摘要
癌性恶液质(cancer cachexia,CC)是恶性肿瘤的特征之一。CC表现为厌食、贫血、蛋白质分解增加、瘦组织群丢失、机体消耗、进行性体重下降、消瘦甚至器官功能受损。其中肌肉等瘦组织群减少,肌肉进行性分解丢失是其最显著的特征,常使病人生活质量下降并导致病人对抗癌治疗的耐受性下降,并最终导致病人的死亡。对CC的治疗目前尚不理想,主要因为对其发生机制了解不够深入。
研究表明癌性恶液质导致的骨骼肌蛋白分解最主要是通过ATP-泛素—蛋白酶体分解途径,而泛素蛋白连接酶(E3)是该途径激活的关键酶。有三种E3组分蛋白在肌肉中特异性表达:Atrogin-1、MuRF1和E3a-Ⅱ。国内外尚无有关E3与CC之间关系的成熟研究,因此深入研究E3在CC中的作用机制意义重大。本课题拟研究肌肉细胞特异性E3在CC发生机制中的作用,探讨其是否是CC理想干预靶点,以期将来用于临床治疗。
课题第一部分,我们研究肌肉细胞特异性E3在恶性肿瘤患者肌肉中的表达情况,并与良性疾病患者进行比较,探讨E3表达上调的临床意义;第二部分,我们通过小鼠CC模型研究肌肉细胞特异性E3以及泛素—蛋白酶体分解系统其他组分在CC发生机制中的作用,从而进一步明确E3在ATP-泛素—蛋白酶体分解途径中的关键地位;第三部分,我们构建了肿瘤坏死因子a(tumor necrosisfactor-alpha,TNF a)诱导的肌肉细胞营养不良模型,并利用慢病毒感染体系构建含有E3(Atrogin-1)siRNA的重组慢病毒,研究通过RNAi技术将Atrogin-1基因沉默后对C2C12肌肉细胞营养不良的保护作用。
我们发现,肌肉细胞特异性E3在恶性肿瘤患者肌肉中特异性高表达;CC小鼠模型能稳定建立,其肌肉中肌肉细胞特异性E3乃至ATP-泛素—蛋白酶体分解系统其他组分特异性高表达,与CC密切相关;TNF a能引起肌肉细胞营养不良,肌肉细胞特异性E3-Atrogin-1基因表达上调,将Atrogin-1基因沉默后能够避免TNF a引起的肌肉细胞营养不良。
我们深入研究肌肉细胞特异性E3以及ATP-泛素—蛋白酶体分解系统其他组分在临床患者、CC小鼠模型骨骼肌中的表达情况,探讨其机制;我们首次采用慢病毒介导的核糖核酸干扰(RNA interference,RNAi)法对Atrogin-1基因进行沉默,成功避免了TNF a引起的肌肉细胞的营养不良,为进一步在临床上对CC进行特异性干预奠定基础。
第一部分肌肉细胞特异性泛素蛋白连接酶在癌症患者肌肉中的表达及意义
【目的】检测肌肉细胞特异性泛素蛋白连接酶(E3)在恶性肿瘤患者骨骼肌中的表达情况,研究其与恶性肿瘤患者营养不良之间的关系,探讨其在CC发生机制中的作用。
【方法】采取21例消化道恶性肿瘤患者和23例良性疾病患者腹壁肌肉组织,采用实时荧光定量PCR(Real time PCR)法和Western Blot法分别测定病人肌肉中Atrogin-1、MuRF1基因mRNA和蛋白水平的表达,探讨其临床意义,并联系两组病人的术前营养状况进行统计分析。
【结果】与良性疾病患者相比,恶性肿瘤患者肌肉中Atrogin-1mRNA、MuRF-1mRNA的表达显著增高(两者P值均＜0.05);恶性肿瘤患者肌肉中Atrogin-1蛋白表达较良性疾病患者明显增强;恶性肿瘤病人术前存在明显营养不良,多项营养相关指标与良性疾病患者相比具有明显的差异(p＜0.05)。
【结论】E3表达上调与营养不良、CC密切相关,E3表达上调在CC发生机制中起重要作用。
第二部分肌肉细胞特异性泛素蛋白连接酶在癌性恶液质小鼠模型中的研究
【目的】检测肌肉细胞特异性泛素蛋白连接酶(E3)以及ATP-泛素—蛋白酶体分解系统其他组分在小鼠CC模型骨骼肌中的表达,并探讨其在CC发生中作用机制。
【方法】利用鼠结肠癌26细胞株(c26)接种BALB/c小鼠,建立CC模型,并设立对照组,观察小鼠每天体重、摄食量和肿瘤大小的变化,测量小鼠最终去瘤体重和腓肠肌重量,Real time PCR、western blot和免疫组化法测定小鼠腓肠肌中Atrogin-1、biuRF1在mRNA和蛋白水平表达,同时检测ubiquitin mRNA和E2_(14K)mRNA的表达情况。
【结果】荷瘤组小鼠有明显恶液质现象。两组小鼠摄食量差异无统计学意义(P＞0.05);荷瘤组去瘤体重和腓肠肌重量明显下降(P＜0.05),荷瘤组E3(Atrogin-1和MuRF1)、ubiquitin和E2_(14K)(在mRNA水平较对照组表达明显增高(P＜0.01),荷瘤组E3蛋白表达也明显升高。
【结论】肌肉细胞特异性E3以及ATP-泛素—蛋白酶体分解途径其他组分在CC小鼠骨骼肌中高表达,与CC明显相关,提示该途径在CC骨骼肌消耗中起重要作用。
第三部分Atrogin-1基因沉默对肌肉细胞营养不良保护作用的研究
【目的】构建TNF a诱导的肌肉细胞营养不良模型,在此基础上研究将Atrogin-1基因沉默对小鼠成肌细胞(C2C12细胞)营养不良的保护作用,以确立Atrogin-1基因是否是恶液质治疗的理想靶点。
【方法】C2C12细胞分化培养96h形成肌管,加入TNF a进行刺激,观察细胞形态的变化,Real time PCR和western blot法分别检测肌管中Atrogin-1mRNA和Atrogin-1蛋白表达。然后设计合成五对Atrogin-1基因siRNA靶序列及一对对照序列,退火形成双链DNA,产物先克隆到pBS-hU6-I载体获得启动子U6,然后克隆到慢病毒核心载体FG12中,重组FG12质粒与包装质粒pRSVREV、pMDLg/pRRE和pHCMV-G共同转染293T细胞,包装病毒,然后用它感染C2C12细胞,将感染后的细胞分化成肌管后,用TNF a进行刺激,观察细胞形态的变化,Real time PCR和western blot法分别检测肌管中Atrogin-1mRNA和Atrogin-1蛋白表达。
【结果】成功构建了TNF a诱导的肌肉细胞营养不良模型,与正常培养组相比,TNF a诱导组肌管萎缩明显。重组慢病毒载体中含大小、序列正确的片段,能成功感染C2C12细胞,与对照组相比,RNAi干预组细胞Atrogin-1mRNA、Atrogin-1蛋白表达下调,肌管无明显萎缩。
【结论】肌肉细胞营养不良模型可通过TNF a诱导成功构建,含Atrogin-1基因siRNA的重组慢病毒载体可以将Atrogin-1基因沉默,从而避免产生TNF a诱导的肌肉细胞营养不良,因此Atrogin-1基因是恶液质治疗的理想干预靶点。
Cancer poses great threaten to human beings,and cancer cachexia(CC) is a common character of many kinds of carcinoma.CC may be presented as anorexia,anemia,protein loss,body expenditure,progressive weight loss and even organ function failure,among which,the most significant character of CC is great loss of skeleton muscle which cannot be interpretated by anorexia.CC can cause poor quality of life,less response to anti-carcinoma therapy,and even death.Up till now,little progress has been made of the treatment of CC,mostly because we know little about the mechanism of it.
Studies have showed that the most important pathway for protein catabolism is ATP-ubiquitin-proteasome system.There are three important enzymes in the system:ubiquitin-activating enzyme(E1), ubiquitin-conjugating enzyme(E2) and ubiquitin-protein ligase(E3). Among the three,E3 is the key enzyme of the pathway.Up till now,there is little mature study about relationship between E3 and CC in the world.So the study of E3 of the mechanism of CC is very important.During this study,we used several molecular biological methods to study the function of muscle specific E3 in the mechanism of CC,to explore whether it is the suitable target for the treatment of CC.
In the first part of the study,we investigated the expression of muscle-specific ubiquitin protein ligase(E3) in muscle of patients with carcinoma or with benign diseases,analyzed the results,and discussed the clinical significance of it.ln the second part of the study,we establish an animal model of cancer cachexia,in muscle of which we investigated the expression of E3 and other components of ATP-ubiquitin-proteasome pathway,so that we could make sure the core position of E3 in the pathway,In the third part of the study,we constructed a malnutrition model of muscle cell induced by the cytokine tumor necrosis factor-a (TNF-a),on which we study the protective effect of Atrogin-1 gene silencing by RNA interference through a lentivirus vector system.
We found that expression of muscle-specific E3 was significantly upregulated in muscle of patients with carcinoma:that the animal model of cancer cachexia was stably established,and expression of E3 and other components of ATP-ubiquitin-proteasome pathway were significantly upregulated in muscle of the model;that significant atrophy of myotubes treated by TNF-a were seen,which could be protected by Atrogin-1 gene silencing.
We first thoroughly investigated the expression of muscle-specific E3 in muscle of patients with carcinoma and animal model.We studied the function of muscle-specific E3 in the mechanism of CO,and successfully protected muscle cells from the state of cachexia by Atrogin-1 gene silencing.All of the results help to find clinical treatment of CC based on muscle-specific E3 in the future.
PARTⅠExpression of muscle-specific Ubiquitin-protein ligases in muscle of patients with carcinoma and its clinical significance.
【Objective】To investigate the expression and clinical significance of muscle-specific E3 in muscle of patients with carcinoma or with benign diseases,study the relation between E3 and malnutrition,and discuss the possible mechanism of its effect in cancer cachexia.
【Methods】21 patients with carcinoma and 23 with benign diseases were enrolled in the trial.The mRNA and protein levels of E3 in muscle of the patients were investigated by real time quantitative PCR and Western Blot,and clinical significance of the results were discussed.The nutritional states of the two groups were analyzed,and the correlation between E3 expression and cancer cachexia was studied.
【Results】There were significant deteriorated nutritional states in patients with carcinoma before operation.The expressions of Atrogin-1mRNA,MuRF1mRNA were significantly increased in muscle of patients in the carcinoma group(P＜0.05),compared with patients with benign diseases,and protein level of Atrogin-1 was also significantly increased.
【Conclusion】Upregulation of expression of E3 was c]osely associated with malnutrition in patients with carcinoma,and it was important in the mechanism of cancer cachexia.
PARTⅡStudy of muscle-specific Ubiquitin-protein ligases in a mouse model with cancer cachexia
【Objective】To investigate expression levels of muscle-specific E3 and other components of ATP-ubiquitin-proteasome pathway in muscle of a mouse model with cancer cachexia,and to study the possible mechanism of E3 in cancer cachexia.
【Methods】BALB/c mice were inoculated subcutaneously with the murine colon-26 adenocarcinoma cell to establish the model of cancer cachexia, and the control group were established.Body weight,food intake and tumor volume were measured daily after inoculation.The mRNA and protein levels of E3(Atrogin-1 and MuRF1) were investigated by real time quantitative PCR,western blot and immunohistochemical technique after day 16 following inoculation,and expression levels of ubiquitin mRNA and E2_(14K) mRNA were also investigated.
【Results】Significant sympotoms of CC were seen in the tumor group.The difference in food intake between two groups was not significant (P＞0.05),whereas the non-tumor body weights and gastrocnemius weights in the tumor group were lost significantly as compared with the control group (P＜0.05).Compared with the control group,the expression levels of E3 (Atrogin-1 and MuRF1) mRNA,ubiquitin mRNA and E2_(14K) mRNA were significantly increased(P＜0.01) in gastrocnemius of the tumor group, and protein level of Atrogin-1 and MuRF1 were also significantly increased.
【Conclusion】The expression levels of E3 and several other components of ATP-ubiquitin-proteasome pathway were significantly increased in muscle of mice inoculated with colon-26 adenocarcinoma cell,and E3 was importmant in mechanism cancer cachexia.
PARTⅢStudy of protective effect of small interfering RNA-induced Atrogin-1 gene silencing on muscle cell malnutrition
【Objective】To construct malnutrition model of muscle cell induced by the cytokine tumor necrosis factor-a(TNF-a),on which we study the protective effect of Atrogin-1 gene silencing,in order to find out whether Atrogin-1 is the ideal target of treatment of malnutrition.
【Methods】C2C12 cell was cultured in growth medium,and differentiation was initiated by replacing the growth medium with differentiation medium to form myotubes.Differentiation was allowed to continue for 96 h before TNF-a was added to induce malnutrition.Then expressed product of Atrogin-1 of myotubes was identified by real time quantitative PCR and Western blot.Five pairs of target siRNA sequences and the control sequences were selected and synthesized according to mouse Atrogin-1 mRNA sequence,after annealing,these double strands were cloned to vector pBS-hU6-I and then to the lentiviral vector FG12.Its length was certificated by digestion with restriction endonuclease and the rightness of the sequence was confirmed by sequecing.The recombinant FG12 vector was cotransfected along with pRSVREV,pMDLg/pRRE and pHCMV-G into HEK293T cells to package lentivirus particles,and viral supernatant was harvested.Then infect C2C12 cells with recombinant lentivirus,and identify the expressed product by real time quantitative PCR and Western blot as before.Myotubes of each group were observed and photographed directly in culture plates without fixation at different time points.
【Results】Significant atrophy of myotubes treated by TNF-a were seen, and the expression levels of Atrogin-1 were upregulated,as compared with the normally cultured group.The results of digestion confirmed the right length of inserted DNA,and the sequencing result was identical with the reported.Compared with the control group,real time quantitative PCR and western blot proved decreased expression of Atrogin-1 in myotubes of the RNAi group,and atrophy of myotubes was not significant after treatment of TNF- a
【Conclusion】Malnutrition model of muscle cell could be constructed by treatment of TNF- a.And a lentivirus RNAi vector containing siRNA of Atrogin-1 gene could be used to protect malnutrition(atrophy) of muscle cells from adverse effect of TNF- a.Atrogin-1 may be the right target of treatment of malnutrition.
引文
[1]Ma G,Alexandar HR.Prevalence and pat.hophysiology of cancer cachexia.In:Bruera E,Portenoy RK,eds:topics in palliative care,vol2[M].New York:Oxford University Press,1998.91-129.
[2]Maltoni M,Nanni O,Pirovano M,et al.Successful validation of the palliative prognostic score in terminally ill cancer patients.Italian Multicenter Study Group on Palliative Care[J].J Pain Symptom Manage,1999,17(4):240-247.
[3]Costelli P,Baccino FM.Cancer cachexia:from experimental models to patient management[J].Curr Opin Clin Nutrition Metab,2000,3(3):177-181.
[4]DeWys W.Management of cancer cachexia[J].Semin Oncol,1985,12(4):452-460.
[5]Anker SD,Coats AJ.Cardiac cachexia:A syndrome with impaired survival and immune and neuroendocrine activation[J].Chest,1999,115(3):836-847.
[6]Argile's JM,Costelli P,Carbo" N,et al.Tumour growth and nitrogen metabolism in the host[J].Int J Oncol,1999,14(3):479-486.
[7]Bossola M,Muscaritoli M,Costelli P,et al.Increased Muscle Proteasome Activity Correlates With Disease Severity in Gastric Cancer Patients[J].Ann Surg,2003,237(3):384-389
[8]Williams A,Sun X,Fischer JE,eta].The expression of genes in the ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer[J].Surgery,1999,126(4):744-749.
[9]Costelli P,Muscaritoli M,Bossola M,et al.IGF-1 is downregulated in experimental cancer cachexia[J].Am J Physiol Regul Integr Comp Physiol,2006,291(3):R674-R683.
[10]Fujita J,rsujinakar,YanoM,et al.Anti-interleukin-6 receptor antibody prevents muscle atrophy in co]on-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways[J].Int J Cancer,1996,68(5):637-643.
[11]Gomes MD,Lecker SH,Jagoe RT,et al.Atrogin-1,a muscle-specific F-box protein highly expressed during muscle atrophy[J].Proc Natl Acad Sci U S A,2001,98(25):14440-14445.
[12]Kwak KS,Zhou X,Solomon V,et al.Regulation of Protein Catabolism by Muscle-Specific and Cytokine-Inducible Ubiquitin Ligase E3 a-Ⅱduring Cancer Cachexia[J].Cancer Res,2004,64(22):8193-8198.
[13]Bodine SC,Latres E,Baumhueter S,et al.Identification of ubiquitin ligases required for skeletal muscle atrophy[J].Science,2001,294(5547):1704 - 1708
[14]Li YP,Chen Y,John J,et al.TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atroginl/MAFbx in skeletal muscle[J].FASEB J,2005,19(3):362-370.
[15]Sacheck JM,Ohtsuka A,McLary SC,et al.IGF-1 stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin-ligases,atrogin-1 and MuRF1[J].Am J Physiol Endocrinol Metab,2004,287(4):E591 - E601.
[16]Sandri M,Sandri C,Gilbert A,et al.Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy[J].Cell,2004,117(3):399-412.
[17]Wong ML,Medrano JF.Real-time PCR for mRNA quantitation[J].Biotechniques,2005,39(1):75-85.
[18]Attwood J,Richardson B.Relative quantitation of DNA methyltransferase mRNA by real-time RT-PCR assay[J].Methods Mol Biol,2004,287:273-283.
[19]Giulietti A,Overbergh L,Valckx D,et al.An overview of real-time quantitative PCR:applications to quantify cytokine gene expression[J].Methods,2001,25(4):386-401.
[20]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods,2001,25(4):402-408.
[21]Tessitore L,Costelli P,Baccino FM.Humoral mediation for cachexia in tumour-bearing rats[J].Br J Cancer,1993,67(1):15-23
[22]Muscaritoli M,MeguidMM,Beverly JL,et al.Mechanism of early tumor anorexia[J].J Surg Res,1996,60(2):389-397
[23]Bossola M,Muscaritoli M,Costelli P,et al.lncreased muscle ubiquitin mRNA levels in gastric cancer patients[J]. Am J Physiol Regul Integr Comp Physiol, 2001, 280(5):R1518-1523.
[24] Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell 1ife[J]. EMBO J, 1998, 17 (24) :7151 - 7160.
[25] Baracos VE , DeVivo C , Hoyle DH, et al . Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma[J]. Am J Physiol, 1995, 268(5Pt1): E996-E1006.
[26] Lorite MJ , Thompson MG, Drake JL, et al . Mechanism of muscle protein degradation induced by a cancer cachectic factor[J].Br J Cancer, 1998, 78(7): 850-856
[27] Stitt TN, Drujan D, Clarke BA, et al. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors[J].Mol Cell, 2004, 14(3) :395-403.
[28] Lecker SH, Jagoe RT, Gilbert A, et al. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[J].FASEB J, 2004, 18(1):39-51
[29] Jagoe RT, Lecker SH, Gomes M, et al. Patterns of gene expression in atrophying skeletal muscles: response to food deprivation[J]. FASEB J, 2002, 16(13):1697-1712.
[30] Tisdale MJ. Loss of skeletal muscle in cancer: biochemical mechanisms[J]. Front Biosci, 2001, 6:D164-D174.
[31] Hasselgren PO, Fischer JE. Muscle cachexia : current concepts of in-tracellular mechanisms and molecular regulation[J].Ann Surg,2001,233(1):9-17.
[32] Darling G, Fraker DL .Jensen JC , et al . Cachectic effects of recombinant human tumor necrosis factor in rats[J]. Cancer Res,1990, 50(13):4008-4013.
[33] Nakashima J, Tachibana M, Ueno M, et al. Association between tumor necrosis factor in serum and cachexia in patients with prostate cancer[J].C1in Cancer Res, 1998, 4(7):1743-1748.
[34] Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene si lencing by RNAi in primary cells [J]. RNA, 2003, 9 (4) :493-501.
[35] Li YP, Schwartz RJ, Waddell ID, et al. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha[J]. FASEB J, 1998, 12(10): 871-880.
[36] Cao Y, Zhao Z, Gruszczynska-Biegala J, et al. Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro[J].Mol Cell Biol, 2003, 23(19): 6725-6738.
[37] Wang Y, Xu L, Duan H, et al. Knocking down type 2 but not type 1 calsequestrin reduces calcium sequestration and release in C2C12 skeletal muscle myotubes[J].J Biol Chem, 2006, 281(22):15572-15581.
[38] Nakashima J, Tachibana M, Ueno M, et al. Tumor necrosis factor and coagulapathy in patient with prostate cancer [J]. Cancer Res, 1995, 55(21): 4881 - 4885
[39] Rando TA, Blau HM. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy [J]. J Cell Biol, 1994, 125(6):1275-1287.
[40] Mayer NJ, Rubin SA. Molecular and cellular prospects for repair, augmentation, and replacement of the failing heart[J]. Am Heart J, 1997, 134(3): 577-586.
[41] Torrente Y, D'Angelo MG, Del Bo R, et al. Extracorporeal circulation as a new experimental pathway for myoblast implantation in mdx mice[J]. Cell Transplant, 1999, 8(3):247-258.
[42] Llovera M, Carbo N , Garcia2Martinez C et al . Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats[J].Biochem Biophys Res Commun, 1996, 221(3):653-655.
[43] Llovera M, Garcia-Martinez C , Lopez-Soriano J et al.Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice[J].Mol Cell Endocrinol, 1998, 142(1-2):183-189.
[44] Guttridge DC, Mayo MW, Madrid LV, et al. NF kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia[J]. Science, 2000, 289: 2363-2366
[45] Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs interference in cultured mammalian cells[J]. Nature, 2001, 441(6836):494-498.
[46] Fire A. RNA - triggered gene silencing[J]. Trends Genet, 1999, 15(9): 358-363.
[47] Catalano C , Azzalin G, Macino G, et al. Gene silencing in worms and fungi [J].Nature, 2000, 404(6775):245.
[48] Sharp PA , Zamore PD. Molecular biology. RNA interfrence[J]. Science, 2000, 287(5462): 2431-2433.
[49] Sharp PA. RNA interfrence-2001[J]. Genes Dev, 2001, 15(5):485-490.
[50] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391 (6669):806-811.
[51] Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotech, 2002, 20 (5): 497-500
[52] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296 (5567): 550-553
[53] Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus mediated RNA interference[J]. Cancer Cell, 2002, 2(3) :243-247
[54] Devroe E, Silver PA. Retrovirus-delivered siRNA[J].BMC Biotechnol, 2002,2: 15
[55] Salvatori G, Ferrari G, Mezzogiorno A , et al. Retroviral vector mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo[J]. Hum Gene Ther, 1993,4(6):713-723
[56] Huard J , Acsadi G, Jani A , et al. Gene transfer into skeletal muscles by isogenic myoblasts[J]. Hum Gene Ther, 1994,5(8):949-958
[57] van Deutekom JC , Floyd SS , Booth DK, et al. Implications of maturation for viral gene delivery to skeletal muscle[J]. Neuromuscul Disord,1998, 8(3 -4) :135-148.
[58] Costantini LC, Bakowska JC, Breake XO, et al. Gene therapy in the CNS[J].Gene Ther, 2000, 7(2): 93-109.
[59] Burns JC, Friedmann T, Driever W, et al. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and effcient gene transfer into mammalian and nonmammalian cells[J]. Proc Natl Acad Sci U S A. 1993 September 1; 90(17): 8033-8037.
[60]Zhu Y,Feuer G,Day S L,et al.Multigene]entivirus vectors based on differentia]splicing and translational control[J].Mol Ther,2001,4(4):375-382.
[61]Iwakuma T,Cui Y,Changh.Self-inactivating lentiviral vectorswith U3 and U5 modil cations[J].J Virol,1999,261(1):120-132.
[62]Cui Y,Iwakuma T,Chang LJ.Contributions of viral splice sites and cis-regulatory elements to lentivirus vector function[J].]Virol,1999,73(7):6171-6176.
[63]Nishitsuji H,Ikeda T,Miyoshi H,et al.Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells[J].Microbes Infect,2004,6(1):76-85
[64]Li M,Rossi JJ.Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells[J].Methods Mol Biol,2005,309:261-272
[1] Anker SD , Coats AJ. Cardiac cachexia: A syndrome with impaired survival and immune and neuroendocrine activation[J]. Chest, 1999, 115 (3) :836-847.
[2]Inui A. Cancer anorexia-cachexia syndrome:current issues in research and management[J].CA Cancer J Clin, 2002, 52(2):72-91
[3] Ma G, Alexandar HR. Prevalence and pathophysiology of cancer cachexia. In : Bruera E , Portenoy RK, eds : topics in palliative care , vol2[M] . New York :0xford University Press ,1998. 91-129.
[4] Maltoni M, Nanni O, Pirovano M, et al. Successful validation of the palliative prognostic score in terminally ill cancer patients. Italian Multicenter Study Group on Palliative Care[J].J Pain Symptom Manage, 1999, 17 (4) :240-247
[5] Tisdale MJ. Cachexia in cancer patients[J]. Nat Rev Cancer, 2002, 2(11): 862-871
[6] Lecker SH, Jagoe RT, Gilbert A, et al. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[J].FASEB J, 2004, 18(1):39-51
[7] Langhans W. Peripheral mechanisms involved with catabolism [J].Curr Opin Clin Nutr Metab Care, 2002,5(4):419-426.
[8] Thompson MG, Palmer RM. Signalling pathways regulating protein turnover in skeletal muscle[J]. Cell Signal, 1998,10(1):1-11
[9] Tisdale MJ . Cancer anorexia and cachexia [J]. Nutrition, 2001, 17(5):438-442.
[10] Williams AB, Decourten-Myers GM, Fischer JE, et al. Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism[J].FASEB J, 1999, 13(11) :1435 - 1443.
[11] Wray CJ, Mammen JM, Hershko DD, et al. Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle[J]. Int J Biochem Cell Biol, 2003, 35(5):698-705.
[12] Llovera M, Carbo N, Lopez-Soriano J, et al. Different cytokines modulate ubiquitin gene expression in rat skeletal muscle[J]. Cancer Lett, 1998, 133(1) :83-87.
[13] Bodine SC, Latres E, Baumhueter S, et al. Identification of ubiquitin ligases required for skeletal muscle atrophy[J]. Science, 2001, 294 (5547):1704-1708.
[14]Sacheck JM,Hyatt JP,Raffaello A,et al.Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases[J].FASEB J,2007,21(1):140-155
[15]Lord JM,Davey J,Frigerio L,et al.Endoplasmic reticulum-associated protein degradation[J].Semin Cell Dev Biol,2000,11(3):159-164
[16]Plemper RK,Wolf DH.Retrograde protein translocation:ERADication of secretory proteins in health and disease[J].Trends Biochem Sci,1999,24(7):266-270
[17]Strous GJ,Govers R.The ubiquitin-proteasome system and endocytosis[J].J Cell Sci,1999,112(Pt 10):1417-1423
[18]Hicke L.Gettin'down with ubiquitin:turning off cell-surface receptors,transporters and channels[J].Trends Cell Biol,1999,9(3):107-112
[19]Laney JD,Hochstrasser M.Substrate targeting in the ubiquitin system[J].Cell,1999,97(4):427-430
[20]Koepp DM,Harper JW,Elledge SJ.How the cyclin became a cyclin:regulated proteolysis in the cell cycle[J].Cell,1999,97(4):431-434
[21]Varshavsky A.The ubiquitin system[J].Trends Biochem Sci,1997,22(10):383-387.
[22]Ciechanover A.The ubiquitin-proteasome pathway:on protein death and cell life[J].EMBO J,1998,17(24):7151-7160.
[23]Kwak KS,Zhou X,Solomon V,et al.Regulation of Protein Catabolism by Muscle-Specific and Cytokine-Inducible Ubiquitin Ligase E3 a-Ⅱduring Cancer Cachexia[J].Cancer Res,2004,64(22):8193-8198
[24]Gomes MD,Lecker SH,Jagoe RT,et al.Atrogin-1,a muscle-specific F-box protein highly expressed during muscle atrophy[J].Proc Natl Acad Sci U S A,2001,98(25):14440-14445.
[25]Tintignac LA,Lagirand J,Batonnet S,et al.Degradation of MyoD mediated by the SCF(MAFbx) ubiquitin ligase[J].J Biol Chem,.2005,280(4):2847-2856.
[26]Skowyra D,Craig KL,Tyers M,et al.F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex[J].Cell,1997,91(2):209-219
[27]Jackson PK,Eldridge AG.The SCF ubiquitin]igase:an extended look[J].Mol Ce11,2002,9(5):923-925.
[28]Li HH,Kedar V,Zhang C,et al.Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex[J].J Clin Invest,2004,114(8):1058-1071.
[29]Baracos rE.Regulation of skeletal-muscle-protein turnover in cancer-associated cachexia[J].Nutrition,2000,16(10):1015-1018.
[30]Baracos VE,DeVivo C,Hoyle DH,et al.Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma[J].Am J Physiol,1995,268(SPtl):E996-E1006.
[31]Temparis S,Asensi M,raillandier Det al.Increased ATP-ubiquitin-dependent proteolysis in skeletal muscles of tumor-bearing rats[J].Cancer Res,1994,54(21):5568-5573.
[32]Strelkov AB,Fields AL,Baracos VE.Effect ofsystemic inhibition of prostaglandin production on protein metabolism in tumor-bearing rats[J].Am J Physiol,1989,257(2 Pt 1):C261-C269
[33]Llovera M,Garcia-Martinez C,Agell Net al.Muscle wasting associated with cancer cachexia is linked to an important activation of the ATP-dependent ubiquitin-mediated proteolysis[J].Int J Cancer,1995,61(1):138-141.
[34]Costelli P,Muscaritoli M,Bossola M,et al.IGF-1 is downregulated in experimental cancer cachexia[J].Am J Physiol Regul Integr Comp Physiol,2006,291(3):R674-R683
[35]Fujita J,Tsujinaka T,Yano M,et al.Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin2dependent proteolytic pathways[J].Int J Cancer,1996,68(5):637-643.
[36]Mackenzie ML,Bedard N,Wing SS,et al.A proinflammatory tumor that activates protein degradation sensitizes rats to catabolic effects of endotoxin[J].Am J Physiol Endocrinol Metab,2005,289(4):E527-533
[37]Lorite MJ,Thompson MG,Drake JL,et al.Mechanism of muscle protein degradation induced by a cancer cachectic factor[J].Br J Cancer,1998,78(7):850-856.
[38]Lorite MJ,Smith HJ,Arnold JA,et al.Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor(PIF)[J].Br J Cancer,2001,85(2):297-302.
[39]Williams A,Sun X,Fischer JE et al.The expression of genes in the Ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer[J].Surgery,1999,126(4):744-749.
[40]Bossola M,Muscaritoli M,Costelli P et al.Increased muscle ubiquitin mRNA levels in gastric cancer patients[J].Am J Physiol Regul Integr Comp Physiol,2001,280(5):R1518-R1523.
[41]Tisdale MJ.Loss of skeletal muscle in cancer:biochemical mechanisms[J].Front Biosci,2001,6:n164-D174.
[42]Hasselgren PO,Fischer JE.Muscle cachexia:current concepts of intracellular mechanisms and molecular regulation[J].Ann Surg,2001,233(1):9-17.
[43]Zoico E,Roubenoff R.The role of cytokines in regulating protein metabolism and muscle function[J].Nutr Rev,2002,60(2):39-51
[44]Mitch WE,Goldberg AL.Mechanisms of muscle wasting.The role of the ubiquitin-proteasome pathway[J].N Engl J Med.1996 Dec 19;335(25):1897-1905
[45]Costelli P,Bossola M,Muscaritoli M,et al.Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin- and Ca(2+)-dependent proteolytic systems in the muscle of tumour-bearing rats[J].Cytokine,2002,19(1):1-5
[46]Li YP,Chen Y,John J,et al.TNF-α acts via p38 MAPK to stimulate expression of the ubiquitin ligase atroginl/MAFbx in skeletal muscle[J].FASEB J,2005,19(3):362- 370
[47]Llovera M,Carbo N,Garcia-Martinez C,et al.Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats[J].Biochem Biophys Res Commun,1996,221(3):653-655.
[48]Llovera M,Garcia-Martinez C,Lopez-Soriano J,et al.Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice[J].Mol Cell Endocrinol,1998,142(1-2):183-189.
[49]Li YP,Lecker SH,Chen Y,et al.TNF- a increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E2_(20k)[J].FASEB J,2003, 17(9):1048 -1057
[50]Du J,Mitch WE,Wang X,et al.G]ucocorticoids induce proteasome C3subunit expression in L6 muscle cells by opposing the suppression of its transcription by NF-kB[J].J Biol Chem,2000,2?5(26):19661 - 19666.
[51]Cai D,Frantz JD,Tawa NE Jr,eL al.IKKbNF-kB activation causes severe muscle wasting in mice[J].Cel],2004,119(2):285-298.
[52]Wyke SM,Russell ST,Tisdale MJ.Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-kappaB activation[J].Br J Cancer,2004,91(9):1742-1750
[53]Hunter RB,Kandarian SC.Disruption of either the Nfkbl or the Bc13gene inhibits skeletal muscle atrophy[J].J Clin Invest 2004,114(10):1504-1511.
[54]Yaron A,Hatzubai A,Davis M,et al.Identification of the receptor component of the IkappaBalpha-ubiquitin ligase[J].Nature,1998,396(6711):590-594
[55]Judge AR,Koncarevic A,Hunter RB,et al.Role for IkappaBalpha,but not c-Rel,in skeletal muscle atrophy[J].Am J Physiol Cell Physiol,2007,292(1):C372-C382.
[56]Murgia M,Serrano At,Calabria E,et al[J].Ras is involved in nerve-activity-dependent regulation of muscle genes[J].Nat Cell Biol,2000,2(3):142-147.
[57]Franch HA,Raissi S,Wang X,et al.Acidosis impairs insulin receptor substrate-l-associated phosphoinositide 3-kinase signaling in muscle cells:consequences on proteolysis[J].Am J Physiol Renal Physiol,2004,287(4):FTO0- F706.
[58]Li BG,Hasselgren PO,Fang CH,et al.Insu]in-like growth factor-Ⅰblocks dexamethasone-induced protein degradation in cultured myotubes by inhibiting multiple proteolytic pathways:2002 ABA paper[J].J Burn Care Rehabil,2004,25(1):112-118.
[59]Léger B,Cartoni R,Praz M,et al.Akt signalling through GSK-3beta,mTOR and Foxol is involved in human skeletal muscle hypertrophy and atrophy[J].J Physiol,2006,576(Pt 3):923-933.
[60]Sacheck JM,Ohtsuka A,McLary SC,et aI.IGF-I stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin-ligases,atrogin-1 and MuRF1[J].Am J Physiol Endocrinol Metab,2004,287(4):E591-E601.
[61]Mitch WE,Bailey JL,Wang X,et al.Evaluation of signals activating ubiquitin- proteasome proteolysis in a model of muscle wasting[J].Am J Physiol,1999,276(5 Pt 1):C1132-C1138.
[62]Dehoux M,Van Beneden R,Pasko N,et al.Role of IGF-1 decline in the induction of atrogin-1/MAFbx during fasting and diabetes[J].Endocrinology,2004,145(11):4806-4812.
[63]Lee SW,Dai G,Hu Z,et al.Regulation of muscle protein degradation:coordinated control of apoptotic and ubiquitin- proteasome systems by phosphatidylinositol 3 kinase[J].J Am Soc Nephrol,2004,15(6):1537-1545.
[64]Stitt TN,Drujan D,Clarke BA,et al.The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors[J].Mol Cell,2004,14(3):395 - 403.
[65]Sandri M,Sandri C,Gilbert A,et al.Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy[J].Cell,2004,117(3):399-412.
[66]Latres E,Amini AR,Amini AA,et al.Insulin-like Growth Factor-1(IGF-1) Inversely Regulates Atrophy-induced Genes via the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Pathway[J].J Biol Chem,2005,280(4):2737-2744
[67]Fang CH,Li BG,Sun X,et al.Insulin-like growth factor Ⅰ reduces ubiquitin and ubiquitin-conjugating enzyme gene expression but does not inhibit muscle proteolysis in septic rats[J].Endocrinology,2000,141(8):2743- 2751
[68]Du,J.,Wang,X.,Miereles,C.,et al.Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions[J].J Clin Invest,2004,113(1):115-123
[69]McFarlane C,Plummer E,Thomas M,et al.Myostatin Induces Cachexia by Activating the Ubiquitin Proteolytic System Through an NF-kB-Independent,FoxO1-Dependent Mechanism[J].J Cell Physiol,2006,209(2):501-514
[70]Barber MD,Ross JA,Fearon KCH.Disordered metabolic response with cancer and its management[J]. World J Surg, 2000, 24 (6) :681-689.
[71] Kotler DP. Cachexia[J]. Ann Intern Med, 2000, 133(8):622-634
[72] Klein S , Simes J , Blackburn GL. Total parenteral nutrition and cancer clinical trials[J]. Cancer, 1986, 58(6):1378-1386.
[73] Mantovani G, Madeddu C, Maccio A, et al. Cancer-related anorexia/cachexia syndrome and oxidative stress:an innovative approach beyond current treatment[J]. Cancer Epidemiol Biomarkers Prev, 2004, 13(10):1651-1659
[74] Carbo N , Lopez-Soriano J , Costelli P, et al . Interleukin-15 antagonizes muscle protein waste in tumour-bearing rats[J].Br J Cance, 2000, 83(4):526-531.
[75] Combaret L , Ralliere C , Taillandier D, et al. Manipulation of the ubiquitin-proteasome pathway in cachexia : pentoxifylline suppresses theactivation of 20S and 26S proteasomes in muscles from tumor-bearingrats [J].Mol Biol Rep ,1999 , 26(1-2):95-101.
[76] Costelli P, Garcia-Martinez C, Llovera M, et al. Muscle protein wastein tumor-bearing rats is effectively antagonized by a beta 2-adrenergicagonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway[J].J Clin Invest,1995, 95(5):2367-2372.
[77] Busquets S, Figueras MT, Fuster G, et al. Anticachectic Effects of Formoterol: A Drug for Potential Treatment of Muscle Wasting[J].Cancer Res, 2004, 64(18):6725 - 6731
[78] Hitt A, Graves E, McCarthy DO. Indomethacin preserves muscle mass and reduces levels of E3 ligases and TNF receptor type 1 in the gastrocnemius muscle of tumor-bearing mice[J]. Res Nurs Health, 2005, 28(1): 56-66
[79] McCarthy DO, Whitney P, Hitt A, et al. Indomethacin and ibuprofen preserve gastrocnemius muscle mass in mice bearing the colon-26 adenocarcinoma. Res Nurs Health[J]. 2004, 27(3):174-184
[80] Smith HJ , Lorite MJ , Tisdale MJ. Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2C12 myoblasts: modulation by eicosapentaenoic acid[J]. Cancer Res, 1999, 59(21):5507-5513.
[81] Whitehouse AS , Smith HJ , Drake JL, et al . Mechanism of attenuation of skeletal muscle protein catabolism in cancer cachexia by eicosapentaenoic acid[J].Cancer Res,2001,61(9):3604-3609.
[82]Smith Hi,Mukerji P,Tisdale MJ.Attenuation of Proteasome-Induced Proteolysis in Skeletal Muscle by B-nydroxy-B-Methylbutyrate in Cancer-Induced Muscle Loss[J].Cancer Res,2005,65(1):277-283
[83]Smith HJ,Khal J,Tisdale MJ.Downregulation of ubiquitin-dependent protein degradation in murine myotubes during hyperthermia by eicosapentaenoic acid[J].Biochem Biophys Res Commun,2005,332(1):83-88.
[84]May PE,Barber A,D' Olimpio JT,et al.Reversal of cancer-related wasting using oral supplementation with a combination of beta-hydroxy-beta-methylbutyrate,arginine,and glutamine[J].Am J Surg,2002,183(4):471-479
[85]nasselgren PO,Fischer JE.The ubiquitin-proteasome pathway:review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions[J].Ann Surg,1997,225(3):307-316