Background:Cardiomyocytes derived from human embryonic stem cells(hESCs)are regulated by complex and stringent gene networks during differentiation.Long non-coding RNAs(lncRNAs)exert critical epigenetic regulatory fun...Background:Cardiomyocytes derived from human embryonic stem cells(hESCs)are regulated by complex and stringent gene networks during differentiation.Long non-coding RNAs(lncRNAs)exert critical epigenetic regulatory functions in multiple differentiation processes.However,the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated.Here,we identified the key roles of ZFAS1(lncRNA zinc finger antisense 1)in the differentiation of cardiomyocytes from hESCs.Methods:A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method,and the number of beating hESCs-derived cardiomyocytes was calculated.Gene expression was analyzed by quantitative real-time PCR(qRTPCR).Immunofluorescence assays were performed to assess the expression of cardiac troponin T(cTnT)andα-actinin protein in cardiomyocytes.Results:qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells,cardiac progenitor cells,and cardiomyocytes.Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs,which was characterized by reduced expression of the cardiac-specific markers cTnT,α-actinin,myosin heavy chain 6(MYH6),and myosin heavy chain 7(MYH7).In contrast,ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes.In terms of the mechanism,we found that ZFAS1 is an antisense lncRNA at the 5′end of the protein-coding gene ZNFX1.Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1.Furthermore,qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation.Conclusion:Altogether,these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1,which may provide a reference for the treatment of heart disease to a certain extent.展开更多
Alternative splicing is a critical process to generate protein diversity.However,whether and how alternative splicing regulates autophagy remains largely elusive.Here we systematically identify the splicing factor SRS...Alternative splicing is a critical process to generate protein diversity.However,whether and how alternative splicing regulates autophagy remains largely elusive.Here we systematically identify the splicing factor SRSF1 as an autophagy suppressor.Specifically,SRSF1 inhibits autophagosome formation by reducing the accumulation of LC3-ⅡI and numbers of autophagosomes in different cell lines.Mechanistically,SRSF1 promotes the splicing of the long isoform of Bcl-x that interacts with Beclinl,thereby dissociating the Beclin1-PIK3C3 complex.In addition,SRSF1 also directly interacts with PIK3C3 to disrupt the interaction between Beclinl and PIK3C3.Consequently,the decrease of SRSF1 stabilizes the Beclinl and PIK3C3 complex and activates autophagy.Interestingly,SRSF1 can be degraded by starvation-and oxidative stresses-induced autophagy through interacting with LC3-Ⅱ,whereas reduced SRSF1 further promotes autophagy.This positive feedback is critical to inhibiting Gefitinib-resistant cancer cell progression both in vitro and in vivo.Consistently,the expression level of SRSF1 is inversely correlated to LC3 level in clinical cancer samples.Our study not only provides mechanistic insights of alternative splicing in autophagy regulation but also discovers a new regulatory role of SRSF1 in tumorigenesis,thereby offering a novel avenue for potential cancer therapeutics.展开更多
基金the National Natural Science Foundation of China[81573434 to BZC]Heilongjiang Touyan Innovation Team Program[BZC],HMU Marshal Initiative Funding(HMUMIF-21018 to BZC)National Nature Science Youth Foudation of China[82000226 to XFG].
文摘Background:Cardiomyocytes derived from human embryonic stem cells(hESCs)are regulated by complex and stringent gene networks during differentiation.Long non-coding RNAs(lncRNAs)exert critical epigenetic regulatory functions in multiple differentiation processes.However,the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated.Here,we identified the key roles of ZFAS1(lncRNA zinc finger antisense 1)in the differentiation of cardiomyocytes from hESCs.Methods:A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method,and the number of beating hESCs-derived cardiomyocytes was calculated.Gene expression was analyzed by quantitative real-time PCR(qRTPCR).Immunofluorescence assays were performed to assess the expression of cardiac troponin T(cTnT)andα-actinin protein in cardiomyocytes.Results:qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells,cardiac progenitor cells,and cardiomyocytes.Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs,which was characterized by reduced expression of the cardiac-specific markers cTnT,α-actinin,myosin heavy chain 6(MYH6),and myosin heavy chain 7(MYH7).In contrast,ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes.In terms of the mechanism,we found that ZFAS1 is an antisense lncRNA at the 5′end of the protein-coding gene ZNFX1.Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1.Furthermore,qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation.Conclusion:Altogether,these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1,which may provide a reference for the treatment of heart disease to a certain extent.
基金This work was supported by the National Natural Science Foundation of China(81830088,81422038,91540110,and 31471235 to Y.W.,81872247 and 31400726 to W.Z.)the Department of Education of Liaoning Province(the"Liaoning Supports High Level Talents Innovation and Entrepreneurship Program"XLYC1802067 to Y.W.)+1 种基金the Department of Science and Technology of Dalian City(the HDalian Supports High Level Talents Innovation and Entrepreneurship Program" 2016RJ02 to Y.W.)the Newton Advanced Fellowship from the Academy of Medical Sciences in UK(JXR11831 to Y.W.).
文摘Alternative splicing is a critical process to generate protein diversity.However,whether and how alternative splicing regulates autophagy remains largely elusive.Here we systematically identify the splicing factor SRSF1 as an autophagy suppressor.Specifically,SRSF1 inhibits autophagosome formation by reducing the accumulation of LC3-ⅡI and numbers of autophagosomes in different cell lines.Mechanistically,SRSF1 promotes the splicing of the long isoform of Bcl-x that interacts with Beclinl,thereby dissociating the Beclin1-PIK3C3 complex.In addition,SRSF1 also directly interacts with PIK3C3 to disrupt the interaction between Beclinl and PIK3C3.Consequently,the decrease of SRSF1 stabilizes the Beclinl and PIK3C3 complex and activates autophagy.Interestingly,SRSF1 can be degraded by starvation-and oxidative stresses-induced autophagy through interacting with LC3-Ⅱ,whereas reduced SRSF1 further promotes autophagy.This positive feedback is critical to inhibiting Gefitinib-resistant cancer cell progression both in vitro and in vivo.Consistently,the expression level of SRSF1 is inversely correlated to LC3 level in clinical cancer samples.Our study not only provides mechanistic insights of alternative splicing in autophagy regulation but also discovers a new regulatory role of SRSF1 in tumorigenesis,thereby offering a novel avenue for potential cancer therapeutics.
