The Role And Mechanism Of Circular RNA CircNfix In Cardiac Hypertrophy

Objective To explore the regulatory role and mechanism of circNfix in Isoproterenol(ISO)induced cardiac hypertrophy.Methods(1)We used the reverse primers of circNfix to perform polymerase chain reaction(PCR)amplification,agarose gel electrophoresis and Sanger sequencing to detect their circular structures and spliced junction sites.Phylop was used to conduct the conservation analysis.The Ribonuclease R(RNase R)was used to verify the stability of circNfix.(2)To explore the function of circNfix,SD rats cardiomyocytes hypertrophy was induced by ISO,and quantitative-time PCR(qRT-PCR)experiments verified the expression of circNfix in hypertrophy cardiomyocytes.Then overexpression and knockdown experiments were performed,and the expression of cardiomyocyte hypertrophy markers brain natriuretic peptide(BNP)and myosin heavy chain(?-MHC)was detected by qRTPCR.The surface area of cardiomyocytes was analyzed by phalloidin staining experiment.In vivo,8-week-old C57 BL / 6 mice were transfected with the circNfix overexpression vector.One week later,the animal model of cardiac hypertrophy was conducted by ISO in mice.Cardiac ultrasonic and HE cell staining were used to observe changes in cardiac function after circNfix transfection.The expression of BNP and ?-MHC in the heart was observed by qRT-PCR,and experiments further confirmed whether circNfix is involved in the regulation of cardiac hypertrophy in vivo.(3)Western Blot(WB)was used to detect the phosphorylation levels of p38 MAPK and CREB proteins when circNfix was overexpressed and knocked down.Results(1)The identification of circ Nfix: PCR amplification experiments using circNfix reverse primer found that cDNA could be used as a template to obtain amplification products,but genome DNA(gDNA)as a template couldn't be obtained,which proved that circNfix has a circular structure.Sanger sequencing confirmed that the sequence at the circNfix spliced junction is correct.circNfix is conservative in humans,mice and rats.Compared with linear mRNA,there was no significant change in the expression of circNfix after RNase R treatment.(2)The regulation role of circNfix: circNfix in ISO-induced cardiac hypertrophy was significantly down-regulated,while transfection with the overexpressed circNfix vector improved the ISO-induced cardiomyocyte hypertrophy.It showed that BNP and ?-MHC expression were downregulated,and phalloidin staining showed decreased surface area of cardiomyocytes.Knockdown of circNfix had the opposite effect.In vivo,overexpression of circNfix can significantly improved cardiac hypertrophy.Cardiac ultrasound showed a marked increase in fractional shortening,and a decrease in the diastolic left ventricular internal diameters.qRT-PCR showed that BNP and ?-MHC expression are down-regulated.Heart HE staining showed that the cells volume decreased.(3)The relationship of circNfix with p38MAPK/CREB pathway: Western Blot showed that the phosphorylation levels of p38 MAPK and downstream molecule CREB were up-regulated during cardiac hypertrophy,while the overexpression of circNfix inhibited the phosphorylation of p38 MAPK and CREB and knockdown of circNfix promoted phosphorylation.Conclusion This study explored the function of circNfix in cardiac hypertrophy using an ISO-induced cardiac hypertrophy disease model,the experiments showed that:(1)circNfix has a circular stable structure and was down-regulated in ISO-induced cardiac hypertrophy.(2)circNfix can inhibit cardiac hypertrophy in vivo and vito.(3)circNfix can inhibit the phosphorylation of p38 MAPK / CREB.