rBTI通过上调PTEN的表达抑制Hep G2细胞增殖

磷酸酶及张力蛋白的同源基因(PTEN) 是一种抑癌基因,可以调控细胞的增殖,与癌症的发生和发展息息相关。本研究采用MTT法和流式细胞术分别检测了重组荞麦胰蛋白酶抑制剂(rBTI)对人肝癌细胞株Hep G2细胞的增殖以及周期的影响。免疫荧光及Western印迹法检测了PTEN和p PTEN的亚细胞定位及蛋白表达的变化。采用qRT-PCR及Western印迹法检测了周期相关蛋白的表达。旨在探究PTEN和p PTEN在rBTI抑制Hep G2细胞增殖和周期阻滞中的作用。结果表明,rBTI能显著抑制Hep G2细胞增殖,将细胞周期阻滞在G0/G1期,并呈时间和剂量依赖性;rBTI作用于Hep G2后,可显著上调PTEN和p-PTEN的表达。同时发现,p-PTEN主要分布于细胞核中,能与核仁发生共定位;周期相关蛋白检测表明,细胞内p53、p21转录水平和蛋白水平均增加。综上所述,rBTI通过上调PTEN的表达,使得细胞周期阻滞于G0/G1期,进而抑制Hep G2细胞的增殖。     

miR-148a通过靶向调节己糖激酶2基因抑制乳腺癌细胞糖酵解和细胞增殖能力

为了探讨miR-148a及己糖激酶2（hexokinase 2,HK2）基因对人乳腺癌细胞糖酵解代谢途径的影响和可能机制,利用实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）检测多种乳腺癌细胞系中miR-148a的表达量,从中筛选miR-148a表达量相对较低的乳腺癌细胞系作为研究对象。再通过观察miR-148a表达量的变化对乳腺癌细胞葡萄糖摄取量、乳酸生成量和细胞增殖指标的影响,以探究miR-148a对乳腺癌细胞糖代谢能力的影响。随后,通过TargetScan在线数据库预测miR-148a和HK2基因的靶向关系,再通过双荧光素酶报告实验、Western免疫印迹以及基因回复实验进行验证,以进一步明确miR-148a和HK2在乳腺癌细胞的糖酵解代谢途径中的作用机制。通过qRT-PCR发现miR-148a在多种乳腺癌细胞系表达降低,尤其是在乳腺癌细胞系MDA-MB231中表达量显著降低（P<0.000 1）。过表达miR-148a使MDA-MB231细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著下降（P<0.01）;而抑制miR-148a表达使MDA-MB231细胞葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著上升（P<0.01）。通过TargetScan在线数据库预测得出,miR-148a与HK2基因3′非编码区（3′-untranslated region,3′-UTR）具有部分结合位点;而双荧光素酶报告实验发现miR-148a与野生型HK2基因的3′-UTR荧光素酶报告载体结合,不与突变型HK2基因的3′-UTR结合。Western免疫印迹检测结果表明,过表达miR-148a使MDA-MB231细胞中HK2蛋白表达量显著下降（P＜0.000 1）,而抑制miR-148a表达则促进HK2蛋白表达量显著上升（P<0.05）。基因回复实验显示,过表达HK2基因使MDA-MB231乳腺癌细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标显著上升（P＜0.01）;将过表达miR-148a载体与过表达HK2载体共转染MDA-MB231细胞,miR-148a逆转了HK2所致的葡萄糖摄取量增加和乳酸生成量上升,并抑制细胞增殖。因此,研究提示,miR-148a可通过靶向抑制HK2基因表达而抑制乳腺癌细胞MDA-MB231糖酵解代谢和细胞增殖。     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

荞麦胰蛋白酶抑制剂的原核表达及纯化

根据文献报道的荞麦胰蛋白酶抑制剂的氨基酸序列及本研究室先前已获得的部分基因序列设计引物,经过RT-PCR扩增,获得荞麦胰蛋白酶抑制剂编码区基因全序列.将该基因克隆到原核表达载体pQE-31中,并转化至大肠杆菌M15,经IPTG诱导表达获得可溶性目的蛋白,其表达量约占菌体总蛋白的25%.该目的蛋白经Ni2 -NTA柱亲和纯化,SDS-PAGE分析显示,在大约9kD处出现明显的目的条带,与预计蛋白分子量大小一致.Western blot鉴定证实,目的蛋白N端带有6个组氨酸标签.活性测定表明,目的蛋白具有专一性的胰蛋白酶抑制剂活性,抑制活性约为77U/mg纯化蛋白.本实验为进一步研究荞麦胰蛋白酶抑制剂结构与功能的关系奠定了基础.     

响应面法优化荞麦胰蛋白酶抑制剂固定化条件

目的:以大肠杆菌表达的重组荞麦胰蛋白酶抑制剂为原材料,研究其固定化方法及条件。方法:以0.2%聚乙烯醇-3%海藻酸钠溶液为载体,CaCl2为固定剂,用物理包埋法对荞麦胰蛋白酶抑制剂进行固定化;在CaCl2浓度、载体与抑制剂体积比以及固定化时间3个单因素基础上,利用响应面法对荞麦胰蛋白酶抑制剂固定化的影响因素进行优化。结果:建立了响应面法优化固定荞麦胰蛋白酶抑制剂的模型,经优化后得到如下最佳固定化条件:CaCl2浓度为5.5%,载体与抑制剂体积比为1.6∶1,固定化时间为31min。在此条件下实际测得固定化抑制剂抑制率为72.4%,而模型预测此条件下的抑制率为74.3%,实测值与理论值相差很小。结论:所建模型拟合程度较高,用该模型优化荞麦胰蛋白酶抑制剂固定化的工艺条件参数准确可信,可为进一步开发胰蛋白酶的应用提供重要参考。     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

Differential phosphorylation of Akt1 and Akt2 by protein kinase CK2 may account for isoform specific functions

Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.     

The role of mitochondria in the aetiology of insulin resistance and type 2 diabetes

Background

The prevalence of type 2 diabetes is rapidly increasing world-wide and insulin resistance is central to the aetiology of this disease. The biology underpinning the development of insulin resistance is not completely understood and the role of impaired mitochondrial function in the development of insulin resistance is controversial.

Scope of review

This review will provide an overview of the major processes regulated by mitochondria, before examining the evidence that has investigated the relationship between mitochondrial function and insulin action. Further considerations aimed at clarifying some controversies surrounding this issue will also be proposed.

Major conclusions

Controversy on this issue is fuelled by our lack of understanding of some of the basic biological interactions between mitochondria and insulin regulated processes in the context of insults thought to induce insulin resistance. Aspects that have not yet been considered are tissue/cell type specific responses, mitochondrial responses to site-specific impairments in mitochondrial function and as yet uncharacterised retrograde signalling from mitochondria.

General significance

Further investigation of the relationship between mitochondria and insulin action could reveal novel mechanisms contributing to insulin resistance in specific patient subsets. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.