Anti-oncogenic PTEN induces ovarian cancer cell senescence by targeting P21

Deletion and mutation of phosphatase and tensin homolog deleted on chromosome10 (PTEN) are closely associated with the occurrence of tumors. Tumor suppressor gene PTEN mutation plays an important role in the pathogenesis of ovarian cancer. However, it has been unclear whether it can regulate the senescence of ovarian cancer cells. We speculated that PTEN might inhibit the occurrence and development of ovarian cancer by promoting the expression of P21. We found that the expression of TRIM39 in human ovarian cancer was significantly diminished. In SKOV3 cells treated with naringin, the expression of TRIM39, which binds P21 and inhibits P21 degradation, was significantly elevated. Real-time polymerase chain reaction (PCR), Western blot, and immunofluorescence were used to detected the expression of PTEN, p21, and TRIM39, β-galactosidase Staining was used to detect cell senescence, Ki67 staining was used to observe cell proliferation, Trim39 interference or overexpression assay was used to detect its function. We speculated that PTEN might promote SKOV3 cell senescence by increasing TRIM39 expression and decreasing P21 degradation. Furthermore, by interfering with TRIM39 in SKOV3 cells, we found that the expression of P21 was downregulated, and the number of senescent SKOV3 cells decreased. With overexpression of TRIM39 in SKOV3 cells, the expression of P21 was upregulated, and the number of senescent SKOV3 cells increased. When naringin, a PTEN agonist, was added to SKOV3 cells in which TRIM39 protein was interfered with, the expression of P21 was significantly lower than that in the control group, and the number of senescent ovarian cancer cells was significantly diminished. Our results indicated that PTEN maintained the stability of P21 and decreased the degradation of P21 by increasing TRIM39 expression, thus promoting the senescence of SKOV3 cells, and PTEN maintained the stability of p21 and promoted the aging of SKOV3 cells might be a novel therapeutic target for ovarian cancer.     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.

Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.     

Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease,potentiates cell proliferation

In this study, we present evidence for the critical role of proteinase-3 (PR3) in the proliferation of myeloid cells via the proteolytic regulation of the cyclin-dependent kinase inhibitor p21(waf1). Expression of recombinant PR3 in rat (RBL) or human (HMC1) mast cell lines increased bromodeoxyuridine incorporation and CDK2 activity compared with RBL and HMC1 cells transfected with an enzymatically inactive PR3 mutant (PR3(S203A)) or with human neutrophil elastase. Western blot analysis of p21(waf1) showed an absence of detectable protein, despite normal levels of p21 mRNA. Ectopic overexpression of p21 restored normal levels of p21 in the RBL/PR3/p21 double transfectants and reverted the proliferative effect of PR3. Inhibition of the 26 S proteasome by lactacystin or of caspases by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not inhibit p21 proteolysis. p21 cleavage correlated with PR3 expression in HMC1 cells infected with recombinant adenoviral vector Ad/PR3. During in vitro studies, purified p21 was cleaved by PR3, resulting in a 10-kDa p21 fragment. Employing double immunofluorescence confocal microscopy, subcellular fractionation, and co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-alpha, which induces PR3 re-expression, we observed that p21 disappeared and was reversed by Pefabloc, a serine proteinase inhibitor. The physiopathological implications of the cleavage of p21 by PR3 have to be determined.     

人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性

成纤维细胞生长因子FGF-21是FGF家族的一个新成员, 最近的研究发现其具有调节血糖的作用, 有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术, 从成人肝脏中克隆人源的FGF-21成熟蛋白基因, 并将其克隆到T载体上, 经测序鉴定后, 将其亚克隆到原核表达载体pSUMO上, 转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后, 用IPTG诱导FGF-21表达, 并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明, FGF-21成熟蛋白基因大小为546 bp, 测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明: 人源FGF-21成熟蛋白大小19.4 kDa, 经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性, 并且GLUT1是FGF-21发挥生物学作用的终末执行单位。     

Analyses of recombinant stereotypic IGHV3-21-encoded antibodies expressed in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells that use IgH encoded by IGHV3-21 and that have a particular stereotypic third CDR (HCDR3), DANGMDV (motif-1), almost invariably express Ig L chains (IgL) encoded by IGLV3-21, whereas CLL that use IGHV3-21-encoded IgH with another stereotypic HCDR3, DPSFYSSSWTLFDY (motif-2), invariably express κ-IgL encoded by IGKV3-20. This nonstochastic pairing could reflect steric factors that preclude these IgH from pairing with other IgL or selection for an Ig with a particular Ag-binding activity. We generated rIg with IGHV3-21-encoded IgH with HCDR3 motif-1 or -2 and IgL encoded by IGKV3-20 or IGLV3-21. Each IgH paired equally well with matched or mismatched κ- or λ-IgL to form functional Ig, which we screened for binding to an array of different Ags. Ig with IGLV3-21-encoded λ-IgL could bind with an affinity of ～ 2 × 10(-6) M to protein L, a cell-wall protein of Peptostreptococcus magnus, independent of the IgH, indicating that protein L is a superantigen for IGLV3-21-encoded λ-IgL. We also detected Ig binding to cofilin, a highly conserved actin-binding protein. However, cofilin binding was independent of native pairing of IgH and IgL and was not specific for Ig with IgH encoded by IGHV3-21. We conclude that steric factors or the binding activity for protein L or cofilin cannot account for the nonstochastic pairing of IgH and IgL observed for the stereotypic Ig made by CLL cells that express IGHV3-21.     

Compound C inhibits clonal expansion of preadipocytes by increasing p21 level irrespectively of AMPK inhibition

AMP-activated protein kinase (AMPK) activation inhibits adipocyte differentiation. This study investigated the effect of compound C (CC), a widely used AMPK inhibitor, on differentiation of 3T3-L1 preadipocytes. CC treatment blocked hormone-induced preadipocyte differentiation due to inhibition of mitotic clonal expansion, which was accompanied by the failure of degradation of p21, a cyclin-dependent kinase inhibitor. CC increased the level of p21 protein, but not its mRNA, in preadipocytes incubated in a hormone-free medium. Cycloheximide decreased the basal p21 level, which was inhibited by CC treatment, supporting the stabilization of p21 by CC. Treatment of AICAR or metformin, AMPK activators, failed to induce p21 or inhibit the ability of CC to increase p21 level. In conclusion, CC inhibits proliferation of preadipocytes as a consequence of an increase in p21 content, which might result from p21 stabilization, and the increase in p21 level by CC might not be associated with AMPK inhibition.     

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.     

Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals

Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site. GP2-PL and GP3-PL inhibited [3H]GDP binding to p21 competitively. Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of [3H]GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation. The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography. The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP. Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16. These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.     

Use of a derivative of Escherichia coli BL21(DE3) for efficient production of three different recombinant proteins

Plasmid instability and growth inhibition of plasmid-bearing cells after induction were encountered when E. coli BL21(DE3) was used as host for the production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody (AhOC x AhCD3), human soluble B lymphocyte stimulator fused with thioredoxin (Trx-hsBLyS) and human parathyroid hormone fused with thioredoxin (Trx-hPTH). A derivative of BL21(DE3), namely, BLRM(DE3), isolated and showing superiority in AhOC x AhCD3 production in our previous work, was further used here for more efficient production of these three different recombinant proteins. By using BLRM(DE3) as host, the simplified one-stage fermentation process was developed, which was more labor-saving and yielded AhOC x AhCD3, comparable to that of the traditional two-stage fermentation process. Also, the plasmid stabilities and production yields of Trx-hsBLyS and Trx-hPTH were dramatically improved by the application of BLRM(DE3) instead of BL21(DE3). A high Trx-hsBLyS yield (about 3.5 g/L) was obtained, which was more than twice as much as that of the recombinant BL21(DE3) strain. The Trx-hPTH yield was improved from about 700 mg/L to 1 g/L. These results further showed the superiority of BLRM(DE3) to BL21(DE3) and suggested its effectiveness for other BL21(DE3)/pET heterologous protein expression systems, which encounter similar problems.     

鼠源成纤维细胞生长因子-21对脂肪细胞糖代谢的作用

成纤维细胞生长因子-21(FGF-21)是FGF家族的成员之一．近年发现FGF-21是一种新的代谢调节因子．从小鼠肝脏克隆FGF-21 cDNA,经测序正确后亚克隆至具有羟胺切割位点的小泛素相关修饰物表达载体上,转化宿主菌Rosetta,得到的转化子经IPTG诱导后获得稳定、高效、可溶的表达产物．表达产物经羟胺切割、透析、复性、柱层析纯化后,在每升宿主菌中可获得4 mg纯度为95%的成熟鼠源FGF-21蛋白,利用葡萄糖氧化酶-过氧化物酶(POD-GOD)法在小鼠3T3-L1脂肪细胞中进行生物学活性检测．结果表明,鼠源FGF-21具有促进脂肪细胞吸收葡萄糖的作用,短期作用(1 h)与胰岛素相似,长期作用(8和12 h)明显优于胰岛素．这一结果为以鼠源FGF-21为模型进一步研究FGF-21的生物学活性及其在糖代谢方面的作用机理奠定了基础．     

Silencing of MicroRNA-21 Confers Radio-Sensitivity through Inhibition of the PI3K/AKT Pathway and Enhancing Autophagy in Malignant Glioma Cell Lines

Radiation is a core part of therapy for malignant glioma and is often provided following debulking surgery. However, resistance to radiation occurs in most patients, and the underlying molecular mechanisms of radio-resistance are not fully understood. Here, we demonstrated that microRNA 21 (miR-21), a well-known onco-microRNA in malignant glioma, is one of the major players in radio-resistance. Radio-resistance in different malignant glioma cell lines measured by cytotoxic cell survival assay was closely associated with miR-21 expression level. Blocking miR-21 with anti-miR-21 resulted in radio-sensitization of U373 and U87 cells, whereas overexpression of miR-21 lead to a decrease in radio-sensitivity of LN18 and LN428 cells. Anti-miR-21 sustained γ-H2AX DNA foci formation, which is an indicator of double-strand DNA damage, up to 24 hours and suppressed phospho-Akt (ser473) expression after exposure to γ-irradiation. In a cell cycle analysis, a significant increase in the G₂/M phase transition by anti-miR-21 was observed at 48 hours after irradiation. Interestingly, our results showed that anti-miR-21 increased factors associated with autophagosome formation and autophagy activity, which was measured by acid vesicular organelles, LC3 protein expression, and the percentage of GFP-LC3 positive cells. Furthermore, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after irradiation. Our results show that miR-21 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy and provide a novel phenomenon for the acquisition of radio-resistance.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Novel events associated with phenotypic reversion of a P element mutant in Drosophila melanogaster.

Transposable P elements have been used extensively for Drosophila mutagenesis. While their mutagenic activity has long been recognized, the mechanisms by which P elements cause mutations are varied and not completely understood. We describe here an experiment to replace a P element at vestigial (vg) that caused a strong mutant phenotype (P[21-3]) with a P element (P[21]) known to produce a very weak phenotype when inserted at vg. In addition to testing the feasibility of P element replacements at vg, our investigation led to the production of 7 new vg alleles and 1 apparent second site suppressor. All the vg21-3 revertants that we recovered had a P element inserted into the first exon of vg at the same location and in the same orientation as the original element in vg21-3, providing a unique opportunity to study the mechanism of transposon mutagenesis. A majority of the revertants arose from a previously described event: internal deletion of P sequences, including the P promoter. In addition, 3 novel reversions of the vg21-3 wing phenotype were recovered. The wings of homozygous vg21r36 flies were normal. However, vg21r36 in combination with a deletion of the vg locus exhibited a strong mutant wing phenotype. This was surprising, because the P element insertion in vg21r36 was very similar to that found in the vg21 allele, which showed only slight nicking of the wings in combination with a deletion. In vg21r4, reversion was caused by a tandem insertion of P[21] and the original P[21-3] element present in vg21-3. Finally, the vg21r7 revertant had a P[21-3] insert at vg and 3 additional P elements elsewhere in the genome. We hypothesize that reversion in the 3 novel cases might be caused by P repressor produced by an element at vg or, in the case of vg21r7, elsewhere in the genome. This raises an interesting aspect of P element evolution. While P transposons produce mutations that might prove deleterious to their host, their success in invading the genome of D. melanogaster may be explained by their ability to silence those same mutations by a range of repressor-producing elements.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

Direct identification of palmitic acid as the lipid attached to p21ras.

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.     

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia coli, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular β-agarase. E. coli DH5α harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5α/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5α/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5α/pTAET21, is an effective vaccine candidate against E. tarda infection.     

vMIP-ⅡN端重组肽的表达纯化及活性鉴定

病毒巨噬细胞炎症蛋白-Ⅱ (viral macrophage inflammatory protein-Ⅱ,vMIP-Ⅱ)由卡波西肉瘤疱疹病毒编码,前期研究证明了vMIP-II N端21肽(NT21MP)选择性的阻断趋化因子CXCR4,从而抑制乳腺癌细胞趋化的活性。通过化学合成法获得编码vMIP-ⅡN末端的基因序列,与pGEX-KG（克隆位点的N 端有谷胱甘肽转移酶GST标签序列）连接构建原核表达载体,重组质粒在大肠杆菌E.coli BL21(DE3)中获得表达,免疫印迹显示重组蛋白GST-NT21MP主要在细菌裂解液上清中表达,可溶性部分经亲和层析、超滤、快速蛋白液相色谱（fast protein liquid chromatography,FPLC）纯化获得高纯度的GST-NT21MP蛋白。利用Transwell趋化试验测定GST-NT21MP的活性。结果显示,重组蛋白GST-NT21MP能够抑制乳腺癌细胞SK-BR-3的趋化活性,可作为治疗乳腺癌转移的潜在性靶向药物。     

The Angiogenic Effect of microRNA-21 Targeting TIMP3 through the Regulation of MMP2 and MMP9

microRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. In vivo, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.