The Effect And Mechanism Of Drosha's Alternative Splicing On Cardiac Structure And Function

BackgroundHeart failure is a complex clinical syndrome caused by structural or functional heart disease that impairs ventricular congestion or drainage.Common causes include myocardial infarction and stress overload.Although there has been some progress in the treatment of heart failure in recent years,but it still has high morbidity and mortality,causing major economic and public health problems to society.The mechanism of the development of heart failure still needs more research to provide theoretical support.MicroRNA?miRNA?is a single-stranded small-molecule RNA with a length of about18-22 nucleotides that has post-transcriptional regulatory activity for gene expression.It is involved in the regulation of various biological processes in the body and islinked withvascular disease.In a mouse model of stress overload-induced heart failure,various miRNAs are abnormally expressed including miR-21,miR-23a,miR-23b,miR24,miR-195,miR-199a,miR-214,miR125b,miR-133a,miR-150,miR-181b,etc.MiRNA's biological function requires a series of protease processing.First,the miRNA gene is transcribed to form a primary miRNA?pri-RNA?,and processed by Drosha enzyme to form a precursor miRNA?precursor miRNA,pre-RNA?,and then cut by Dicerto form mature miRNAs and then combine with target genes in the form of RNA-induced silencing complex?RISC?to promote their degradation or translation inhibition.RNA alternative splicing is another universal regulatory mechanism for post-transcriptional gene expression regulation.The genes of eukaryotes are by exons and introns.Both are transcribed into precursor messenger RNA?pre-m RNA?under the action of transcription factors.These introns of the pre-m RNAs are cut by the action of the RNA spliceosome,and the exons are connected to form mature m RNAs,which in turn provide m RNA templates for protein translation.However,the definition of many exons is not absolute,but is regulated by a series of internal and external environments of the cell,so that the same precursor RNA can form different m RNA subtypes under different conditions,which may translate into significant different protein isoforms.Drosha is a key enzyme for miRNA synthesis and maturity.After consulting related databases and literatures,we found that Drosha has alternative splicing and has two subtypes of Drosha-L/Drosha-S.Combining a variety of miRNAs that are abnormally expressed in heart failure,we speculate that Drosha's alternative splicing may be involved in regulating the structure and functions of the heart.To this end,we designed this study to explore the role of Drosha's alternative splicing in cardiac structure and function,and further explore the regulatory mechanism of Drosha's alternative splicing in cardiac structure and function.Part?Abnormal transformationof Drosha's alternative splicing in myocardial infarction and pressure overload heart failure modelsObjective:To verify the existence of alternative splicing in Drosha and explore its abnormal transformation in myocardial infarction and stress overload heart failure.Methods:1.RNA was extracted from rat tissues and the expression of the key enzymes in miRNA synthesis was detected by polymerase chain reaction?PCR?agarose gel electrophoresis.2.Model of myocardial infarction was constructed by coronary artery ligation;model of stress overload heart failure was constructed by Transverse Aortic Constriction?TAC?.The expression of cardiac function markers was detected by Real-time Quantitative PCR?RT-q PCR?and the hematoxylin-eosin staining?HE?was used to detect the changes in cardiac structure.3.The expression of Drosha-L and Drosha-S in myocardial infarction and heart failure models was detected by RT-q PCR and the change of Drosha-L/S ratio was calculated.Results:The related literatures and databases were consulted to confirm that Drosha mainly has two subtypes of Drosha-L and Drosha-S.The difference lies in the jump of Exon7,and the existence of alternative splicing in rat tissues and hearts was verified by PCR.Electrocardiogram showed R wave elevation and ST segment elevation in myocardial infarction rats,HE staining and detection of cardiac function markers showed that myocardium thickened in the TAC group,and the expression of ANP,BNP and MYH7increased,indicating that the modelswere successfully constructed.Drosha-L/S decreased significantly at 8 and 12 hours after myocardial infarction,and Drosha-L/S increased significantly in TAC heart failure rats.Conclusion:There are alternative splicing forms of Drosha's RNA splicing.Drosha Exon 7 jumps generate two subtypes of Drosha named Drosha-L and Drosha-S.Drosha's alternative splicing occurs abnormally in the state of myocardial infarction and heart failure.Part?The impact of Drosha's alternative splicing on the structure and function of the heartObjective:To investigate the effects of Drosha's alternative splicing transformation on cardiac structure,cardiac function,and myocardial cell proliferation.Methods:1.Drosha Exon 7 knockout rats were obtained by CRISPR-Cas9 gene knockout technology,and different genotype knockout rats were obtained by hybridization.The genotype was identified by genome identification and PCR of cardiac tissue.2.Cardiac function was detected by cardiac ultrasound and/or hemodynamic methods in rats of different genotypes and rats of stress overload heart failure,and cardiac function markers were detected by RT-q PCR.HE staining was used to detect the structure of the rats'heart.3.Immunohistochemical method was used to detect the expression of PCNA in rats with different genotypes at 4 weeks of age.4.Western blot was used to detect the expression of Drosha Exon 7 knockout rat cardiomyocyte proliferation-related proteins in different genotypes of different age and pressure overload models.Results:1.CRISPR-Cas9 was used to construct Drosha Exon 7 knockout rats.Genome identification could confirm the rat genotype.2.Cardiac ultrasound showed no significant difference in cardiac function in 4-week-old rats.Hemodynamics suggested that there was no significant difference in heart LVSP,+dp/dtmax,and-dp/dtmax between 14-week-old adult Drosha Exon 7 knockout rats compared with wild-type rats.Lvedp of Drosha Exon 7^+/-and Drosha Exon 7^-/-was significantly reduced,suggesting a reduction in preload.RT-q PCR results showed that the expression of cardiac function markers in Drosha Exon 7 knockout rats at 4 weeks and 14 weeks was reduced.HE staining showed no significant difference in the size of myocardial cells and wall thickness in different genotypes of rats at 4 and 14 weeks of age.3.Cardiac ultrasound showed that EF,FS,and LVPW of 1-year-old Drosha Exon 7 knockout rats were significantly higher than those of wild type.Hemodynamics showed that LVSP?+dp/dtmax and-dp/dtmax of 1-year-old Drosha Exon 7 knockout rats were significantly higher than wild-type rats,and ANP and BNP were significantly reduced by RT-q PCR.The HE staining results showed that the myocardial cells size of Drosha Exon7 knockout rats increased significantly,and the thickness of heart wall increased.4.The survival rate of Drosha Exon 7^-/-TAC was significantly lower than that of wild type.Hemodynamic results showed that LVSP,+dp/dtmax,-dp/dtmax of Drosha Exon 7knockout rats in pressure overload model were significantly lower than those of wild type rats,and ANP and BNP were significantly increased by RT-q PCR results.HE staining showed that the myocardial cells of Drosha Exon 7 knockout rats were significantly enlarged,and the wall thickness of heart wall was significantly higher than that of wild type rats.5.PCNA immunohistochemical staining revealed that PCNA expression in Drosha Exon 7 knockout rats was reduced.The results of cell cycle and proliferation-related proteins measured by WB method showed that the expressions of Cyclin A and PCNA were reduced in 4-week-old Drosha Exon 7 knockout rats.The expression of Cyclin A,Cyclin B,and PCNA in Drosha Exon 7 knockout rats of 14-week-oldand pressure overload models increased.There was no significant difference in the expressions of Cyclin A?Cyclin B?Cyclin D2?Cyclin D3?PCNA and BCL2 in rats of different genotypes at 1 year of age.Conclusion:Drosha Exon 7 knockout does not affect cardiac development and can reduce cardiac preload in adult rats.Drosha Exon 7 knockout can ameliorate heart failure caused by aging through myocardial hypertrophy.Drosha Exon 7 knockout promotes the development of heart failure by aggravate myocardial hypertrophy in the pathological situation of pressure overload heart failure.Drosha Exon 7 knockout can inhibit the proliferation of cardiomyocytes in 4-week-old rats and promote the proliferation of cardiomyocytes in 14-week-old rats.The pathological conditions of pressure-overloaded heart failure also maintain the role of promoting proliferation,but disappear with age to 1year old.Part?Exploration of the mechanism of Drosha's alternative splicing in regulating cardiac structure and functionObjective:To explore the mechanisms by which Drosha's alternative splicing regulates cardiac structure,cardiac function,and myocardial cell proliferation.Methods:1.The effect of Drosha Exon 7 knockout on miRNAs expression in 4-week-old rats was detected by RT-q PCR.2.RNA-seq was used to detect the differentially expressed genes by Drosha Exon 7 knockoutin 4-week-old rats,and the related signal pathway changes were analyzed by bioinformatics methods.3.Western blot was used to detect the expression of Drosha Exon 7 knockout rat cardiac hypertrophy-related signaling pathway proteins in different genotypes of different age and pressure overload models.Results:1.RT-q PCR was used to detect the expression of miRNAs in 4-week-old ratsof different genotypes for 4 weeks.It was found that knockout of Drosha Exon 7 can reduce the expression of various miRNAs with different functions.2.RNA-seq results were analyzed by bioinformatics and screened genes were verified by RT-q PCR to found that differential genes of 4-week-old Drosha Exon 7 knockout and wild type rats were mainly enriched in cell cycle,and these genes were regulated by various miRNAs.3.WB method was used to detect the expression of myocardial hypertrophy-related signaling pathway proteins in 4-week-old,14-week-old,1-year-age and pressure overload heart failure rats.The results showed that the expression of MKK6,P-MKK3/6,and P-P38 in 4-week-old Drosha Exon 7 knockout rats was significantly lower than that of wild type.The expressions of MKK6,MKK3,m TOR and P-m TOR in 14-week-old Drosha Exon 7 knockout rats were significantly reduced,while the expressions of SRC,P-ERK and AKT were significantly increased;the expression of P-AKT is different in heterozygotes and homozygotes,and the expression in Drosha Exon 7^+/-is reduced while there is no significant difference in Drosha Exon 7^-/-.The expression of P-PLC?,PKA,P-PKA,SRC,and P-P38 in Drosha Exon 7 knockout rats at 1 year oldwas significantly reduced,while the expressions of ERK and P-ERK were significantly increased;P-AKT,m TOR and P-m TOR expression levelsin Drosha Exon 7^+/-were significantly higher than those of wild type,but there was no significant difference in Drosha Exon 7^-/-.In the model of pressure overload heart failure,the expressions of P-PKA,P-ERK,MKK6,MKK3,P-MKK3/6,P-P38,MAPKAPK2,P-MAPKAPK2 and P-m TOR in Drosha Exon 7 knockout rats were all obvious increased,the expression levels of P-PLC?and SRC significantly decreased;ERK expression of Drosha Exon 7^-/-decreased,m TOR expression increased without significant differences in Drosha Exon 7^+/-;AKT expression of Drosha Exon 7^+/-was significantly reduced while Drosha Exon 7^-/-was not significantly changed.Conclusion:The abnormal transformation of Drosha's alternative splicing altered the expression of multiple miRNAs.These miRNAs work together to regulate the proliferation of cardiomyocytes by regulating cell cycle signaling pathways.Drosha alternative splicing may participate in regulating the structure and function of the heart through multiple miRNAs.MAPK and AKT signaling pathways participated in the regulation of myocardial hypertrophy by Drosha alternative splicing.Different changes in the P38 MAPK signaling pathway may be the main reason for the difference between physiological and pathological myocardial hypertrophy caused by Drosha Exon 7 knockout.