Change Of MiRNA Expression Patterns On Dysferlinopathies And Effect Of MiR-708 On Myoblast Growth

ObjectiveTo study the relationship of mi RNA and dysferlinopathies, changes of mi RNAsexpression patterns in dysferlinopathies patients’ tissue were tested. On the basis ofcytological experiments and bioinformatics website, the effect of mi R-708 mimic on thegrowth myoblast was detected.Method1.Cases of dysferlinopathies’ patients were choosed by clinical feature,hematoxylin-eosin staining, creatase histochemisty, specific staining. And muscletissue with nonspecific changes were choosed as contrast. Part of tissues weretaken to mi RNA array analysis.2.Use the bioinformatics website to predict mi RNA that effect on DYSF gene,combined with mi RNA array results, choose the mi RNA that close todysferlinopathies and the gene related to proliferation and differentiation ofmyoblast, Taqman probe real-time quantitative PCR(RT-PCR) was used to detectthe expression of these mi RNAs in human muscle tissues and C2C12 duringproliferation and differentiation.3.Synthesised mi R-708 mimic and transfected it into C2C12. Then optical microscopewas used to observe the growth of C2C12, CCK-8 was used to detecte the effect ofmi R-708 mimic on proliferation; flow cytometry was used to detecte cell cycle andapoptosis; RT-PCR and Western blot were used to detecte related gene expressionof C2C12 during proliferation and differentiation.Results1.Immunohistochemistry suggested that: the sarcolemma protein of dysferlin showed alinear expression in normal muscle cells, and in the dysferlinopathies muscle tissues,the expression of dysferlin was significantly decreased or disappeared.Pathologicalchanges of dysferlinopathies group under light microscope were as follows: musclefibers showed mild to severe atrophy with multiple circular hypertrophy,scatteredrimmed vacuoles, necrosis of individual muscle fibe, the core fiber increased,interstitial fibrosis hyperplasia.2. The results of mi RNA array showed that in dysferlinopathies group, 116 mi RNAup-regulated and 176 mi RNA down regulated. Part of abnormal expression ofmi RNA is related to pathophysiological process of the regulation of the actincytoskeleton formation, intracellular enzyme activity, cell metabolism and MAPK,Wnt signal pathway. Combined with microarray and target gene prediction results,three mi R(-122,-346,-708) were closely related to DYSF gene. Mi R-122,-346 wentdown, mi R-708 rose up.3.Compared to muscle tissues of control group, the expression level of mi R-122、-346、-708 is higher in 9 cases of dysferlinopathies patients(The P values were 0.04,0.04, 0.03), and the expression pattern of mi R- 708 in dysferlinopathies patient’stissue was consistent with the result of the array.4.Detected the expression of mi R-122,-346,-708 during C2C12 myoblasts growth.The expression level of mi R-122, display,-346,-708 is low in the proliferativephase, At early stage of differentiation mi R-122,-346,-708 were increased, butwith the prolongation of differentiation time of C2C12,the expression level ofmi R-122 did not change, mi R-346 and mi R-708 downregulated,and mi R-708 ismore obviousiy decreased, reached the proliferative level.5.Transfected C2C12 cells with mi R-708 mimic, By optical microscope we observedthat:myoblast cell proliferation was inhibited. CCK-8 analysis showed thatnhibition rate of mimic group was(15.40 ± 0.0052)%,(P=0.004), significantlyhigher than that in NC group(2.87± 0.0034)%. Flow cytometry results showed thattransfected with mi R-708 mimic, the cell cycle was significantly changed, S phaseshortened(20.87%), G1 prolonged(68.64%), but S phase and G1 phase of NCgroup proportion were 45.66%, 42.69%; the apoptosis index had no obviouschange between mimic group and NC group. RT-PCR and Western blot resultsshowed that:compared to the negative control group,expression of MYOD,MYOG,MHC, DYSF was not significantly changed in mi R-708 mimic group.Conclusions1.Dysferlinopathies may cause the expression pattern changes of mi RNA, which mayinvolved in pathophysiological process of the regulation of the actin cytoskeletonformation, intracellular enzyme activity, cell metabolism and MAPK, Wnt signalpathways.2.Mi R-708 may be the regulational molecule of myoblast in proliferation period, butthere is no significant correlation with differentiation and apoptosis of myoblast.The mechanism of mi R-708’ abnormal expression in dysferlinopathies need to befurther discussed.