| Brachial root avulsion leads to spinal motoneurons death. Previous studies have shown that avulsion induced a cascade of molecular and cellular events including changes in the expression of genes and the phosphorylation of signaling molecules in cell death-related pathways. Based on the m RNA microarray analysis of the avulsion-injured spinal cord in the previous studies, those genes with downregulation have been proved to be required for promoting neuronal survival and axonal regeneration, but those genes with upregulation have been proved to be involved in apoptosis and DNA damage. However, the molecular mechanism of avulsion-induced abnormal gene expression during motoneuron degeneration is still unclear. Micro RNAs(mi RNA) are small non-coding RNAs that modulate protein expression levels by antagonizing m RNA translation. Individual mi RNA targets and blocks hundreds of protein-coding genes that regulate many biological processes in neuronal lesions. An increasing number of studies have demonstrated that mi RNAs in the spinal cord are altered after spinal cord injury. However, little is known regarding in the spinal cord during avulsion. There are also emerging evidences that alterations in RNA metabolism in the spinal cord are spatial-specific and time-specific. Up to now, the mechanism of the avulsion-injured motoneurons apostosis is unknown, neither the expression profile of thoses mi RNAs targeting the genes for neuronal survive, axonal regeneration, and neuronal apoptosis in avulsion-injured spinal cord. Therefore, we chose two time points, the 3 days post-lesion when the RNA stability in the spinal cord lost and the 14 days post-lesion when motoneurons death began, to study the mi RNA functions in avulsion injured motoneurons death. Part I. Time-specific micro RNA expression patterns in adult rats following the brachial plexus root avulsionObjectives: To compare the mi RNAs expression patterns in ipsilateral vs contralateral spinal cord following unilateral brachial plexus roots avulsion of the adult rats.Methods: The animal model of cervical spinal cord avulsion injuries was established using the adult Spraque-Dawley rats. The microarrays of ipsilateral and contralateral spinal cord tissues were applied and compared on the 3rd day and 14 th day after injury. The expression levels of the altered mi RNAs together with the predicted target genes were chosen to be vertified by using realtime RT-PCR. The spatial expression patterns of the predicted target genes of the altered mi RNAs in avulsion-injured spinal cord were studied and confirmed by using the immunofluorescence methods.Results:(1)The spatial-specific expression patterns of the altered mi RNAs in the spinal cord were shown by comparing mi RNAs levels between the ipsilateral ventral horn and the contralateral ventral horn of the spinal cord at the same time point. The results showed that 40 of mi RNAs were upregulated but 23 of them were downregulated on the 3rd day, later on, 25 of mi RNAs were upregulated but 18 of them were downregulated on the 14 th day post-lesion.(2)The time-specific expression patterns of the altered mi RNAs were shown by comparing mi RNAs levels in the ipsilateral ventral horn of the spinal cord between the 3rd day and the 14 th day post-lesion. The results showed that 10 of mi RNAs demonstrated increase; but only mi R-466c-3p presented a sustained decrease on both time points. There were 4 mi RNAs upregulated on the 3rd day but downregulated on the 14 th day. Moreover, 5 mi RNAs upregulation and 5 downregulation occureed on the 14 th day compared to those on the 3rd day post-lesion.(3)The results of the q RT-PCR confirmed the upregulations of the mi R-146b-5p and mi R-31a-3p on the 14 th day, downregulations of mi R-466c-3p on both time points in ipsilateral vs contralateral spinal cord. Also, the q RT-PCR confirmed the downregulation of mi R-376b-3p and mi R-137-3p and upregulation of the mi R-144-3p in ipsilateral spinal cord on the 14 th day compared to those on the 3rd day after injury.(4)The results of the prediction of the target genes of the altered mi RNAs by the bioinformatics software showed that activating transcription factor 3(ATF-3) and c-jun were potentially target genes of mi R-484 and mi R-324-3p, which were downregulated on the 3rd day. The calcium-activated neutral protease-2(Calpain-2) was predicted to be a potential target gene of the mi R-137-3p which downregulated, and GFAP(glial fibrillary acidic protein) was predicted to be a potential target gene which downregulated mi R-335 on the 14 th day. The time-specific and spatial-specific expressions of the above targeted genes were confirmed by the immunofluorescence analyses of the injured spinal cord. The results showed that both ATF-3 and c-jun were upregulated and expressed in the nuclei of the injured motoneurons on the 3rd day. On the 14 th day after injury; the increase expressions of the calpain-2 were checked in the cytoplasma of the injured motoneurons, and GFAP overexpression also confirmed in astroglial cells of the ipsilateral spinal cord.Conclusion: We explored mi RNA expression profiles in the spinal cord of the adult rats and described the pattern of mi RNA expression following spinal cord injury induced by a unilateral brachial roots avulsion surgery. Our results indicate that the alterations in mi RNAs may contribute to the mechanism of injured motoneuron degeneration Part II. Mi R-137-3p targets the calpain-2 proteinObjective: To vertify the inhibiton effect of mi R-137-3p on the expression of calpain-2 protein in the differentiated PC12 cells.Methods: The mi R-137-3p lentivirus was constraucted by Gi Kai gene company. The cultured differentiated PC12 cells were treated by the GFP(green fluorescent protein) labeled lentivirus with different MOI. The efficiency of the lentivirus transfection was evaluated by checking GFP particle density under fluorescence microscopy. The safety concentration of lentivirus was determined by measuring the viability of the cells by CCK-8 assay. Then, a set of the differentiated PC12 cells were randomly divided into three subgroups treated by culture medium only(normal), mi R-137-3p lentivirus in culture medium(mi R-137-3p), or negative control lentivirus in culture medium(NC). Then mi R-137-3p and calpain-2 m RNA levels in different treated PC12 cells were detected by fluorescence quantitative PCR, and the calpain-2 and n NOS protein levels were measured by western-blot analysis. The confirmation of the mi R-137-3p targeting calpain-2 protein was carried out by the luciferase activity analysis of the cultured HEK293 cells co-transfected with mi R-137-3p mimic and a firefly luciferase reporter plasmid carrying the wild-type(WT) or the mutant(MUT) 3’UTR of calpain-2. Another set of the cultured differentiated PC12 cells were subdivided into thress subgroups and treated by culture medium only, the calpain-2 si RNA, or the random-sequence si RNA. Then, the expression patterns of calpain-2 and n NOS proteins were detected by immunofluorescence. The calpain-2 and n NOS m RNA and protein levels were measured and compared among the three subgroups.Results:(1)At the 36 h and 60 h t ime points following GFP-labled negative control-lentivirus treatment, the GFP particles were detected both in the cytoplasm and in the nuclei of the differentiated PC12 cells.(2)Compared to the viability of the differentiated PC12 cells in PC12 subgroup, the cell viability in the mi R-137-3p subgroup was 99.66%, and in the NC subgroup was 99.83%. Statistic analysis showed that the difference in cell viability was not significant among the three subgroups. The optimal concentration of lentivirus was MOI 100+polybrene.(3)If we set number 1 as the mi R-137-3p m RNA level and the calpain-2 m RNA level in PC12 subgroup, the level of the mi R-137-3p m RNA was 757.51±73.23 in mi R-137-3p subgroup, and 1.20±0.22 in the NC subgroup. The level of the calpain-2 m RNA was 0.28±0.09 in mi R-137-3p subgroup, and 1.23±0.32 in the NC subgroup. The m RNA leve differences of both mi R-137-3p and calpain-2 were statistically significant between the mi R-137-3p and NC subgroups(all p<0.05),however, no significant differences occurred between the NC and the PC12 subgroups.(4)Western-blot results also showed significant differences in the protein levels of calpain-2 after treatment of the mi R-137-3p lentivirus treatment. If the level of caplain-2 protein in PC12 subgroup was set as 1, it was 0.51±0.07 in mi R-137-3p subgroup and 1.03±0.12 in NC subgroup. Meanwhile in the same way, the level of the n NOS protein was set as 1 in PC12 subgroup. It was 0.53±0.03 in mi R-137-3p subgroup and 1.14±0.15 in NC subgroup. The differences of the protein levels of both calpain-2 and n NOS were statistically significant between mi R-137-3p subgroup and NC subgroup(all p<0.05), while there was no significant difference between PC12 group and NC group.(5)The result of the luciferase activity analysis showed that mi R-137-3p repressed the activity of the luciferase reporter containing the wild-type 3’UTR of calpain-2.(6)Immunofluorescent double labeling showed that calpain-2 and n NOS proteins were co-expressed in the cytoplasma of the differentiated PC12 cells. The q PCR results showed that the level of the calpain-2 m RNA was 0.64±0.08 in calpain-2 si RNA subgroup and 1.02±0.24 in random-sequence si RNA subgroup if it was set as 1 in PC12 subgroup. The n NOS m RNA level in calpain-2 si RNA subgroup was 0.43±0.002 and in random-sequence si RNA subgroup was 1.04±0.12 if it was set as 1 in PC12 subgroup. Western results showed that the protein levels of both calpain-2 and n NOS were downregulated by the calpain-2 si RNA treatment. If the protein level of both calpain-2 and n NOS was set as 1, the protein level of calpain-2 was 0.48±0.07 in calpain-2 si RNA subgroup and 1.15±0.16 in random-sequence subgroup, meanwhile, the n NOS protein level was 0.25±0.09 in calpain-2 si RNA subgroup and was 1.09±0.16 in random-sequence subgroup. The differences between calpain-2 si RNA and random-sequence si RNA subgroups were statistically significant in the protein level of both calpain-2 and n NOS(all p<0.05), while there was no significant difference between PC12 group and random-sequence si RNA subgroups.Conclusion: Lentivirus can steadily be transfected into the differentiated PC12 cells and effectively upregulated the expression of mi R-137-3p. It indicates that lentivirus pathway is feasible to neuronal-like cells. Calpain-2 was confirmed to be a target gene of mi R-137-3p and calpain-2 si RNA can effectively knockdown the calpain-2 gene and cause the downregulation of the n NOS gene both at m RNA and at protein levels. Part III. Overexpression of mi R-137-3p protect the PC12 cells from the oxidative stress in vitroObjective: To explore the effect of mi R-137-3p--calpain-2--n NOS pathway in oxidative stress induced apoptosis of the PC12 cells in vitroMethods: Hydrogen peroxide(H2O2)-induced apoptosis in PC12 cells(PC12+ H2O2) was established and followed by the treatment of the mi R-137-3p lentivirus in culture medium(mi R-137-3p+H2O2), negative control lentivirus in culture medium(NC+H2O2), and the culture medium as normal control(PC12 subgroup). Then CCK8 assay, the q PCR, the western-blot, and the calpain activity assay were used to test the cell viability of the treated PC12 cells, the m RNA levels of mi R-137-3p, the m RNA and protein levels of the calpain-2 and n NOS gene, and the enzyme activity of the calpain-2 of the treated PC12 cells, respectively.Results:(1)All of the PC12 cells underwent oxidative stress showed a lower m RNA level of the mi R-137-3p but a higher m RNA level of the calpain-2 and n NOS genes when compared to that of the normal control(all p<0.05). If the m RNA level of mi R-137-3p, calpain-2 or n NOS was set as 1 in normal control PC12 cells, the m RNA level of mi R-137-3p was decreased to 0.38±0.21. meanwhile, the m RNA levels of calpain-2 and n NOS were increased to 7.64±0.40 and 5.84±0.27significantly(all p<0.05).(2)With the H2O2 treatment, the cell viability was decreased in all of the H2O2 treated cells as compared to that of the normal control cells(all p<0.05). The cell viability was 91.38%±0.27% in mi R-137-3p+H2O2, which significantly higher than 86.32%±0.32% in PC12+H2O2, and 86.94%±1.82% in NC+H2O2 subgroups, respectively(all p<0.05). There was no s ignificant difference between the PC12+H2O2 and NC+H2O2 subgroups.(3)Overexpression of the mi R-137-3p by lentivirus transfection significantly decreased the protein levels of calpain-2 and n NOS. If the protein level of the calpain-2 and n NOS was set as 1 in normal control PC12 cells subgroup, the calpain-2 protein level was 1.34±0.11 in mi R-137-3p+ H2O2, 4.31±0.76 in PC12+ H2O2, and 3.85±0.65 in NC+ H2O2 subgroups. The n NOS protein level was 1.24±0.42 in mi R-137-3p+ H2O2, 2.80±0.69 in PC12+ H2O2 and 2.65±0.29 in NC+H2O2 subgroup. The levels of calpain-2 and n NOS were significantly decreased in mi R-137-3p+ H2O2 subgroup when compared to those in PC12+ H2O2 and NC+ H2O2 subgroup(all p<0.05), while there was no significant difference between PC12+ H2O2 and NC+ H2O2 subgroups.(4)The activity of calpain enzyme in mi R-137-3p lentivirus treated cells was 1.58±0.69 fu/mg,which was significantly lower than 3.57± 0.76 fu/mg in the NC treated cells and 3.54±0.17 fu/mg in normal control cells. After H2O2 treatment, the activity of calpain enzyme was increased to 9.58 ±0.62 fu/mg in PC12+ H2O2, 9.68±0.23 fu/mg in NC+H2O2, and 3.30±0.64 fu/mg in mi R-137-3p + H2O2 subgroups. It was significantly decreased in the mi R-137-3p + H2O2 subgroup when compared to that in NC+ H2O2 subgroup(p<0.05).Conclusion: We collected PC12 oxidative damage cells induced by H2O2, finding that the level of mi R-137-3p was downregulated but calpain-2 and n NOS was upregulated. Over-expression of mi R-137-3p could relive cell apoptosis through inhibition of calpain-2 and n NOS. Part IV. Overexpression of the mi R-137-3p in rats’ spinal cord in vivo reduced lesion-induced motoneurons death following the brachial plexus roots avulsionObjective: To explore the role of mi R-137-3p-calpain-2-n NOS pathway in avulsion-induced motoneurons death in vivoMethods: Adult rats were performed with 5 roots avulsion of the right brachial plexus(Av). One week later, the injured rats were operated again with the mi R-137-3p lentivirus, negative control-lentivirus microinjected into the ipsilateral ventral horns of the injured C7 spinal cord segment. Injections were placed at 0.5mm lateral to the midline at a depth of 1.3mm to target motor neurons in the ventral horn. Injections were made using a heat-pulled glass capillary needle(tip diameter 60um) to limit damage to the spinal cord. For injection site, a volume of up to 2ul of lentivirus(105 TU) was slowly infused over 5 min. Then, the expression levels of mi R-137-3p m RNA, Calpain-2 protein in the spinal cords of the mi R-137-3p lentivirus(mi R-137-3p+Av), NC-lentivirus(NC+Av), Saline(Av) treated and normal rats were detected and by PCR, western-blot and Immunofluorescence histochemistry, and NADPH-d(Nicotinamide adenine dinucleotide phosphate diaphorase) after 2 to 4 weeks of the injury. The n NOS positive motoneuron and survival motoneurons were quantify by NADPH-d plus neutral red staining.Results:(1)Immunofluorescence double labeling demonstrated that GFP labeled lentivirus were transfected in the cytoplasma of the motoneurons which were also Ch AT(Choline acetyltransferase) positive in the ipsilateral ventral horns in the cross-sections of the C7 spinal segments after 2w and 4w of avulsion.(2)q PCR results showed that the expression level of mi R-137-3p m RNA was 0.49±0.05 in Av, 6.09±0.86 in mi R-137-3p+Av, and 0.34±0.08 in NC+Av subgroups at 2w after avulsion if it was set as 1 in normal spinal cord. The difference between mi R-137-3p+Av and Av subgroups was statistically significant(p<0.05), while there was no significant difference between Av and NC+Av subgroups.(3)Western-blot results showed that the protein level of calpain-2 in normal spinal cord was 4.46±0.20 in Av group, 0.44±0.14 in mi R-137-3p+Av, and 4.91±1.04 in NC+Av subgroups at 2w after avulsion if it was set as 1 in normal spinal cord. The difference between mi R-137-3p+Av and Av subgroups were statistically significant(p<0.05), while there was no significant difference between Av and NC+Av subgroups.(4)The n NOS positive motoneurons were only detected in lesioned ventral horns. The number of the n NOS positive motoneurons was 47.83%±3.13% at 2w 13.21%±2.60%at 4w in Av, 18.78%±6.37 % at 2w and 19.51%±5.71 % at 4w in mi R-137-3p+Av, and 44.28%±13.14%at 2w and 17.81%±2.92% at 4w in NC+Av sungroups at after avulsion. At 2w of injury, n NOS positive motoneurons in mi R-137-3p+Av subgroup was significantly lower than that in Av subgroup(p<0.05), while there was no significant difference between Av and NC+Av subgroups. At 4w of injury there was no significant difference among three subgroups.(5) The number of surviving motoneuron in C7 spinal segments was 66.90%±1.08% at 2w and 49.13%±16.26% at 4w in Av, 86.07%±3.56% at 2w and 78.04%±7.14% at 4w in mi R-137-3p+Av, and 65.67%±6.63% at 2w and 56.12%±3.33% at 4w in NC+Av subgroups after avulsion. A significant increase number of the surviving motoneurons in the mi R-137-3p+Av subgroup was observed when compared to those in the other two subgroups both at 2w and 4w after avulsion(all p<0.05)., while there was no significant difference between Av and NC+Av subgroups(p>0.05).Conclusion: In root-avulsion of the brachial plexus of the adult rats, microinjection of mi R-137-3p lentivirus into the ventral horns can effectively transfected the mi R-137-3p genes into the cytoplasm of the ipsilateral injured motoneurons. The mi R-137-3p lentivirus did upregulated the expression of mi R-137-3p m RNA and downregulated the calpain-2 and n NOS protein levels in injured spinal cords at 2w after treatment. Moreover, overexpression of mi R-137-3p genes reduced the avulsion-induced motoneurons death at 2w and 4w after injury. Summary 1. We explored the mi RNA expression profiles using a brachial root avulsion rat model and described the pattern of mi RNA expression following spinal cord injury. 2. The calpain-2 was confirmed to be a target gene of mi R-137-3p both in PC12 cells and in the adult spinal cord in the present study,. 3. The present result explored the mi R-137-3p-calpain2-n NOS pathway playing a provital role in the oxidative stress of the differentiated PC12 cells. 4. In the brachial roots avulsion of the adult rats, microinjection of mi R-137-3p lentivirus into the ventral horns can effectively transfected the mi R-137-3p genes into the cytoplasm of the ipsilateral injured motoneurons. 5. Upregulation of the mi R-137-3p in avulsion-injured spinal cord was demonstrated to reduce the injured motoneurons death. The mi R-137-3p-calpain2-n NOS mechanism can reduce avulsion-induced motoneuronns death. The mi R-137-3p could be used as a m olecular target in the treatment of the neuronal diseases caused by the oxidative stress and trauma. |
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