| PART Ⅰ EXPRESSION OF LNCRNA NRON IN TOF EMBRYONIC HEART AND CELLULAR DISTRIBUTIONBackground: Previous studies have shown that lncRNA NRON is associated with cardiomyopathy,heart failure and tumorigenesis.In the cytoplasm,it can be used as a scaffold in complex with other proteins to participate in protein inactivation,transport,or as a "sponge" to isolate mi RNAs that are involved in the regulation of protein degradation.These studies have focused on the relationship between LncRNA NRON and post-developmental heart disease,and so far,no literature has reported the relevance of lncRNA NRON to the developing heart and congenital heart disease(CHD).Tetralogy of Fallot(TOF)is the most phenotypic and common type of complex congenital heart diseases and can be used as a good model for studying diseases related to heart development.Objective: This part of the study will clarify the correlation between lncRNA NRON and developing heart and TOF,and clarify its distribution in cardiomyocytes,which is conducive to further study of its function.Methods:(1)The hearts of C57BL/6J mice at embryonic stage(E9.5,E10.5,E11.5,E13.5,E14.5),postnatal 7 days(P7)and adult(Adult)were collected,RNA was extracted and RT-PCR was performed to amplify lncRNA NRON,DNA gel electrophoresis was performed and lncRNA NRON was constructed temporal expression profile.(2)RT-qPCR was used to detect the difference in the expression of lncRNA NRON at different times in C57BL/6J mice,so as to clarify the correlation with the timing of heart development in mouse.(3)Eight normal embryonic hearts and eight TOF fetal hearts each with a mean gestational age of 20 weeks were collected,and the expression of lncRNA NRON was detected by RT-qPCR after RNA extraction from right ventricular tissue to observe whether there was a difference between the two groups to clarify the correlation between lncRNA NRON and TOF.(4)For studying its role in the cardiomyocyte nucleus,the localization of NRON was examined in human cardiomyocytes by RNAFish and subcellular fractionation assay.Results:(1)The transcription of lncRNA NRON starts at day E10.5of mouse embryonic development and is stably expressed from embryonic stage to adulthood thereafter.(2)The expression of lncRNA NRON was not uniform,showing an increasing trend from E10.5 days to P7,and then gradually decreasing thereafter until adulthood.(3)Compared with the normal control group,lncRNA NRON showed significantly lower expression in the embryonic heart of TOF group,and the difference was statistically significant(P<0.0001).(4)In h ESC-induced differentiated cardiomyocytes(h ESC-CM,HCM)and AC16 human cardiomyocytes,80%of NRON was localized in the nucleus.Conclusion:(1)The timing of lncRNA NRON expression has partial overlap with the critical time window of heart valve development in mouse and may be involved in heart development.(2)The low expression of lncRNA NRON and the occurrence of TOF are correlated.(3)The lncRNA NRON is mainly distributed in the nucleus of cardiomyocytes,laying the foundation for the study of its role in the nucleus.PART Ⅱ KNOCKOUT OF LNCRNA NRON INHIBITS HESC DIFFERENTIATION TO NORMAL CARDIOMYOCYTESBackground: The development of congenital heart disease is inextricably linked to abnormal myocardial differentiation.Previous studies have shown that abnormalities in core transcription factors(NKX2.5,HAND1,GATA4)and signaling pathways(Wnt signaling pathway,TGF-β signaling pathway)related to myocardial differentiation are associated with the development of TOF,suggesting that myocardial differentiation abnormalities may play a dominant role in the development of TOF,and any factors that lead to myocardial differentiation abnormalities may be a cause of TOF.Any factor leading to abnormal myocardial differentiation may be a cause of TOF.In the first part of the study,we found that lncRNA NRON was correlated with cardiac development and TOF.Objective: In the second part,we will investigate the effect of knocking out NRON on myocardial differentiation using the property that human embryonic stem cells(hESCs)can differentiate toward the myocardium.Methods :(1)The hESCs lncRNA NRON knockout cell lines(NRON-KO hESCs)and empty control cell lines(empty-vector hESCs)were constructed using epi CRISPR technology,and the homozygote and heterozygous strains were selected by gel electrophoresis of the internal primer PCR product(238 bp)and external primer(292bp),and the external primer PCR product was sequenced to verify the knockout success.(2)The two constructed cell lines were induced to differentiate in vitro,and the differentiation differences were observed by electron microscopy and cellular immunofluorescence.(3)RT-qPCR was performed to detect the expression differences of marker genes at different differentiation periods.Results:(1)Two homozygotes and forty-five heterozygous were successfully selected and the sequencing results of the external primer PCR products were consistent with the sequences of the ligated fragments after knockout,proving that the knockout was successful.(2)Cells in the empty-vector hESCs group began to see the first clusters of beating cardiomyocytes at day 8 of induction differentiation,and by day 12 almost 90% of the cells could see beating.Cell immunofluorescence confirmed that only a few cells were visible with c Tn T labeling,and the amount of fluorescence was significantly lower than that of the control group.(3)RT-qPCR results showed that the expression of the early mesodermal marker genes T and MIXL1 was significantly suppressed(P<0.001);the transcript levels of the core cardiac development transcription factor genes NKX2.5,MEF2 C and GATA4 were significantly lower than those of controls at days 4,6 and 8(P<0.001),resulting in the induction of differentiation of expression of cell-associated myocardial markers(ANP,BNP,α-MHC,β-MHC,TNNT2 and TNNT3)almost disappeared(P<0.0001).Conclusion:Knockout NRON affects the differentiation of hESCs into cardiomyocytes and prevents hESCs from differentiating into functional cardiomyocytes.The lncRNA NRON is essential for hESCs differentiation to cardiomyocytes.PART Ⅲ THE LNCRNA NRON ACTIVATED THE WNT/Β-CATENIN SIGNALING PATHWAY BY PROMOTING SHP2 NUCLEAR ACCUMULATIONBackground:The shp2 protein dephosphorylates Parafibromin in the nucleus and promotes its complex formation with β-catenin,thereby activating the Wnt classical signaling pathway.The in vitro differentiation protocol we adopted was able to complete the differentiation of hESCs into myocardium by using small molecules to modulate the Wnt/β signaling pathway,and the efficiency of myocardial cell differentiation was as high as 98%.The results of the first two parts of the study suggest that lncRNA NRON is mainly localized in the nucleus of cardiomyocytes,and hESCs cannot differentiate into functional cardiomyocytes after knockout of lncRNA NRON.Based on the differentiation scheme,it is speculated that knockout of lncRNA NRON may have interfered with the Wnt/β-catenin signaling pathway and resulted in the inability of hESCs to differentiate into cardiomyocytes.Objective:To validate the regulation of Wnt/β-catenin signaling pathway by lncRNA NRON and the proteins that may be involved,the third part will further investigate how NRON is involved in the regulation of cardiomyocyte development and its potential mechanisms.Methods:(1)RT-qPCR was performed to detect the transcript levels of Wnt/β-catenin signaling pathway downstream target genes,including cyclin D1,Mycand Axin2,in the knockout and control groups at 4 days of induction of differentiation.(2)NRON overexpression plasmid vector(pc DNA-NRON)was constructed to detect the regulatory effect of lncRNA NRON on Wnt/β-catenin signaling pathway using dual luciferase reporter gene.Atthe same time,the NRON-KO hESCs and empty vector hESCs cell lines were extracted from RNA and sent for sequencing.(3)Extracted nuclear proteins from AC16 human heart cells for RNA Pulldown and mass spectrometry(MS)to search for proteins interacting with lncRNA in the nucleus,and validated the results by western blot.(4)Western blot was used to detect the difference in the expression of NRON interacting proteins between the control group and the knockout group.(5)The effects of protein inhibitors on the lncRNA Nron-activated Wnt/β-catenin signaling pathway were detected using dual luciferase reporter genes.(6)LncRNA NRON was overexpressed in AC16 human cardiomyocytes,and the changes of protein localization in cells were detected by laser confocal microscopy..Results:(1)Compared with empty vector hESCs,the downstream target genes cyclin D1,Myc and Axin2 of the Wnt/β-catenin signaling pathway showed significant downregulation in NRON-KO hESCs,with statistically significant differences(P<0.001),while the expression of β-catenin was not affected(P> 0.05).(2)The TOPflash activity in HEK293 T cells transfected with both pc DNA-NRON and β-catenin was increased 2-fold compared with cells transfected with β-catenin only,and the difference was statistically significant(P<0.01).(3)RNA-seq and KEEG enrichment analysis of cells in the knockout and control groups revealed that genes down-regulated in expression in the NRON-KO hESCs cell line were enriched in the Wnt signaling pathway.(4)RNA Pulldown-MS revealed that shp2 protein specifically binds to NRON,which was supported by WB.(5)Dual luciferase reporter assays showed that the relative TOPflash luciferase activity was sharply decreased by more than 10-fold after the addition of TNO155,a potent inhibitor of shp2,and the difference was statistically significant(P<0.001).(6)WB analysis of shp2 showed no expression difference between empty-vector hESCs and NRON-KO hESCs.(7)Laser confocal results showed that shp2 accumulated mainly in the nucleus in cells transfected with pc DNA3.1-NRON,yet it accumulated in the cytoplasm in cells transfected with the empty-vector.Conclusion:(1)LncRNA NRON can activate the classical Wnt signaling pathway.(2)The lncRNA NRON interacts with shp2 in the nucleus and is involved in regulating myocardial differentiation by promoting nuclear aggregation of shp2 to activate the Wnt/β-catenin signaling pathway. |
PDF Full Text Request