The Study Of Denervation On The Expression Changes Of Muscle Specific Micrornas In Atrophied Skeletal Muscle Of Mice

Objective:to explore the expression changes of muscle special microRNAs(miR-1,miR-133,miR-206 et al )in the atrophied skeletal muscle resulted in by the denervation,and to establish the foundation to futher to study their molecular mechanisms on the denervated skeletal muscle and the significance on forensic identity source scienceMethod:Experimental animal:male healthy C57 mice weighing 20±1g of clean degree and of 12 weeks old were selected in this study. These mice were provided by the experimental animal center at Third Military Medical University.Grouping and model building:30 C57 mice were used and divided randomly into 2 groups:one of which was the normal control group without mice be disposed(n=6);the other group was the control group(n=24)whichwas divided averagely into 4 time points(n=6),mice of the group were operated on sterile condition after they were anesthetized by injecting 1% pentobarbital sodium at the standard of 40 mg/kg drug dose: cutting longitudinally the pars dorsalis of the right hindlimb of mice, Seperating bluntly musles between the biceps femoris and vastus lateralis,exposing the sciatic nerve, cutting approximate 5mm sciatic nerve at the position of the piriformis, lower edge 2mm, exposing freely the proximal end,suturing the distal end after be returned 180°in the sarolemma of both the biceps femoris and the gastrocnemius muscle ,and suturing in turn each layer tissue, which built the model of the atrophying gastrocnemius muscle of mice from the denervation. The sciatic nerve of the left hindlimb of mice wes not cut after being exposed.Detecting the percentage Of the relative wet weight of gastrocnemius muscle: Killing separately mice on 0d(the normal control group).3d 7d 14d and 28d after being operated,cuting and seperating in turn the skin and subcutaneous tissue of bilateral hindlimbs by ophthalmmic scissors, peeling carefully the sarolemma of gastrocnemius muscle,cutting and obtaining integral bilateral gastrocnemius muscle,balancing their wet weight immediately by the electronic analytical balance,computing the relative wet weight of muscle and its percentage,and finally drawing the changing curve of percentage of the relative wet weight of muscle.Observed by microscope:After the muscle which was used in Northern blot analysis was cut-out,the rest muscle was disposed by the following processes: fixed with 4% formaldehydum polymerisatum, paraffin-embedded, and making sections of 5μm thickness by selecting the serial Section of muscle belly of gastrocnemius muscle. The sections were dyed with HE and observed by microscope.Detecting the percentage of CSA of muscle fibers: We measured CSA of 50 muscle fibers in each section and automatically computered their average with HPIAS-1000 image analysis system.After the percentage of CSA was computered with the computer, the changing curve of percentage of .it was drawn.Northern blot analysis:Total RNA samples were extracted using RNAiso reagent(Takara). (concrete methods refering to the specification).The used probes were oligonucleotide DNA sequences of reverse complementary of mir-1,133,206 and U6( ( miR-1:5′-TACATACTTCTTTACATTCCA -3′;miR-133: 5′-CAGCTGGTTGAAGGGGACCAAA-3′;miR-206 : 5′-CCACACACTTCCTTACATTCCA -3′;U6 : 5′-ATATGGAACGCTTCAATT-3′)and were labeled by usingγ-32P-ATP endlabeled method. 20μg of total RNA samples were electrophoresed on denaturing 15% polyacrylamide gels and electroblotted for 1h onto Hybond-N+ membranes(Amersham) on the condition of constant-current(400mA). The membranes were UV-crosslinked on the condition of 1200μJ for 1min,baked on the condition of 80℃for 1h, hybridized withγ-32P-ATP endlabeled oligonucleotide DNA probes on the condition of 42℃for 24 h,and autoradiographied on the condition of -70℃for 48 h after being washed.Images were captured on film.and executed gray scale scanning by Quantity One. Grayscales of 3 moleculars in different time points were compareed with that of internal reference(U6) ( the result was expressed using 1 on 0 day of denervation, quantized value of each molecular in each time points was expressed using multiple of the former).Result:Differences of bilateral gastrocnemius muscle in normal control group:There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance(P﹤0.05).Effects of denervation on the percentage of relative wet weight and CSA of muscle fibers of gastrocnemius muscle:After the sciatic nerve had been cut for 3 days,the percentage of relative wet weight of gastrocnemius muscle decreased significantly compared with the control group, and the ratio decreased gradually with the time of denervation prolonging,which the atrlphic degree of gastrocnemius muscle of mice aggravated gradually with time of denervation prolonging(P﹤0.001).The same holds ture with effects of CSA of muscle fibers. The percentage of CSA of muscle fibers decreased also gradually with the time of denervation prolonging,and its decreased degree was more significant than that of relative wet weight of gastrocnemius muscle(P﹤0.001).Effects of denervation on morphology of gastrocnemius muscle:High power microscopic(200) ,pathological sections showed that cytoplasm of myocyte started to lose in the early stage of the denervation,the diameter decreased,and Nucleus showed signs of active metabolism(forexample, volume of Nucleus changed big, clear nucleolus, Nucleus lied in the center of cells,and so on),which could be the compensatory reaction of myocyte of denervation. With the time of denervation prolonging,the diameter and CSA of muscle fibers decreaded significantly, cytoplasm pyknosis obviously, the phenomenon of nuclear ingression was more and more widespread,nucleus increased, collagen fibers hyperplasiaed obviously, inflammatory cells invaded generally,and so on.Effects of denervation on expressions of muscle specific microRNAs:Compared with the normal control group,expressions of muscle specific microRNAs appeared a series of changes with the time of denervation prolonging. The expression of miR-206 was obviously up-regulated with the time denervation prolonging, and on the twenty-eighth days, its abundance was over 5 times higher than that of normal muscle; however, those of miR-1 and miR-133 decreased firstly and then gradually recovered with the time elapsing,and on the twenty-eighth days,their abundance are still lower than that of normal muscle.Conclusion:1.With the time denervation prolonging, the expressions of muscle specific microRNAs appeared significant changes,therefore,we speculate that they could play a role to a certain degree during the denervated atrophy of skeletal muscle,which offered new view to explore the mechanisms of skeletal muscle atrophy in the level of microRNAs.2.With the time denervation prolonging,the percentage of relative wet weight of gastrocnemius muscle and CSA of muscle fibers degreased significantly,which proved that the mouse model of sciatic nerve resection was successfully established;3.There were significant differences in wet weight and CSA of muscle fibers of bilateral gastrocnemius muscle in normal C57 mice.According to statistic analysis, the differences had statistical significance..