| Objective:Myotonic dystrophy type 1,(DM1)is a common autosomal dominant myopathy,the most common form of adult-onset muscular dystrophy,with multiple systemic diseases and progressive deterioration.Muscular atrophy is one of the main symptoms of clinically common muscle system involvement,which seriously affects the quality of life of patients.Skeletal muscle satellite cells(SSCs)play an important role in muscle regeneration Decreased proliferative capacity and reduced number of SSCs can lead to muscle regeneration disorders,which can lead to muscle atrophy.Muscle biopsy in DM1 patients showed a decrease in the proliferative capacity of SSC,but the mechanism is unknown.Muscleblind-like protein 1(MBNL1)plays an important role in the pathogenesis of DM1,and MBNL1 is responsible for the regulation of RNA splicing.However,the role of MBNL1 is not only reflected in the shearing of RNA molecules,MBNL1 can regulate the proliferation of neural stem cells,and can promote the growth of neurites.Mice knocked out of MBNL 1 gene showed symptoms of muscle atrophy.We have previously used the gene editing technology TALEN to eliminate the toxic RNA of neural stem cells and cardiomyocytes derived from DM1 patients,and successfully reversed the phenotype of MBNL1.However,the role of MBNL1 in the proliferation of skeletal muscle satellite cells in DM1 patients has not been studied.The mammalian target of rapamycin(mTOR)plays an important role in cell growth,proliferation,differentiation,metabolism,autophagy,and protein synthesis.mTOR plays an important role in the proliferation of neural stem cells and the growth of neurites in DM1 patients.Proliferation and autophagy are closely related.In the DM1 Drosophila model,overexpression of mTOR inhibits autophagy to rescue the reduced means flight area.At the same time,MBNL1 can regulate the mTOR signaling pathway.In summary,we hypothesized that the abnormal function of MBNL1 may affect the autophagy level of skeletal muscle satellite cells and affect its proliferation by affecting the mTOR signaling pathway.The human induced pluripotent stem cell(iPSC)derived from the patient’s somatic cells carries all the genetic information of the DM1 patient,and maintains the pluripotent differentiation characteristics,and can be differentiated into SSC.The establishment of a skeletal muscle satellite cell disease model in DM1 patients undoubtedly provided a good tool for studying the pathogenesis of muscle atrophy in DM1 patients.This study aimed to investigate whether dysfunction of MBNL1 affects the proliferation of skeletal muscle satellite cells by affecting the mTOR signaling pathway in a model of patient-derived skeletal muscle satellite cellsPART Ⅰ DM1 SKELETAL MUSCLE SATELLITE CELLS EXHIBIT A PROLIFERATION DEFECTMethods:iPSC derived from normal human cells(DM1-04iPSC),iPSC derived from somatic cells of DM1 patients(DM1-03iPSC),DM1-03 iPSC(DM1-13-3iPSC)with toxic RNA eliminated by TALEN gene editing,successfully differentiated into skeletal muscle satellite cell DM1-04SSC,DM1-03SSC and DM1-13-3SSC.Immunofluorescence staining showed that the skeletal muscle satellite cells successfully expressed the markers Pax7 and MyoD;the proportion of Pax7+MyoD+positive cells can be used to compare the proliferative capacity of the three-lineage cells.(Antigen Ki-67,Ki67)Immunofluorescence staining was used to compare the difference in the proportion of positive cells in the three groups of satellite cells.Cell counting Kit(CCK-8 Kit)was used to detect the proliferation of three-line satellite cells;(Antigen Ki-67,Ki67)Immunofluorescence staining was used to compare the difference in the number of positive cells in the three groups of satellite cells.Through above methods,the proliferative ability of normal human,DM1 patient source and genetically modified satellite cells was compared,and whether the SSC derived from DM1 patient was consistent with the patient muscle biopsy result,and there was a proliferation defect.The RNA fluorescence in situ hybridization detected the disappearance of DM1-13-3SSC toxic RNA.Western blot was used to determine the MBNL1 protein expression in the three-line SSC.To detect the proliferation of the three-line SSC,and to explore whether genetic modification can improve the proliferation of skeletal muscle satellite cells in DM1 patients1.Skeletal muscle satellite cell marker paired box protein Pax-7(Paired box protein Pax-7,Pax7)and myogenic differentiation factor MyoD(Myogenic Differentiation 1,MyoD)immunofluorescence staining to confirm the establishment of disease models2.The cell counting Kit(CCK-8 Kit)was used to compare the OD450 values of DM1-03SSC and DM1-13-3SSC after MBNL1 overexpression,and the proliferation ability was compared3.Immunofluorescence staining,comparing the proportion of three lines of SSC Ki67 positive cells4.In situ hybridization of nucleic acid molecules to detect SSC toxic RNA,and to confirm whether toxic RNA disappeared in DM1-13-3SSC5.Western blot was used to determine the expression of MBNL1 protein in three lines of SSCResults:1.There were no differences in morphology among the three iPSC lines,all of which successfully differentiated into SSCs(DM1-04,DM1-03,DM1-13-3)and expressed the cell markers Pax7 and MyoD.The proportion of proliferating SSCs(Pax7+MyoD+)in DM 1-03 group was lower than that in DM1-04 group(P<0.05),while that in DM1-13-3 group was higher than that in DM1-03 group but was lower than that in DM1-04 group(P<0.05)2.CCK8 assay showed that the OD450 value of DM 1-03 S SC was lower than that of DM1-04 group(P<0.05),while that of DM1-13-3 group was higher than that of DM1-03 group(P<0.05),but lower than that of DM1-04 group(P<0.05)3.The Ki67 immunofluorescence staining showed that the proportion of Ki67 positive cells in the DM1-03SSC group was significantly lower than that in the DM1-04SSC(P<0.05),while the proportion in the DM1-13-3 group was higher than that in the DM1-03 group(P<0.05),but lower than the DM1-04 group(P<0.05)4.The results of fluorescence in situ hybridization showed that no toxic RNA was found in the nucleus of DM1-13-3SSC,and toxic RNA was found in DM1-03SSC5.Western blot analysis showed that nuclear MBNL1 protein expression in DM1-03 SSC was higher than that in DM1-04 SSC(P<0.05),while that in DM1-13-3 SSC was lower than that in DM1-03 SSC(P<0.05)However,no significant difference was found between the DM 1-04 and DM1-13-3 SSC(P>0.05)Conclusions:DM1-04iPSC,DM1-03iPSC,DM1-13-3iPSC three-line pluripotent stem cells can be differentiated into skeletal muscle satellite cells;DM1-03SSC has proliferation defects;TALEN gene editing eliminates toxic RNA of DM1-13-3SSC,and enhances it’s proliferative capacity,but it’s proliferative capacity is still lower than that of DM1-04SSCPART Ⅱ THE ROLE OF MBNL1 IN THE PROLIFERATION OF DM1 SKELETAL MUSCLE SATELLITE CELLSMethods:1.Adenovirus transfected DM1-03SSC and DM1-13-3SSC to overexpress MBNL1,and the overexpression effect was verified by Western blot and Quantitative real time polymerase chain(qrt-PCR)2.Ki67 immunofluorescence was conducted to compare the proportion of Ki67 positive cells after MBNL1 was overexpressed in DM1-03SSC and DM1-13-3SSC3.The cell counting Kit(CCK-8 Kit)was used to compare the OD450 values of DM1-03SSC and DM1-13-3SSC after MBNL1 overexpressed,and the proliferation ability was compared4.Western blot analysis was used to detecet the autophagy level in three cell lines and the levels of phosphorylation of mTOR(p-mTOR),autophagy-related protein microtubulesas sociated protein light chain 3(LC3)and P62 after overexpression of MBNL1 in DM1-03SSC and DM1-13-3SSC5.DM1-03SSC,DM1-04SSC and DM1-13-3SSC were transfected with mRFP-GFP-LC3 adenovirus to detect the autophagy level of three cell lines.The autophagic flux of DM1-03SSC and DM1-13-3SSC after MBNL1 overexpression was also detectedResults:1.Western blot analysis showed that the expression levels of MBNL1 in DM1-03SSC and DM1-13-3SSC after MBNL1 overexpressed were significantly higher than those in DM1-03SSC group(P<0.05);PCR results showed that MBNL1 increased significantly at the RNA level in DM1-03SSC and DM1-13-3SSC after MBNL1 overexpressed(P<0.05)2.Ki67 immunofluorescence showed that the proportion of Ki67-positive cells in DM1-03 and DM1-13-3 groups was enhanced after MBNL1 overexpression(P<0.05),but was still lower than that in the DM1-04 group(P<0.05)3.The CCK8 assay showed that the OD450 values of DM1-03 and DM1-13-3 groups was enhanced after MBNL1 overexpression(P<0.05),but was still lower than that in the DM1-04 group(P<0.05)4.Western blot analysis of the autophagy level of three-line cells showed that:the ratio of LC3Ⅱ/LC3Ⅰ in DM1-03 SSC was significantly higher than that in DM1-04 SSC(P<0.05),while that in DM1-13-3 SSC was significantly lower than that in DM1-03 SSC(P<0.05)but higher than that in DM1-04 SSC(P<0.05).The levels of p-mTOR/mTOR and P62 in DM1-03 SSC were significantly lower than those in DM1-04 SSC(P<0.05),while those in DM1-13-3 SSC were significantly higher than those in DM1-03 SSC(P<0.05)but lower than those in DM1-04 SSC(P<0.05)5.GFP-mRFP-LC3 showed that:the GFP/mRFP per cell in DM1-03 SSC was significantly lower than that in DM1-04 SSC(P<0.05),while that in DM1-13-3 SSC was higher than that in DM1-03 SSC(P<0.05)but lower than that in DM1-04 SSC(P<0.05).The GFP/mRFP per cell in DM1 SSC was increased after MBNL1 overexpression but was still lower than that in DM1-04 SSC(P<0.05).Additionally,the GFP/mRFP per cell in DM1-13-3+Ad-MBNL1 SSC was higher than that in DM 1-03+Ad-MBNL1 SSC(P<0.05).Conclusions:Overexpression of MBNL1 significantly increased the proliferation of DM1-03SSC,DM1-13-3SSC,increased the relative expression of p-mTOR,and inhibited the autophagy level of DM1-03SSC and DM1-13-3SSCPARTⅢ RELATIONSHIP BETWEEN AUTOPHAGY AND PROLIFERATION OF DM1 SKELETAL MUSCLE SATELLITE CELLSMethods:1.Adenovirus transfected DM1-03SSC and DM1-13-3SSC to overexpress mTOR,and the effect was detected by western blot and real-time quantitative polymerase chain reaction.2.After overexpression of mTOR,western blot was used to detect changes in autophagy-related proteins LC3,P62 and PCNA levels3.After overexpression of mTOR,CCK8 detect changes in proliferative capacityResults:1.After mTOR overexpression in DM1-03SSC and DM1-13-3SSC,western blot and PCR results showed that mTOR was significantly elevated in protein and mRNA levels after overexpression of mTOR in DM1-03SSC and DM1-13-3SSC(P<0.05)2.After overexpression of mTOR,western blot analysis showed that the levels of LC3Ⅱ/LC3Ⅰ in DM1-03 and DM1-13-3 SSC decreased after mTOR overexpression(P<0.05),and there were no differences from DM1-04 SSC(P>0.05).The expression of P62 and proliferating cell nuclear antigen(PCNA)in DM1-03 and DM1-13-3 SSC increased after mTOR overexpression but was still lower than that in DM1-04 SSC(P<0.05).There was no difference in LC3Ⅱ/LC3Ⅰ between DM1-03+Ad-mTOR and DM1-13-3+Ad-mTOR SSC(P>0.05).The expression of PCNA and P62 in DM1-13-3+Ad-mTOR SSC were higher than those in DM1-03+Ad-mTOR SSC(P<0.05)3.After overexpression of mTOR,CCK8 assays showed that the OD450 values of DM1 SSCs was enhanced after mTOR overexpression,but the OD450 values was still lower than that in DM1-04 SSCs(P<0.05)The OD450 values of the DM1-13-3+Ad-mTOR group was higher than that in the DM1-03+Ad-mTOR group(P<0.05)Conclusions:These results suggest that inhibiting autophagy activation by mTOR overexpression promotes DM1 SSCs proliferation,and this effect was more pronounced in the genome modification groupPARTⅣ OVEREXPRESSION OF MBNL1 CAN PROMOTE THE PROLIFERATION OF DM1 SKELETAL MUSCLE SATELLITE CELLS BY INHIBITING AUTOPHAGY VIA THE MTOR PATHWAYMethods:1.After MBNL1 overexpression,DM1-03 and DM1-13-3 were treated with 50 nM rapamycin and divided into DM1-03SSC,DM1-03SSC+AdMBNL1,DM1-03SSC+AdMBNL1+Rapamycin groups And DM1-13-3SSC is also grouped.Western blot was used to determine p-mTOR/mTOR and autophagy-related protein LC3,P62,proliferation-related index PCNA,and to compare the changes in cell proliferation and autophagy levels after treatment with rapamycin after MBNL1 overexpression2.CCK8 was used to determine the OD 450 values of each group of cells,and the change of OD 450 values of each group caused by rapamycin treatment after MBNL1 overexpression was compared3.Ki67 immunofluorescence staining was used to determine the proportion of Ki67 positive cells in each group,and whether the effect of MBNL1 overexpression on SSC proliferation was affected after rapamycin blocked the mTOR pathwayResults:1.Western blot showed that the level of LC3Ⅱ/LC3Ⅰ was increased(P<0.05),the levels of p-mTOR/mTOR,PCNA,and P62 in DM1-03+Ad-MBNL1 SSC were significantly reduced after rapamycin treatment(P<0.05).There were no significant differences in the levels of LC3Ⅱ/LC3Ⅰ,p-mTOR/mTOR,PCNA,and P62 between the DM 1-03+Ad-MBNL1+rapamycin and DM1-03 groups(P>0.05).The level of LC3Ⅱ/LC3Ⅰ was increased(P<0.05),the levels of p-mTOR/mTOR,PCNA,and P62 in DM 1-13-3SSC+Ad-MBNL 1 were significantly reduced after rapamycin treatment(P<0.05).There were no significant differences in the levels of LC3Ⅱ/LC3Ⅰ,p-mTOR/mTOR,PCNA,and P62 between the DM1-13-3+Ad-MBNL1+rapamycin SSC and DM1-13-3 SSC groups(P>0.05)2.The OD 450 values of DM1-03+Ad-MBNL1+rapamycin SSC were significantly lower than those in DM1-03+Ad-MBNL1 SSC(P<0.05),whereas there were no significant differences between DM1-03+Ad-MBNL1+rapamycin SSC and DM1-03 SSC(P>0.05).The OD 450 values of DM1-13-3+Ad-MBNL1+rapamycin SSC were significantly lower than those in DM1-13-3+Ad-MBNL1 SSC(P<0.05),whereas there were no significant differences between DM1-13-3+Ad-MBNL 1+rapamycin SSC and DM1-13-3 SSC(P>0.05)3.The results of Ki67 immunofluorescence staining showed that the proportion of Ki67 positive cells in DM1-03+Ad-MBNL1+rapamycin SSC group was significantly lower than that in DM1-03+Ad-MBNL1 SSC(P<0.05),whereas there was no significant difference between DM1-03+Ad-MBNL1+rapamycin SSC and DM1-03 SSC(P>0.05).The proportion of Ki67 positive cells in DM1-13-3+Ad-MBNL1+rapamycin SSC was significantly lower than that in DM1-13-3+Ad-MBNL1 SSC(P<0.05),whereas there was no significant difference between DM1-13-3+Ad-MBNL1+rapamycin SSC and DM1-13-3 SSC(P>0.05)Conclusions:MBNL1 reverses the proliferation defect of skeletal muscle satellite cells in myotonic dystrophy type 1 by inhibiting autophagy via the mTOR pathway. |
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