| Objective To determined the specific miRNAs in the spinal cord after transsection and find its targets in PC12,Hela cells.Methods:1.6 adult rats were randomly devided into sham operated group(n=3) and operated group(n=3).At 3 days post operation,RNA were extracted from the spinal cord and the liver tissues for miRNA array analysis;2.total miRNAs were hybridized with miRNA array chip,then the results obtained from spinal cord and liver were compared between that of the control and the experiment group to find the specific miRNA in injuried spinal cord;3.according to primary proteomics results, TargetScan was employed to predict the possible targets of miR-138;4.To authenticate those results,target proteins obtained from thoracic segments of spinal cord in innocent rats,were investigated with immunohistochemistry;5.PC12,Hela cells were devided into 3 groups,including nomal group,lipofectamin group(control group) and miR-138 group.At 24h,48h and 72h after transfection,the numbers of PC12,Hela cell in all groups were counted;6.at 24h,48h and 72h post transfection, MTT assay were performed in all groups to evaluate the cell viability and to calculate the inhibition ratio;7.expressional changes and intracellular locations of the target proteins were detected by using immunocytochemistry and Western Blot;8.RT-PCR were used to investigate the mRNA levels of targets;9.data analysis was performed using SPSS13.0. Results.1.miR-138 was specially reduced in the spinal cord of cord transected rats;2. there were 343 targets predicted with TargetScan;assocaited with the primary proteomics results,5 targets were determined in this study;they were synuclein, beta(Sncb);vimentin(Vim);tropomyosin 4(Tpm4);voltage-dependent anion channel 2(VDAC2) and neurotrophic tyrosine kinase,receptor,type 2(NTRK2,TrkB).3. Sncb,Vim,Tpm4,TrkB and VDAC2 were detected in thoracic segments of nomal spinal cord;4.compared with that of nomal group,the numbers of PC12 cells of lipofectamin group decreased at 48h after transfection,while compared with that of the lipofectamin group,cells of miR-138 group were reduced after 24h,48h and 72h post operation.Howerver,similar phenomenon was not observed in Hela cell. lipofectamin group cells decreased at 72h after transfection.There were no significant differences between miR-138 group and lipofectamin group.5.compared with that of the nomal group,OD values of lipofectamin group decreased at 48h and 72h in PC12 cell,while at 24h,48h in Hela cell.At 72h post operation,compared with that of the control group,cell viabilities were inhibited in miR-138 group both in PC 12 and Hela cell.However,at 24h and 48h post operation,viabilities of Hela cell were promoted. The hibition ration of PC12 were -6.29275%at 24h,22.62443%at 48h and 22.11101%at 72h post cord transcection,for Hela cell they were -15.6846%, -17.299%and 7.813765%;6.Analyzed with immunocytochemistry,Tpm4 and TrkB were detected in both cell,while Vim were limited in Hela cell.Scnb and VDAC2 were not found in both cell.The immunoreactive(IR) of Tpm4 was located in cytoplasm,and immunostain of Vim were detected in cytoplasm and process,while TrkB IR could be found in cytoplasm and nucleolus.The ananlysis of grey scals value revealed that TrkB level decreased in Hela miR-138 group,compared with that of control group.7.The protein levels of Tpm4,Sncb and VDAC2 were detected in PC12 by using Western Blot,and Tpm4,VDAC2 in Hela cell.Protein expression of Sncb decreased in PC12 miR-138 group,while VDAC2 protein level decrease in Hela cell compared with that of control group.8.In both cell,mRNA expressions of Vim, Tpm4 and VDAC2 were amplified,but Sncb and TrkB could not detected.There were no significant differences of mRNA expression pattern between miR-138 group and control group.Conclusion.1.miR-138 might take an important part in spinal cord injury;2. miR-138 could inhibit the viability of PC12 and Hela cell;3.Sncb,VDAC2 and TrkB might be the potential targets of miR-138;4.The regular methods of miR-138 might be translation restrain,but not degradation of mRNA. |
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