Muscle-specific Ubiquitin-protein Ligase Is Important In Cancer Cachexia: Based On Basic And Clinincal Studies

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.