| ObjectiveTo study the difference of type ? and type ? muscle fibers of paraspinal muscles between the concave and convex side of main thoracic curve in adolescent idiopathic scoliosis and its regulatory mechanism.Methods1.Slicing and staining analysis of paraspinal muscle samplesThe paraspinal muscle samples of 46 patients with AIS were collected and stained with ATPse after frozen section.The muscle fiber parameters were measured and compared between the concave and convex side and among different levels.At the same time,the correlation between muscle fiber parameters and clinical data of the patients was analyzed.2.Transcriptome sequencing and verification analysis of paraspinal musclesThe paraspinal muscle samples of 5 AIS patients with clinical parameters matched were selected for transcriptome high-throughput sequencing to screen the differentially expressed genes between the concave and convex side,and the expression of the six genes with the largest absolute value of Log2 Fold Change in the sequencing was verified by large sample size RT-q PCR and Western-blot.Meanwhile,the correlation between the differences of gene m RNA expression between the concave and convex side and muscle fiber parameters were analyzed.3.Exploring the mechanism of differential expression of MYOZ2 between the concave and convex side from an epigenetic perspectiveThe existence of Cp G island near MYOZ2 promoter was predicted by Meth Primer software so as to explore the possibility of methylation regulating the differential expression of MYOZ2 between the concave and convex side.The differentially expressed MicroRNAs between the concave and convex side were screened by MicroRNA high-throughput sequencing,and the expression of the five MicroRNAs with the largest absolute value of Log2 Fold Change in the sequencing were verified by large sample size RT-q PCR.In the meantime,the correlations between the differences of MicroRNAs expression between the concave and convex side and muscle fiber parameters were analyzed to explore the possibility of MicroRNA regulating the differential expression of MYOZ2.Results1.The proportion,cross-sectional areas of type ? muscle fibers,and strength factors on the convex side of AIS were significantly larger than those on the concave side,while the proportion of type ? muscle fibers on the convex side was significantly lower than that on the concave side.The proportion of type ? muscle fibers on the convex side of main thoracic curve in AIS was significantly higher than that on the concave side,while the proportion of type ? muscle fibers on the convex side was significantly lower than that on the concave side.The difference indexs of type ? and ? muscle fiber between the concve and convex side in the apical region were lower than those at the level of upper and lower end vertebrae,and the odds ratio of ? / ? muscle fibers on the convex side in the apical region was significantly higher than that at the level of upper and lower end vertebrae.The strength factor of the concave side in the apical region was significantly lower than that at the level of the upper and lower end vertebrae.2.The difference index of type ? and ? muscle fibers in the apical region of main thoracic curve in AIS was negatively correlated with Cobb angle,AVT and annual progression of curvature,while the odds ratio of ?/ ? muscle fibers on the convex side was positively correlated with Cobb angle,AVT and annual progression of curvature,but the odds ratio of ?/ ? muscle fibers on the concave side was negatively correlated with Cobb angle,AVT and annual progression of curvature.3.32 differentially expressed genes between the concave and convex side were screened by m RNA high-throughput sequencing,of which the expressions of 17 genes were higher on the convex side than those on the concave side,and the expreesions of 15 genes were higher on the concave side.Large sample size RT-q PCR and Western-blot showed that only the expression of MYOZ2 was significantly different between the concave and convex side,which was consistent with the results of sequencing.In addition,the difference of MYOZ2 expression betwenn the concave and convex side was negatively correlated with the difference index of type ? and type ? muscle fibers,and positively correlated with the odds ratio of ? / ? muscle fibers on the convex side.4.Utilizing software prediction,it was found that there was no Cp G island near the MYOZ promoter.5.Utilizing MicroRNA high-throughput sequencing,21 differentially expressed MicroRNAs between the concave and convex side were screened,of which the expression of 10 MicroRNAs were higher on the convex side than those on the concave side,and 11 MicroRNAs were higher on the concave side.Large sample size RT-q PCR verification showed that only the expressions of mi R-499-5p and mi R-133a-3p were significantly different between the concave and convex side,which was consistent with the sequencing results.In addition,the difference of mi R-499-5p expression between the concave and convex side was positively correlated with the odds ratio of ? / ? muscle fibers and the strength factor on the convex side.The difference of mi R-133a-3p expression between the concave and convex side was negatively correlated with the difference index of type ? and type ? muscle fibers.Conclusion1.For patients with AIS,there was mutual transformation between type ? and type ? muscle fibers of main thoracic curve,especially in the apical region.Although the transformation may be a secondary change under the different tension load between the concave and convex side,it is closely related to the progress of curvature in AIS.2.The expression of MYOZ2 on the convex side of main thoracic curve was significantly different from that on the concave side in AIS,and the difference of MYOZ2 expression between the concave and convex side was closely related to the mutual transformation between type ? and ? muscle fibers.3.There was a significant difference in the expression of mi R-499-5p and mi R-133a-3p between the concave and convex side of main thoracic curve,and the differences of mi R-499-5p and mi R-133a-3p expression between the concave and convex side were closely related to the mutual transformation between type ? and ? muscle fibers.To sum up,starting from the pathology of paraspinal muscles in AIS,our team found the mutual transformation of muscle fibers of main thoracic curvature,and further proposed that mi R-499-5p and mi R-133a-3p may regulate the transformation of muscle fibers by affecting the differential expression of MYOZ2 between the concave and convex sides.The concept of treatment aiming at muscle fiber transformation provides a new idea and direction for the prevention and control of AIS progression. |
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