The Role And Mechanism Of LncRNA XIST In Cardiac Hypertrophy

Background:Cardiac hypertrophy is the physiological adaptive response of the heart to maintain cardiac function against cardiac physiological and pathological overloading.Cardiac hypertrophy is mainly characterized by an increased cardiomyocyte size and heart mass.However,sustained pathological stress will lead to pathological cardiac hypertrophy,which is mainly manifested by enlarging cardiomyocyte size,cardiac fibrosis,cardiomyocyte death,increased collagen synthesis,and myofibroblasts activation.Together,these alterations result in maladaptive cardiac remodeling,which ultimately leads to heart failure and sudden death.Therefore,understanding of the pathogenesis of cardiac hypertrophy are helpful to prevent the progression of cardiac hypertrophy in the early stages and treat heart failure.Long non-coding RNAs(LncRNAs)are a sort of RNA transcripts with over 200 nucleotides and lack an open reading frame,thereby cannot encode proteins.At present,many studies have shown that lnc RNAs are involved in the regulation of various physiological and pathological processes of the heart,and are closely related to the pathogenesis of various cardiovascular diseases.More and more studies have shown that lnc RNAs are important regulatory factors in the progression of cardiac hypertrophy.LncRNA XIST is the main regulator of mammalian X chromosome transcriptional silencing and is a characteristic molecular change in the pathophysiological process of cardiac hypertrophy.However,the role and underlying mechanism of XIST in the occurrence and development of cardiac hypertrophy has not yet been fully elucidated.Object:Our research aims to explore the roles of XIST in the occurrence and development of cardiac hypertrophy and investigate the potential molecular mechanism.Method:Transverse aortic constriction(TAC)was used to establish the animal model of pathological cardiac hypertrophy.To evaluate whether the modeling was successfully established,we conducted histopathology and morphometric analysis and assayed the HW/BW ratio of mice,echocardiography,and the expression of cardiac hypertrophyrelated genes(ANP、BNP、β-MHC)using qPCR and Western Blot.In addition,the levels of XIST,miR-126 a,and IGF-1 in the myocardium were detected at the same time.Cell surface measurement is also conducted by assaying immunofluorescence staining,protein/DNA ratio in Ang Ⅱ-treated HL-1 cell cardiac hypertrophy model.Cardiomyocyte hypertrophy indicators,ANP、BNP,and β-MHC are also detected by qPCR and Western Blot.To evaluate the effect of XIST on Ang Ⅱ-induced cardiac hypertrophy,HL-1 cells were transfected with si-XIST and cell surface measurement is conducted by immunofluorescence staining,protein/DNA ratio,and the expressions of ANP,BNP,β-MHC,and IGF-1 are detected by qPCR and Western Blot.The potential binding sites of XIST and miR-126 a are predicted by bioinformatic assays,and are confirmed by the luciferase reporter assay.To evaluate the effect of miR-126 a on Ang Ⅱ-induced cardiac hypertrophy,HL-1 cells were transfected with miR-126 a mimics,cell surface measurement is conducted by immunofluorescence staining,protein/DNA ratio,the expressions of ANP,BNP,and β-MHC are detected by qPCR and Western Blot.Bioinformatics predicts that potential binding sites between IGF-1and miR-126 a,and the luciferase reporter assay confirms the relation between IGF-1and miR-126 a.The expressions of Akt and p-Akt were detected by Western Blot in Ang Ⅱ-treated HL-1 cells that transfected with si-XIST.Result:(1)Establishment of in animal models of cardiac hypertrophy:After four weeks of TAC,the volume of the heart was significantly increased in the TAC group compared with the Sham group.Histological analysis revealed that the cross-sectional area of the heart was substantially enlarged in the TAC group compared with the Sham group.Echocardiography analysis demonstrated that LVEDd and LVPWd in the TAC group were significantly higher than in the Sham group.The levels of EF and FS were lower in the TAC group than that in the Sham group.A higher HW/TL ratio was found in TAC mice than that of Sham mice.The expressions of hypertrophic markers atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),and β-myosin heavy chain(β-MHC)were significantly increased in the TAC group.(2)Establishment of in cell models of cardiac hypertrophy:HL-1 cells were exposed to Ang Ⅱ at a concentration of 150 nmol/L for 24 hours to induce cardiomyocyte hypertrophy.Compared with the control group,the cell surface area,protein/DNA ratio,and the expressions of hypertrophic markers were increased in the Ang Ⅱ group.(3)The expression of XIST was up-regulated both in vivo and in vitro models of cardiac hypertrophy.PCR analysis demonstrated that si-XIST transfection resulted in a significant decrease in XIST expression in HL-1cells.Immunofluorescence staining for α-actinin displays that silence XIST diminished the enlargement of the cell surface area induced by Ang Ⅱ.Similarly,silence XIST also reduced the protein/DNA ratio in Ang Ⅱ treated HL-1 cells.Furthermore,silence XIST could also inhibit the expressions of hypertrophic markers(ANP、BNP、β-MHC)in Ang Ⅱ treated HL-1cells.(4)The expression of miR-126 a was downregulated both in vivo and in vitro models of cardiac hypertrophy.There is a negative correlation between the expression of miR-126 a and XIST in the AngⅡ-induced cell model of cardiac hypertrophy.The luciferase reporter assay confirms that XIST could directly bind with miR-126 a.(5)PCR analysis demonstrated that miR-126 a mimics transfection led to a marked increase in miR-126 a expression in HL-1cells.Overexpression of miR-126 a could reverse the increase in cell surface area induced by Ang Ⅱ.Similarly,overexpression of miR-126 a could decrease the protein/DNA ratio and inhibit the expressions of hypertrophic markers(ANP,BNP,β-MHC)in Ang Ⅱ-treated HL-1cells.(6)The expression of IGF-1 was upregulated both in vivo and in vitro models of cardiac hypertrophy.Inhibition of XIST or over-expression of miR-126 a could inhibit the expression of IGF-1 in Ang Ⅱ treated HL-1 cells.The luciferase reporter assay confirms that IGF-1 could directly bind with miR-126 a.(7)In Ang Ⅱ-treated HL-1 cells,the protein level of p-Akt was significantly increased,while silenced XIST decreased the protein level of p-Akt.Conclusion:In animal and cell models of cardiac hypertrophy,LncRNA XIST and IGF-1expression were up-regulated,while miR-126 a expression was down-regulated.Silencing XIST or overexpression of miR-126 a can inhibit Ang Ⅱ-induced cardiomyocyte hypertrophy,suggesting that XIST exerts deteriorating effects in AngⅡ-induced cardiomyocyte hypertrophy.LncRNA XIST promotes the development of cardiac hypertrophy to regulate the IGF-1/Akt pathway by sponging miR-126 a.Innovation and significanceThis study confirmed that LncRNA XIST could promote cardiac hypertrophy.Furthermore,XIST could activate the IGF-1/Akt signaling pathway by competitively binding miR-126 a in the progression of cardiac hypertrophy,which provided a novel therapeutic strategy for cardiac hypertrophy and heart failure.