| Objective Present study analyzes the mechanism of sciatic nerve conditioning injury(SNCI)promoting repair fo dorsal column lesion(DCL) through bioinformatics on mi RNomes level and find out the key micro RNA(mi RNA). then further study the target gene of key mi RNA and investigate effect of key mi RNA on neurite growth of dorsal root ganglion(DRG) neurons via regulating the expression of key mi RNA on cellular level. At last, try to regulate neurite regrowth of DRG neurons via applying?-Phenylethyl isothiocyanate(PEITC).Method1. Microarray 3.0 is used to detect expression profile of mi RNAs of rat DRG tissues in SNCI group and simple dorsal column lesion(SDCL) group. And find out the key mi RNA which play a vital role in SNCI promoting repair of DCL through bioinformatics.2. RT-q PCR is applied to validate the data of microarray. mi RBase, Target Scan and document retrieval are combined together to predict the target gene of key mi RNA.3. Validate the target gene of key mi RNA on m RNA level and protein level through RT-q PCR and Western Blot.4. Isolate and culture DRG neurons from neonate Wistar rat(<24h). Inhibit the expression of key mi RNA and inhibit the activity of its target protein, and then apply immunofluorescence to investigate the neurite regrowth of DRG neurons.5. Administer different doses of PEITC on DRG neurons and select the best dose via using immunofluorescence to investigate the neurite regrowth of DRG neurons.RT-q PCR and Western Blot are applied to validate the mechanism of PEITC on mi RNA level and protein level.6. statistical analysis is preformed via SPSS 18.0. Measurement data are expressed as mean ± SD. ANOVA and q test are used to analyze comparison among multiple groups. The comparison between two independent samples are applied student's t test. P < 0.05 is considered significant.Result1. Compared with control group, there were 681 mi RNAs differentially expressed at least once in the adult rat DRGs of each check point of SNCI group or SDCL group. Among all the altered mi RNAs, mi R-17-5p was upregulated at 3rd, 7th and14 th day post DCL in SDCL group and the fold change is 4.17, 2.98 and 5.12 respectively. However, in SNCI group, mi R-17-5p was downregulated at 3rd, 7th and 14 th day post DCL, and the fold change are-3.31,-2.96 and-3.55 respectively.2. The downregulation of mi R-17-5p in SNCI group is dependent on the synergy of SNCI and DCL.3. Inhibiting mi R-17-5p in DRG neurons can enhance the expression of growth-associated protein 43(GAP-43) and neurite regrowth.4. Through mi RBase, Target Scan and document retrieval, present study predicts that the putative target gene of mi R-17-5p is signal transducer and activator of transcription 3(STAT3).5. Compare with control group, in SNCI group the expression of STAT3 and p-STAT3 proteins are upregulated at 4th, 3rd, 7th and 14 th day post DCL, but in SDCL group, these two proteins are downregulated at 3rd, 7th and 14 th day post DCL. STAT3 protein and p-STAT3 protein had opposite trend with mi R-17-5p.6. mi R-17-5p can modulate p-STAT3 level through regulating the expression of STAT3 protein, and further affect the expression of GAP-43 and neurite regrowth of DRG neurons.7. 10 ?M PEITC can downregulate the expression of mi R-17-5p in DRG neurons effectively, and promote the neurite regrowth of DRG neurons through mi R-17-5p/STAT3/GAP-43 axis.Conclusion1. Among all the altered mi RNAs, mi R-17-5p was upregulated at 3rd, 7th and 14 th day post DCL in SDCL group and the However, in SNCI group, mi R-17-5p was downregulated at these three check points.2. The downregulation of mi R-17-5p in SNCI group is dependent on the synergy of SNCI and DCL.3. The target gene of mi R-17-5p is STAT3. STAT3 play a vital role in neurite regrowth of DRG neurons thtough mi R-17-5p/STAT3/GAP-43 axis.4. PEITC promote the neurite regrowth of DRG neurons through mi R-17-5p/STAT3/GAP-43 axis. |
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