RNAi-mediated Gene Downregulation Of Proteasome Subunit RC2 And Ubiquitin Ligase MAFbx Can Ameliorate Rat Denervated Skeletal Muscle Atrophy

It is a challenging medical problem to delay intrinsic muscle atrophy of hand resulted from peripheral motor nerve injury. Function loss of hand intrinsic muscle means the core function loss of upper extremity, which brings a lot of burden of mind and economy for patients, their family, and society. So, strengthen of basis research and exploration of the nature of denervation muscle atrophy have considerable theoretical and practical importance.Evidences from many experiments that denervated skeletal muscle fibers undergo rapid muscle atrophy within the first 2 weeks after denervation have shown that muscle wasting occurs mainly through an acceleration of proteolysis, and that the increase in protein degradation is largely caused by upregulated expression and activity of the ubiquitin-proteasome pathway. It has been confirmed that muscle protein breakdown can be reduced by treatment with a proteasome inhibitor in vivo during sepsis and denervation. RNA interference (RNAi) is the process of a sequence-specific post-transcriptional gene-silencing induced by double-stranded RNA (dsRNA) homologous to the silenced gene. The use of RNAi for gene therapies is widely studied, especially in viral infections, cancers, and other disorders. We assume that inhibiting increased activity of the ubiquitin-proteasome pathway by RNAi might ameliorate denervated skeletal muscle atrophy. So, we select two genes encoding proteasome subunit RC2 and ubiquitin ligase MAFbx from ubiquitin-proteasome pathway, which have close relations with protein catabolism and skeletal muscle atrophy from previous reports, and explore the effect on preventing rat denervated skeletal muscle atrophy by RNAi. The aim of the study was to find a new method to delay denervated skeletal muscle atrophy.Part Ⅰ Expression Pattern and Significance of Proteasome Subunit RC2 and Ubiquitin Ligase MAFbx in Rat Denervated Skeletal MuscleObjective To observe expression of proteasome subunit RC2 and ubiquitin ligase MAFbx in rat denervated skeletal muscle, and investigate the molecular mechanism of skeletal muscle wasting. Methods A model of denervated gastrocnemius at the right lower limb was established in 48 rats. They were randomly assigned to eight groups with 6 in each. The control group was zero day after postoperation, the experiment group were one day, and 2, 3, 4, 5, 6, 14 days after postoperation. The subsequent changes in RC2 and MAFbx mRNA levels were determined by real-time quantitative RT-PCR and RC2 protein levels by Western blot.Results Expression of RC2 mRNA in rat denervated gastrocnemius was upregulated gradually, peaked in three days, then decreased (respectively, 4.98, 0.42-fold at 3, 14 days; P<0.05). The changes of protein levels were similar to that of mRNA levels, but amplitude of changes was lower (respectively, 2.70, 0.35-fold at 3, 14 days; P<0.05). The level of MAFbx mRNA was clearly increased after denervation, reached a maximum level by 2 days, remained at a high level during one week, then decreased gradually (respectively, 15.3, 8.7, 3.7-fold at 2, 6, 14 days; P<0.05). Amplitude of changes of MAFbx mRNA was higher than that of RC2 mRNA.Conclusions After denervation, gene expression of RC2 and MAFbx was upregulated, especially MAFbx, which support the concept that denervated-induced muscle atrophy is mainly caused by the ubiquitin-proteasome proteolytic pathway. Inhibiting upregulated gene expression of RC2 and MAFbx by RNAi should begain early after denervation. Part Ⅱ Cloning and Sequence Analyzing of theRecombinant Plasmid of Proteasome Subunit RC2 Gene and Ubiquitin Ligase MAFbx Gene for RNA InterferenceObjective To Clone the recombinant plasmid affecting gene RC2 andMAFbx translation by RNAi and analyze the nucleic acid sequence of therecombinant for further searching new gene therapy method of atrophy ofdenervated skeletal muscle.Methods Three pairs of DNA template coding siRNA of RC2 andMAFbx gene, respectively, were designed and synthesized. They werenamed RC2- Ⅰ , RC2-Ⅱ, RC2-Ⅲ and MAFbx-Ⅰ , MAFbx-Ⅱ, MAFbx-Ⅲ. The complement form was obtained by annealing and inserted intovector pSilencer2.0-U6, and the recombinant plasmid were transformedinto Topo 10 strain. Finally, the recombinant plasmid identified byrestriction enzyme was used for sequence analysis.Results The plasmid expressing siRNA was successfully construstedand the aim sequence was obtained.Conclusions The plasmid expressing siRNA of RC2 and MAFbx genewas successfully cloned, which are informative for further study in vitro.Part Ⅲ Downregulation of Proteasome Subunit RC2Gene and Ubiquitin Ligase MAFbx Geneby RNA Interference in VitroObjective To evaluate inhibitory effect of RC2 and MAFbx in L6 cells by RNAi, which would be further explored as a new way for intervention of skeletal muscle atrophy of denervation.Methods L6 rat myoblasts was used as target cells. The plasmid pEGFP-N1 and the plasmid expressing siRNA were cotransfected into the cultured cells by transfection reagent lipofectamine 2000, through which transfection efficiency was checked. Then, the plasmid expressing siRNA were transfected. 48 hours and 72 hours after transfection, changes inRC2 and MAFbx mRNA levels and RC2 protein levels were determined by real-time quantitative PCR and Western blot, respectively.Results The plasmids of siRNA RC2-Ⅰ , RC2-Ⅱ and RC2-Ⅲ have no inhibitory effect on RC2 expression in mRNA and protein levels in L6 rat myoblasts at 48 h (P>0.05). 72 h after transfection, the plasmid of siRNA RC2- Ⅰ could specifically inhibit RC2 expression, leading to 75 % and 68 % reduction, respectively (P<0.05), But the other two, RC2-Ⅱ and RC2-Ⅲ, have no inhibitory effect (P>0.05). The plasmids of siRNA MAFbx-Ⅰ,MAFbx-Ⅱ, MAFbx-Ⅲ could specifically inhibit MAFbx expression in mRNA levels, leading to 22%, 47%, 25% reduction at 48 h (P<0.05) and 55%, 81%, 47% at 72 h (P<0.05), respectively.Conclusions Gene expression of RC2 and MAFbx can be substantially inhibited in vitro by plasmid containing siRNA RC2- Ⅰ and MAFbx- Ⅱ, respectively, which are informative for further study in vivo. Not all siRNAs are equally effective.Part Ⅳ RNAi-mediated Gene Downregulation of Proteasome Subunit RC2 and Ubiquitin Ligase MAFbx by Electroporation Ameliorate Rat Denervated Muscle AtrophyObjective To explore the treatment effect on delaying of rat denervated muscle atrophy by RNAi-mediated gene downregulation of proteasome subunit RC2 and ubiquitin ligase MAFbx by electroporation.Methods A plasmid encoding green florescent protein (GFP) was first used to test efficiency of electroporation and potimize parameters for electroporation in vivo. A model of denervated long extensor muscle of toes at the right lower limb was established in 18 rats. They were randomly divided into two groups: Electroporation group (group EP) and Non-electroporation group (group NEP) with 9 in each. Plasmid pEGFP-N1 (50μl, 0.8μg/μl) solution was injected using a 27-gauge needle in a proximal to distal direction inside long extensor muscle oftoes after operation. Then, two implanted stainless steel needles were used as electrodes and connected to the generator and an electrical field was applied to the muscle. A voltage of 200 V per centimetre was applied in 10 100μs square wave pulses at 1 Hz using a BTX ECM 830 electroporator. Samples were collected 1 week, 2, 3 weeks after the injection of plasmid DNA. The changes of GFP protein levels after 1 week were determined by Western blot. A model of denervated long extensor muscle of toes at the right lower limb was established in 72 rats. They were randomly assigned to one denervated control group (group CON) and two gene therapy groups (group RC2 and group MAFbx) with 24 in each. The unoperated sides of the treated animals also served as controls. Six normal rats were treated as normal control group (group Normal). Three different siRNA plasmid solution containing RC2- Ⅰ, MAFbx- Ⅱ, CON (50μl , 0.8μg/μl)was injected and transfected by electroporation as methods mentioned above, respectively. The changes of RC2 and MAFbx mRNA levels and RC2 protein levels after 3 days were determined by real-time quantitative PCR and Western blot, respectively. On postoperative 2, 3 and 4 weeks, the rate of wet muscle weight preservation, mean diameter of muscle fiber and mean cross-section area of muscle fiber and muscle protein content were checked and then compared between group CON and group RC2 or group MAFbx, respectively. The differences between groups were analyzed by one-way ANOVA. Ultrastructural changes of muscle fiber were observed at 2, 3, 4 weeks postoperation.Results GFP plasmid was efficiently deliverd into muscle by electroporation and robust GFP expression in muscle could be observed more than three weeks. Histology shows that injected plasmid DNA diffuses extensively in muscle tissue. In contrast, in the absence of electric pulses, GFP expression was restricted to the vicinity of the path of the DNA-injecting needle. RC2 mRNA and protein levels of group RC2 in 3 days was significantly inhibited compared to that of group CON, 35% and 25%, respectively (P<0.05). MAFbx mRNA levels of group MAFbx in 3 days was significantly inhibited 33% compared to that of group CON (P<0.05). The rate of wet muscle weight preservation, meandiameter of muscle fiber and mean cross-section area of muscle fiber, muscle protein content were significantly greater in group RC2 than those of group CON on postoperative 2, 3 weeks (P<0.05), but on 4 weeks, there was no significant difference between two groups. In contrast, the parameters mentioned above were significantly greater in group MAFbx than those of group CON on postoperative 2, 3, 4 weeks (P<0.05). Ultrastructural changes of muscle fiber in group RC2 and group MAFbx was lighter than those of group CON on 3 weeks. Changes in group MAFbx were lighter than those in group CON on 4 weeks.Conclusions Electrotransfer drastically increases the efficiency of i.m. gene transfer, compared to only direct injection. Therefore, i.m. electrotransfer may be crucial in obtaining a therapeutic response. RNAi-mediated gene transfer by electrotransfer can partially inhibit gene expression of RC2 and MAFbx in vivo, through which can reduce muscle protein breakdown and ameliorate morphological degeneration of denervated muscle atrophy. Compared to RC2, MAFbx is likely to play a key role in the generation of denervated muscle atrophy.