痕疙瘩中eIF2a表达的阳性率明显高于增生性瘢痕组织和正常皮肤组织(P<0.05),miR-557和eIF2a在不同病变最大径、不同增殖指数和有无伴随症状中的比较差异有统计学意义(P<0.05)。瘢痕疙瘩中miR-557与eIF2a呈负相关性(r=-0.69,P=0.016)。双荧光素酶报告基因实验显示miR-557与eIF2a具有靶向关系。结论miR-557在瘢痕疙瘩中的表达下降,eIF2a的表达升高,均与临床及组织病理特征有关,miR-557与eIF2a具有靶向负调控关系。

Type 2 Possibility Factor Rotation in No-Data Problem

Uemura [1] discovered a mapping formula that transforms and maps the state of nature into fuzzy events with a membership function that expresses the degree of attribution. In decision theory in no-data problems, sequential Bayesian inference is an example of this mapping formula, and Hori et al. [2] made the mapping formula multidimensional, introduced the concept of time, to Markov (decision) processes in fuzzy events under ergodic conditions, and derived stochastic differential equations in fuzzy events, although in reverse. In this paper, we focus on type 2 fuzzy. First, assuming that Type 2 Fuzzy Events are transformed and mapped onto the state of nature by a quadratic mapping formula that simultaneously considers longitudinal and transverse ambiguity, the joint stochastic differential equation representing these two ambiguities can be applied to possibility principal factor analysis if the weights of the equations are orthogonal. This indicates that the type 2 fuzzy is a two-dimensional possibility multivariate error model with longitudinal and transverse directions. Also, when the weights are oblique, it is a general possibility oblique factor analysis. Therefore, an example of type 2 fuzzy system theory is the possibility factor analysis. Furthermore, we show the initial and stopping condition on possibility factor rotation, on the base of possibility theory.

Initial and Stopping Condition in Possibility Principal Factor Rotation

Uemura [1] discovered the mapping formula for Type 1 Vague events and presented an alternative problem as an example of its application. Since it is well known that the alternative problem leads to sequential Bayesian inference, the flow of subsequent research was to make the mapping formula multidimensional, to introduce the concept of time, and to derive a Markov (decision) process. Furthermore, we formulated stochastic differential equations to derive them [2]. This paper refers to type 2 vague events based on a second-order mapping equation. This quadratic mapping formula gives a certain rotation named as possibility principal factor rotation by transforming a non-mapping function by a relation between two mapping functions. In addition, the derivation of the Type 2 Complex Markov process and the initial and stopping conditions in this rotation are mentioned. .

circEIF6调节miR-129-5p/TRAF6轴对胰腺癌细胞增殖、凋亡和吉西他滨敏感性的影响

目的:探讨环状RNA真核起始因子6(circEIF6)调节miR-129-5p/肿瘤坏死因子受体相关因子6(TRAF6)轴对胰腺癌细胞增殖、凋亡和吉西他滨(GEM)敏感性的影响。方法:将胰腺癌细胞SW1990分为对照组(正常培养)、si-NC组(转染si-NC)、si-circEIF6组(转染si-circEIF6)、si-circEIF6+inhibitor-NC组(si-circEIF6和inhibitor-NC共转染)、si-circEIF6+miR-129-5p inhibitor组(si-circEIF6和miR-129-5p inhibitor共转染);RT-qPCR检测circEIF6、miR-129-5p的表达水平;MTT法检测细胞增殖能力;流式细胞术检测细胞凋亡;免疫印迹检测TRAF6、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3(caspase-3)的表达;MTT法检测不同浓度GEM对细胞增殖抑制率的影响;双荧光素酶报告基因实验分别验证circEIF6和miR-129-5p、miR-129-5p和TRAF6的关系。结果:与对照组、si-NC组比较,si-circEIF6组circEIF6表达、OD490值、TRAF6、PCNA蛋白表达降低,miR-129-5p表达、细胞凋亡率、Bax、caspase-3蛋白表达升高;与si-circEIF6组、si-circEIF6+inhibitor-NC组比较,si-circEIF6+miR-129-5p inhibitor组OD490值、TRAF6、PCNA蛋白表达升高,miR-129-5p表达、细胞凋亡率、Bax、caspase-3蛋白表达降低;细胞增殖抑制率在GEM处理下以剂量依赖性的方式显著增加;与对照组比较,GEM处理下细胞的增殖抑制率、凋亡率显著升高,敲低circEIF6可提高GEM处理下细胞的增殖抑制率、凋亡率,而miR-129-5p inhibitor可减弱敲低circEIF6发挥的上述作用;circEIF6靶向负调控miR-129-5p的表达,miR-129-5p靶向负调控TRAF6的表达;双荧光素酶报告实验证实miR-129-5p与circEIF6和TRAF6存在靶向关系。结论:敲低circEIF6可能通过靶向miR-129-5p下调TRAF6表达,进而抑制胰腺癌细胞增殖、促进细胞凋亡、提高胰腺癌细胞对GEM的敏感性。

TFAP2A基因重组真核表达载体的构建及鉴定

目的构建转录因子增强子结合蛋白-2α的真核表达载体并进行鉴定,为TFAP2A的后续研究提供有效工具。方法采用限制性内切酶BamHⅠ和HindⅢ对真核表达载体CV702质粒进行酶切获得线性化载体;通过PCR扩增制备TFAP2A的cDNA片段,将线性化载体和TFAP2A的cDNA扩增产物进行体外环化连接,构建CV702-TFAP2A重组质粒。将连接产物进行细菌转化,挑取平板上的单克隆进行PCR鉴定,对阳性克隆进行测序及结果分析。将CV702-TFAP2A重组真核表达载体转染乳腺癌细胞MDA-MB-231,通过Western blot检测TFAP2A的表达效率。结果菌落PCR扩增和测序结果显示,CV702-TFAP2A重组真核表达载体构建成功;Western blot结果与Image J灰度值分析结果显示,与CV702对照载体相比,转染CV702-TFAP2A重组质粒的MDA-MB-231中的Flag-TFAP2A蛋白相对表达量更高(P<0.05)。结论成功构建CV702-TFAP2A的重组真核表达载体,证实转染CV702-TFAP2A的细胞中Flag-TFAP2A的表达水平升高。

肝细胞肝癌组织中WDR4和EIF2A的表达及临床预后价值

目的探讨肝细胞肝癌(HCC)组织中WD重复结构域4(WDR4)、真核翻译起始因子2A(EIF2A)的表达情况及临床预后意义。方法收集2019年1月—2020年1月南京大学医学院附属金陵医院收治的90例HCC患者的临床资料。利用R语言(4.2.1版本)分析癌症基因组图谱(TCGA)数据库下载的327对HCC癌组织和癌旁组织中WDR4 mRNA、EIF2A mRNA表达的转录组测序数据。免疫组织化学检测HCC癌组织、癌旁组织中WDR4、EIF2A表达。分析HCC癌组织WDR4、EIF2A表达与临床病理特征的关系。Kaplan-Meier法分析WDR4、EIF2A表达对HCC患者预后的影响。Cox回归模型分析HCC预后的影响因素。结果HCC癌组织WDR4 mR-NA、EIF2A mRNA表达高于癌旁组织,差异有统计学意义(P均<0.05)。HCC癌组织中WDR4、EIF2A表达阳性率高于癌旁组织,差异有统计学意义(P均<0.05)。HCC癌组织WDR4 mRNA与EIF2A mRNA表达呈正相关(r=0.404,P<0.001)。HCC癌组织中WDR4蛋白与EIF2A蛋白表达呈正相关(r=0.667,P<0.05)。肿瘤最大径>5 cm、TNM分期Ⅲ期及浸润型HCC癌组织中WDR4、EIF2A阳性率分别高于肿瘤最大径≤5 cm、TNM分期Ⅰ-Ⅱ期及非浸润型癌组织,差异有统计学意义(P均<0.05)。WDR4阳性组3年累积生存率明显低于WDR4阴性组,差异有统计学意义(P=0.016)。EIF2A阳性组3年累积生存率明显低于EIF2A阴性组,差异有统计学意义(P=0.022)。肿瘤TNM分期Ⅲ期、肿瘤最大径>5 cm、WDR4阳性、EIF2A阳性是HCC患者不良预后的独立危险因素。结论HCC组织中WDR4、EIF2A表达升高,两者与HCC不良临床病理特征有关,是评估HCC预后的肿瘤标志物。

Different Eukaryotic Initiation Factor 2Be Mutations Lead to Various Degrees of Intolerance to the Stress of Endoplasmic Reticulum in Oligodendrocytes

Vanishing white matter disease （VWM）, a human atitosomal recessive inherited leukoencephalopathy, is due to mutations in eukaryotic initiation factor 2B （elF2B）. elF2B is responsible for tile initiation of protein synthesis by its guanine nucleotide exchange lhctor （GEF） activity. Mutations ofelF2B impair GEF activity at different degree. Previous studies implied improperly activated unlblded protein response （UPR） and endoplasmic reticulum stress （ERS） participated in the pathogenesis ofVWM. Autophagy relieves endoplasmic reticulum load by eliminating the unfolded protein. It is still unknown the effects of genotypes on the pathogenesis. In this work, UPR and autophagy flux were analyzed with different mutational types. Methods： ERS tolerance, reflected by apoptosis and cell viability, was detected in human oligodendrocyte cell line transfected with the wild type, or different mutations of p. Argl 13 His, p. Arg269＊ or p. Ser610-Asp613del in el F2 Be. A representative U PR-PERK component of activating transcription lhctor 4 （ATF4） was measured under the basal condition and ERS induction. Autophagy was analyzed the flux in the presence of lysosomal inhibitors. Results： The degree of ERS tolerance varied in different genotypes. The truncated or deletion mutant showed prominent apoptosis cell viability declination after ERS induction. The most seriously damaged GEF activity ofp. Arg269＊ group underwent spontaneous apoptosis. The truncated or deletion mutant showed elevated ATF4 under basal as well as ERS condition. Decreased expression of LC3-1 and LC3-11 in the mutants reflected an impaired autophagy flux, which was more obvious in the truncated or deletion mutants alter ERS induction. Conclusions： GEF activities in dilt;erent genotypes could influence the cell ERS tolerance as well as compensatory pathways of UPR and autophagy. Oligodendrocytes with truncated or deletion inutants showed less tolerable to ERS.

真核翻译起始因子4γ2调控N-乙酰氨基半乳糖转移酶1 mRNA对胃癌细胞恶性表型的影响

目的探讨真核翻译起始因子4γ2(EIF4G2)通过调控N-乙酰氨基半乳糖转移酶1(GALNT1)mRNA对胃癌进展的影响。方法通过生物信息数据库分析GALNT1、EIF4G2在胃癌细胞和胃癌组织中的表达及与胃癌患者预后的关系,并分析胃癌组织中EIF4G2、GALNT1表达的相关性。采用实时荧光定量聚合酶链反应(RT-qPCR)检测GALNT1 mRNA、EIF4G2 mRNA在人胃黏膜上皮细胞GES-1和人胃癌细胞(AGS细胞等)中的表达水平,并采用蛋白质印迹法(Western blot)检测GALNT1、EIF4G2蛋白表达水平。建立干扰GALNT1和过表达EIF4G2的AGS细胞株,采用CCK-8法和克隆形成实验检测细胞增殖能力,采用TUNEL实验检测细胞凋亡能力,采用划痕实验和Transwell实验分别检测细胞迁移和侵袭能力,采用Western blot检测凋亡和转移相关蛋白表达水平,采用RIP实验和RNA下拉实验检测GALNT1 mRNA与EIF4G2的结合关系。结果胃癌组织和胃癌细胞中的GALNT1、EIF4G2水平分别高于正常组织和人胃黏膜上皮细胞,差异有统计学意义(P<0.001),且与胃癌患者预后差相关;胃癌组织中,EIF4G2表达与GALNT1表达呈正相关。敲低GALNT1后,AGS细胞活力降低,克隆形成数、迁移和侵袭细胞数减少,凋亡细胞数增加,Bcl-2、MMP2、MMP9蛋白表达降低,Bax、cleaved-caspase3、cleaved-caspase9蛋白表达增加,差异有统计学意义(P<0.001)。EIF4G2可与GALNT1 mRNA结合,促进GALNT1 mRNA、GALNT1蛋白表达及GALNT1 mRNA稳定性,差异有统计学意义(P<0.001)。共转染sh-GALNT1-1与oe-EIF4G2可逆转sh-GALNT1-1对AGS细胞增殖、凋亡、迁移和侵袭的作用,差异有统计学意义(P<0.05或P<0.01或P<0.001)。结论EIF4G2可通过稳定GALNT1 mRNA促进胃癌细胞增殖、迁移、侵袭并抑制细胞凋亡,其或可成为胃癌临床诊疗的新靶点。

YAP通过调控EEF1A2的表达促进肝癌细胞的迁移和侵袭

目的:研究Yes相关蛋白(yes-associated protein,YAP)通过调控真核细胞翻译延伸因子1A2(eukaryotic translation elongation factor 1 alpha 2,EEF1A2)的表达促进肝癌细胞迁移和侵袭的机制。方法:通过建立YAP和EEF1A2干扰表达或过表达的SMMC-7721和SK-HEP1肝癌细胞模型,采用Transwell实验检测细胞迁移和侵袭能力的改变;应用反转录和实时定量PCR(reverse transcription-quantitative real-time polymerase chain reaction,RT-qPCR)和Western blot法检测相关基因的mRNA和蛋白表达水平;利用染色质靶向酶切检测(cleavage under targets and release using nuclease,CUT&RUN)-qPCR实验检测YAP能否与EEF1A2基因的启动子序列结合。结果:EEF1A2在肝癌组织中高表达,并与肝癌细胞的迁移能力呈正相关;EEF1A2可以促进低迁移能力的SMMC-7721细胞和高迁移能力的SK-HEP1细胞迁移和侵袭,调控细胞中上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志物Snail和E-cadherin的表达;YAP可以上调EEF1A2的表达而促进肝癌细胞的迁移和侵袭;CUT&RUN-qPCR实验结果显示YAP可以与EEF1A2基因的启动子序列结合。结论:YAP可以通过与EEF1A2基因的启动子序列结合,促进EEF1A2的表达,进一步促进肝癌细胞的迁移和侵袭。

木犀草素介导PERK/eIF2α/CHOP信号通路改善新生大鼠坏死性小肠结肠炎的作用研究

目的探讨木犀草素介导蛋白激酶RNA样内质网激酶(PERK)/真核翻译启动因子2α(eIF2α)/CCAAT增强子结合蛋白同源蛋白(CHOP)信号通路改善新生大鼠坏死性小肠结肠炎(NEC)的作用及相关机制。方法取新生SD大鼠30只,其中24只采用缺氧冷刺激法建立NEC大鼠模型,按随机数字表法分为木犀草素17.5 mg/kg组、木犀草素35 mg/kg组、木犀草素70 mg/kg组、模型组,另6只大鼠设为正常组(仅予0.1 mL 0.9%氯化钠溶液干预处理),1次/d,连续灌胃给药4 d。取大鼠小肠上皮细胞系IEC-6细胞采用脂多糖刺激法建立NEC细胞模型并分为木犀草素5μmol/L组、10μmol/L组、20μmol/L组、模型组,取未经脂多糖处理的IEC-6细胞设为正常组(仅予0.9%氯化钠溶液干预处理);另取建模后的IEC-6细胞分为GSK26064140.5μmol/L组、GSK26064141.0μmol/L组、GSK26064142.0μmol/L组、GSK26064144.0μmol/L组、模型组(仅予等体积0.9%氯化钠溶液干预处理),采用噻唑蓝法筛选细胞存活率最高的药物浓度(木犀草素20μmol/L,GSK26064144.0μmol/L)作为后续实验浓度,增设木犀草素20μmol/L+GSK26064144.0μmol/L组。采用HE染色观察大鼠肠道组织病理学变化,采用原位末端转移酶标记法、ELISA法、Western blot法分别检测木犀草素对大鼠肠道组织凋亡率,大鼠肠道组织、血清及IEC-6细胞中TNF-α、IL-6水平,以及大鼠肠道组织及IEC-6细胞中PERK/eIF2α/CHOP信号通路相关因子表达水平的影响。结果与模型组比较,木犀草素35 mg/kg组、木犀草素70 mg/kg组大鼠肠道组织损伤均有不同程度的改善,且细胞凋亡率均明显较低(均P<0.05)。与模型组比较,木犀草素35 mg/kg组、木犀草素70 mg/kg组大鼠肠道组织中TNF-α、IL-6水平明显较低(均P<0.05),木犀草素17.5 mg/kg组、木犀草素35 mg/kg组、木犀草素70 mg/kg组大鼠肠道组织中p-PERK/PERK、p-eIF2α/eIF2α、CHOP表达水平以及血清中TNF-α、IL-6水平均明显较低(均P<0.05),木犀草素20μmol/L组IEC细胞中TNF-α、IL-6以及p-PERK/PERK、p-eIF2α/eIF2α、CHOP表达水平均明显较低(均P<0.05);与木犀草素20μmol/L组比较,木犀草素20μmol/L+GSK26064144.0μmol/L组IEC-6细胞中p-PERK/PERK、p-eIF2α/eIF2α、CHOP表达水平均明显较低(均P<0.05)。结论木犀草素可有效改善新生大鼠NEC,其作用机制可能与下调TNF-α、IL-6以及p-PERK/PERK、p-eIF2α/eIF2α、CHOP表达水平进而抑制炎症反应有关。

健脾补土方对脑缺血再灌注大鼠缺血侧皮质Claudin-5、Occludin、p-BAD、Bcl-2、Caspase-3蛋白表达的影响

目的探讨健脾补土方减轻脑缺血再灌注大鼠神经元损伤的可能作用机制。方法将80只SD大鼠随机分为假手术组10只、手术组70只,手术组经模型制备后评价,剔除不成功的及死亡大鼠,二次分组,分为模型组、依达拉奉组、健脾补土方高、中、低剂量组,每组各13只。手术组采用大脑中动脉栓塞法制备脑缺血再灌注模型(middle cerebral artery occlusion,MCAO),干预7天,取材。取材前每组选取10只,采用国际公认的大鼠脑卒中后神经功能缺损评分法(mNss评分)进行神经功能缺损评分。取材部位为缺血侧脑皮质组织。每组随机选取3只采用苏木素—伊红(hematoxylin-eosin,HE)染色法观察病理形态改变;每组随机选取其中5只采用Western blot法检测紧密连接蛋白-5(Claudin-5)、闭合蛋白(Occludin)表达量;剩余5只采用免疫组化法检测磷酸化相关死亡启动因子(phospho-Bcl-xL/Bcl-2 asociated death promoter,p-BAD)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)和半胱天冬氨酸蛋白酶-3(Caspase-3)蛋白表达量。结果(1)造模后,与假手术组比较,模型组大鼠神经功能缺损评分明显上升,经药物干预后,各组神经功能缺损评分均有所下降(P<0.05),其中以依达拉奉组与健脾补土方高剂量组下降最为明显;(2)造模后,模型组大鼠较假手术组病理改变严重,出现组织大片坏死,经药物干预后,各组病理改变均有所改善,依达拉奉组和健脾补土方高剂量组尤为明显;(3)与模型组比较,各药物干预组脑组织缺血皮质区Occludin、Claudin-5蛋白表达均有所上调,差异有统计学意义(P<0.05),其中以依达拉奉组和健脾补土方高剂量组最为显著;(4)与模型组比较,各药物干预组脑缺血皮质组织p-BAD、Bcl-2蛋白表达均明显增加,Caspase-3蛋白表达有所降低(P<0.05),其中依达拉奉组与健脾补土方高剂量组最为显著。结论健脾补土方可能通过上调p-BAD、Bcl-2、Claudin-5、Occludin蛋白表达,下调Caspase-3蛋白表达,从而改善脑缺血再灌注大鼠神经功能缺损症状及脑组织病理改变,进而促进缺血脑组织损伤修复。

miR-429对乳腺癌细胞增殖和迁移的影响及其机制

目的探讨miR-429对乳腺癌细胞增殖和迁移的影响及其机制。方法采用实时荧光定量PCR(RT-qPCR)法和免疫印迹法,分别检测乳腺癌MCF-7、MDA-MB-231、MDA-MB-468、BT-549细胞及正常乳腺上皮细胞MCF-10A的eIF4E mRNA和蛋白相对表达量。在MCF-7细胞中转染miR-429 mimics(A组)、mimics NC(B组)、miR-429 inhibitors(C组)及inhibitors NC(D组),采用RT-qPCR法、CCK8法、细胞划痕实验和免疫印迹法检测A~D组细胞miR-429表达情况、细胞增殖和迁移情况、eIF4E mRNA和蛋白相对表达量。在293T细胞中转染eIF4E-3′UTR-Wt+miR-429 mimics(E组)、eIF4E-3′UTR-Wt+mimics NC(F组)、eIF4E-3′UTR-Mut+miR-429 mimics(G组)、eIF4E-3′UTR-Mut+mimics NC(H组),检测E~H组细胞相对荧光素酶活性,分析miR-429对eIF4E的靶向调控作用。结果RT-qPCR与免疫印迹检测结果显示,MCF-7、MDA-MB-231、MDA-MB-468、BT-549细胞中的eIF4E mRNA以及蛋白的相对表达量均明显高于正常乳腺上皮细胞MCF-10A(F=74.414、1981.243,P<0.01)。RT-qPCR法检测结果显示,A组细胞的miR-429相对表达量明显高于B组(t=25.390,P<0.01),而eIF4E mRNA相对表达量明显低于B组(t=-6.363,P<0.05),C组细胞miR-429相对表达量明显低于D组(t=-4.652,P<0.05),而eIF4E mRNA相对表达量明显高于B组(t=-2.928,P<0.05)。CCK8实验检测结果显示,与B组细胞相比,A组细胞第24、48、72小时时的增殖活力明显降低(F=26.148~40.997,P<0.01);与D组细胞相比,C组细胞第24、48、72小时增殖活力明显增高(F=6.410~82.593,P<0.05)。细胞划痕实验显示,A组细胞愈合率明显低于B组(t=-22.584,P<0.01),C组细胞愈合率明显高于D组(t=11.464,P<0.01)。免疫印迹法检测结果显示,A组细胞中eIF4E蛋白相对表达量明显低于B组(t=-20.355,P<0.01),C组细胞eIF4E蛋白相对表达量明显高于D组(t=3.622,P<0.01)。双荧光素酶报告基因实验结果显示,各组细胞相对荧光素酶活性比较差异具有显著性(F=366.823,P<0.05),而且E组细胞相对荧光素酶活性显著低于F组(t=-42.961,P<0.01)。结论miR-429或通过靶向调控eIF4E基因进而抑制乳腺癌细胞的增殖和迁移。