Identification Of Micrornas In The Soleus And Extensor Digitorum Longus And The Founction Of MIR-143-3P On Myogenesis

For the past few years, microRNAs (miRNAs) are found to have important roles in regulating the stability and translation of mRNA at the posttranscriptional level. More and more researches have demonstrated that miRNAs take part in regulating lots of biological processes related to muscle development, however, there are no researches about the functionon of miRNAs on regulating fast or slow myofibers development. Thus, in this study, in order to find the miRNAs during soleus (SL) and extensor digitorum longus(EDL) development, solexa sequencing was used to identify the miRNAs in SL and EDL. In addition, we explored the potential function of these miRNAs.There was significant difference in miR-143-3p expression between SLand EDL, The mature sequence of miR-143-3p is highly conserved in human、rat、pig and other species. Up to now, there has no research on the role of miR-143-3p in regulating the growth of skeletal muscle.In this experiment,we used C2C12myoblasts as experimental material, exploreing the function of miR-143-3p during myogenesis.The major results of the experiment are as follows:1. A total of10,601,731and13,006,035high quality reads were obtained from the SL and EDL small RNA libraries, respectively. The length of the small RNA sequence was mainly for22nt,which accounted for79.78%and81.93%of the total high quality reads;A total of23,439,862clean reads were obtained from the two libraries after removing the reads of low quality sequences and other contaminants. There were182,638and105,405unique reads among the total number of the clean sequences between the SL and EDL small RNA libraries;There were42,573common sequences reads from the unique sequences between the SL and EDL libraries. The rate of matched with known miRNAs in SL and EDL library were57.17%and45.14%, respectively,while the rate of not annotated small RNAs were13.46%and12.85%,respectively.2. There were258unique mature miRNAs had been identified. Among the258miRNAs,199of the miRNAs expressed in SL,205expressed in EDL. There were length and sequence heterogeneity existed among the identified miRNAs; The phenomemon about the editing of bases in the seed sequence of the miRNAs were widespread. 3. The analytic data of the expression for258miRNAs showed that there were247miRNAs coexpressed in the two libraries,7miRNAs were detected only in the SL sRNA library and in the EDL sRNA library were11miRNAs were detected; There were21miRNAs expressed signifeicant differentially in SL and EDL,10miRNAs were up-regulated and11were down-regulated in SL. The expression rangeability was widely, the number of sequencing reads were changed from several reads to several million reads.4. In accordance with the measuer of miRNA biogenesis,69short RNA produced45potential novel miRNAs,which could form stable step-loop structure. The reads about the novel miRNAs were very low, only a few reads on average.5. miR-143-3p was widely existed in all the tissues analyzed, but the expression level were different. It expressed highest in heart, followed by fat, muscle and liver, the kidney and spleen volume is low reletivelly.6. The expression level of miR-143-3p was upregulated during C2C12cells differentiation; Overexpression of miR-143-3p in C2C12cells, cells differentiation was promoted significantly,meanwhile, the expression of MyoG and MyHC mRNA level were greatly upregulated;The results of Immunofluorescence staining showed that there were more myotubes compared with control.In a word, this research had extended the number of known porcine miRNAs and offtered a full analysis of miRNAs expressed in SL and EDL. In addition, we had explored the function of miR-143-3p during myoblast differentiation.