| BackgroundAutophagy is an evolutionarily conserved biological process that is lysosomal-mediated and maintains cellular metabolism and internal environment homeostasis by degrading intracellular proteins and damaged organelles.Autophagy can be roughly divided into macro-autophagy,micro-autophagy,and chaperone-mediated autophagy.Macro-autophagy is commonly known as autophagy.Autophagy is an inherent biological behavior of cells.Cardiomyocytes have autophagy behaviors under normal circumstances,and autophagic activity would be changed in response to stress,ischemia,and hypoxia.Autophagy is involved in the occurrence of various cardiovascular diseases,which influences the maintenance of cardiac function and the prognosis of the heart diseases.Normal levels of autophagic activity can protect cells from environmental stimuli,but excessive autophagy and insufficient autophagy can lead to cardiovascular diseases.Cardiac hypertrophy is a common pathological process of a variety of heart diseases.Early cardiac hypertrophy is an adaptive change of cardiomyocytes in response to overload stress,which can maintain the normal function of the heart.However,increased oxygen consumption of cardiomyocytes could reduce heart function and lead to myocardial ischemia.Studies have found that there is a close relationship between cardiomyocytes autophagy and cardiac hypertrophy.Although there have been many studies on cardiomyocytes autophagy and cardiac hypertrophy,the specific regulation mechanisms of autophagy and hypertrophy are still unclear.Long non-coding RNAs(long non-coding RNAs,lnc RNAs),as a class of newly discovered endogenous RNA molecules,have been the focus of research in recent years.They are over 200 bases in length and have no ability to encode proteins.Lnc RNAs were previously considered to be the "transcription noise" of genes and do not have biological functions.With the application and development of next-generation high-throughput sequencing technology,a large number of lnc RNAs with rich biological functions have been continuously discovered.Increasing studies have revealed that lnc RNAs are widely involved in the regulation of gene expression and play a vital role in the occurrence and development of cell proliferation,differentiation,apoptosis,and autophagy.Lnc RNAs can regulate gene expression through multiple levels by gene imprinting,chromatin remodeling,splicing regulation,m RNA degradation and translation regulation,and participate in the regulation of pathophysiological processes.The abnormal expression of lnc RNAs is related to a variety of human diseases,including tumors,neurodegenerative diseases,metabolic diseases and cardiovascular diseases.Transcribed ultra-conserved noncoding RNAs(T-UCRs)are a special class of lnc RNAs transcribed from ultra-highly conserved sequences and highly conserved among different species.The role of T-UCRs in the development of tumors has been reported.However,the research of T-UCRs in the development of cardiovascular diseases is still in its infancy.Therefore,further exploration research on the molecular mechanism of T-UCRs in cardiomyocytes autophagy and hypertrophy will enrich and expand the regulatory network of cardiomyocytes autophagy and hypertrophy,and provide new directions and basis for the development of prevention and treatment drugs of cardiovascular diseases.Aims1.Construct cardiomyocytes autophagy and cardiac hypertrophy model induced by angiotensin II and screen dysregulated lnc RNAs/m RNAs to determine the target lnc RNAs.2.Identify the role of uc.88 involved in regulating cardiomyocytes autophagy and cardiac hypertrophy.3.Clarify the molecular mechanism of uc.88 inhibiting cardiac hypertrophy by promoting cardiomyocytes autophagy.Methods1.Expression profile of lnc RNAs/m RNAs during cardiomyocytes autophagy and cardiac hypertrophyNeonatal rat cardiomyocytes were isolated and cultured from by collagenase digestion combined with differential adherence methods.Subsequently,0.1umol/L of angiotensin II was used to stimulate the myocardial cells for different times(12 hours,24 hours,36 hours,and 48 hours),and the changes of cardiomyocytes cell surface area,hypertrophic genes expression levels and autophagic activity after angiotensin II stimulation were detected to identify the best time of angiotensin II to induce cardiomyocytes autophagy and hypertrophy.Total RNA was collected from cardiomyocytes after normal culture and angiotensin II stimulation,and differentially expressed lnc RNAs and m RNAs in cardiomyocytes autophagy and hypertrophy were identified by lnc RNA array.Then,GO analysis and pathway analysis were applied to determine the biological processes that these differentially expressed genes during cardiomyocytes autophagy and hypertrophy induced by angiotensin II might be involved in.2.Functional exploration of uc.88 in cardiomyocytes autophagy and cardiac hypertrophyThe expression of uc.233,uc.88 and uc.378 was detected by q RT-PCR after angiotensin II stimulated primary neonatal rat cardiomyocytes.In rat,AC16 and mouse cardiomyocytes,the expression level of uc.88 was detected by q RT-PCR,and then the subcellular localization of uc.88 was determined by nuclear separation method and q RT-PCR.Primary neonatal rat cardiomyocytes were transfected by interfering RNA targeted uc.88 and angiotensin II was used to induce cardiomyocytes autophagy and hypertrophy after measuring the transfection efficiency.The expression of autophagic marker proteins BECN1,LC3II/I was detected by western blot,and autophagosomes in cardiomyocytes were observed by transmission electron microscopy,and the GFP-m RFP-LC3 dual fluorescence tracer system was used to detect autophagy flow in cardiomyocytes to explore the role of uc.88 in cardiomyocytes autophagy.To determine the function of uc.88 in cardiomyocytes hypertrophy induced by angiotensin II,the changes of cardiomyocytes cell surface area and the expression of hypertrophy markers Nppa and Nppb were detected.3.The mechanism of uc.88 inhibiting cardiac hypertrophy by promoting cardiomyocytes autophagyIt was determined by biological information analysis method that only TANK exists in the area near the uc.88 gene locus.The expression level of TANK in rat,AC16 and mouse cardiomyocytes was detected by q RT-PCR.QRT-PCR and Western blot were used to detect the expression changes of TANK at the m RNA and protein levels after inhibition of uc.88.The expression of uc.88 was detected by q RT-PCR after overexpression/inhibition of TANK.RIP technology was applied to determine the internal relationship between uc.88 and TANK.Transfection of primary neonatal rat cardiomyocytes with adenovirus system to overexpresses TANK and angiotensin II was used to induce cardiomyocytes autophagy and hypertrophy after measuring the transfection efficiency.The expression of autophagic marker proteins BECN1,LC3II/I was detected,and autophagosomes in cardiomyocytes were observed by transmission electron microscopy,and the GFP-m RFP-LC3 dual fluorescence tracer system was used to detect autophagy flow.The changes of cardiomyocytes cell surface area and the expression of hypertrophy markers Nppa and Nppb were detected in order to determine the function of TANK in cardiomyocytes hypertrophy.Subsequently,simultaneous inhibition of uc.88 and TANK,or inhibition of uc.88 and overexpression of TANK in cardiomyocytes,then angiotensin II was used to induce cardiomyocyte autophagy.The expression of autophagic markers and the number of autophagic flux were detected to determine the changes of cardiomyocytes autophagic activity.Western blot was applied to identify the changes of related autophagy signaling pathways.In angiotensin-induced cardiomyocyte hypertrophy model,uc.88 small interfering RNA+ autophagy activator(Rapa)or uc.88 small interfering RNA+ autophagy inhibitor(3-MA)were used to intervene in cardiomyocytes.The changes of cardiomyocytes cell surface area,hypertrophic markers and autophagic markers were detected to investigate the mechanism of uc.88 regulating cardiac hypertrophy.Finally,a mouse cardiac hypertrophy animal model constructed was constructed by angiotensin II slow-released pump in order to identify the administration of uc.88 on the process of cardiac hypertrophy.Results1.Differently expressed lnc RNAs/m RNAs during cardiomyocytes autophagy and hypertrophyNeonatal rat cardiomyocytes were successfully isolated from 1-to 2-day-old Sprague-Dawley rats by collagenase digestion combined with differential adherence methods.After angiotensin II stimulation of primary cardiomyocytes for 12 hours,the expression levels of autophagic proteins BECN1 and LC3II/I were significantly increased.Transmission electron microscopy results showed that the number of autophagosomes in cardiomyocytes was also increased after angiotensin II stimulation,and the surface area of cardiomyocytes did not change significantly at this time,which suggested that the cardiomyocytes autophagic model was successfully constructed.After 48 hours of angiotensin II stimulation,the surface area of cardiomyocytes increased significantly and cardiomyocytes autophagic activity was maintained at a high level.A total of 1249 significantly differently expressed lnc RNAs and 197 differentially expressed m RNAs were screened during cardiomyocytes autophagy through the lnc RNA array.Among them,43 were differentially expressed T-UCRs,and most of the differentially expressed lnc RNAs were intergenic lnc RNAs.There were 1577 significantly differently expressed lnc RNAs and 496 m RNAs differentially expressed during cardiomyocytes hypertrophy.Among them,59 differentially expressed T-UCRs were screened.Most of the differentially expressed lnc RNAs were intergenic lnc RNAs.Among them,uc.88,uc.233 and uc.378 were differentially expressed during both of cardiomyocytes autophagy and hypertrophy.The expressions of uc.88 and uc.233 are upregulated and the expression of uc.378 is downregulated.The results of GO analysis and pathway analysis indicated that the differentially expressed genes during cardiomyocytes autophagy were mainly related to cellular energy and metabolic pathways,and the differentially expressed genes during cardiomyocytes hypertrophy were mainly related to epithelial-mesenchymal transition and insulin secretion regulatory pathways.2.Functional study of uc.88 in cardiomyocytes autophagy and hypertrophyAfter angiotensin II stimulated the primary rat cardiomyocytes for 12 hours,the expression level of uc.88 was significantly increased.With the extension of the stimulation time,the expression of uc.88 was stable and showed a time-dependent change.After angiotensin II stimulation,the expression of uc.233 first increased,then decreased,and then gradually increased.The expression of uc.378 first decreased and then increased,and its expression gradually decreased after 24 hours of stimulation.In AC16 cardiomyocytes and mouse cardiomyocytes,the expression of uc.88 was increased after angiotensin II stimulation,and increased by a time-dependent manner and was consistent with uc.88 expression in primary rat cardiomyocytes.Nuclear separation test combined with q RT-PCR analysis revealed that uc.88 was distributed in cytoplasm and nucleus.After inhibiting the expression of uc.88 in primary rat cardiomyocytes,the expression levels of autophagy proteins BECN1 and LC3II/I were significantly reduced,and the number of autophagosomes and autophagic streams was significantly reduced.After silencing of uc.88,the surface area of cardiomyocytes increased significantly,and the expression levels of Nppa and Nppb were also increased significantly.3.The mechanism of uc.88 inhibiting cardiac hypertrophy by promoting cardiomyocytes autophagyUsing bioinformatics analysis method,only TANK exists in the area near the uc.88 gene locus.After angiotensin II stimulated primary rat cardiomyocytes,AC16 cardiomyocytes,and primary mouse cardiomyocytes,q RT-PCR results showed that the expression of TANK was stable and consistent among different species,increasing by time-dependent manner.In human and rat cardiomyocytes,inhibition of uc.88 could reduce TANK expression.However,after overexpression/inhibition of TANK,the expression of uc.88 in cardiomyocytes did not change.RIP assay showed that uc.88 could specifically combine with TANK.After overexpression of TANK,the expression of BECN1 and LC3II/I was increased in cardiomyocytes,and the number of autophagosomes and autophagic flow was also increased.The surface area of cardiomyocytes was decreased,and the expression of Nppa and Nppb was also decreased.By inhibition of uc.88 and TANK together,the expression of BECN1 and LC3II/I in cardiomyocytes was significantly reduced,and the number of autophagosomes was also significantly reduced.After inhibition of uc.88 and overexpression of TANK,the expression of BECN1 and LC3 II / I in cardiomyocytes was increased.The number of autophagosomes was increased.Meanwhile,AMPK and m TOR signaling pathways were activated.Autophagy activator Rapa could promote uc.88-induced autophagy and inhibit angiotensin II-induced cardiac hypertrophy,and autophagy inhibitor 3-MA inhibited uc.88-induced autophagy and promoted angiotensin II-induced cardiac hypertrophy.Finally,an angiotensin II sustained-release pump was used to construct an animal model of cardiac hypertrophy in mice.Injection of uc.88 inhibitor could aggravate the process of cardiac hypertrophy in mice.Conclusions1.Angiotensin II(0.1umol/L)stimulated the primary rat cardiomyocytes for 12 hours to successfully establish a model of cardiomyocytes autophagy;after 48 hours of stimulation,a cardiac hypertrophy model was successfully established.2.A total of three T-UCRs(uc.88,uc.233 and uc.378)were differentially expressed in both of cardiomyocytes autophagy and hypertrophy,and the expression of uc.88 was stable and increased in a time-dependent manner after angiotensin II stimulation.3.Inhibition of uc.88 could inhibit angiotensin II-induced cardiomyocytes autophagy,and promote angiotensin II-induced cardiac hypertrophy.4.Overexpression of TANK promoted angiotensin II-induced cardiomyocytes autophagy,and inhibited angiotensin II-induced cardiac hypertrophy.5.uc.88 could promote cardiomyocytes autophagy by mediating TANK expression,which inhibited cardiac hypertrophy induced by angiotensin II. |
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