Study On The Regulatory Effect Of MiR-1976 Overexpression On Cav1.2 And Cav1.3

Objective:The application of qRT-PCR and Western blot combined with the ECG detection results of experimental rabbits and transgenic mice to explore the relationship between miR-1976 and pace-making gene Cav1.2 and Cav1.3.Then,explore the molecular mechanism of age-related sinoatrial node dysfunction.Method:1.From Kunming Medical University experimental animal department,Chenggong Area,Kunming,acquired a total of 15 healthy Japanese white rabbits of either gender,divided into 5 experimental groups according to age,respectively,in 6 months,1 year,2 years,3 years,4 years group.The rabbits have been grouped would be stoped feeding 12 hours before the experiment.In the experiment,anesthesia with the same time and the same dose isoflurane,and print ECG recording,then draw the material from rabbits’sinoatrial node in animal operation room.And stored that in tanks of liquid nitrogen,protect RNA and protein from degradation.Final,experiment with sinoatrial node use qRT-PCR and Western blot,obtain the experimental results,combined with the ECG data analysis.2.We purchased 12 miR-1976 over-express C57BL/6 mice from Cyagen Biosciences Inc,3 males and 4 females(Born on November 18,2015),4 males and 1 female(Born on November 30,2015),They were raised in Kunming Medical University experimental animal department(SCXK(Yunnan)K2015-0005,SYXK(Yunnan)K2015-0002),Acquired F2 and F3 generation.The transgenic mice tail,the tissue was identified by DNA extraction and PCR,get the wild type,heterozygous and homozygous mice.The reproduction of F2 wild type,heterozygous and homozygous mice were phenotypic observation and recording of ECG,and the data will be summarized.Result:1.With the increase of rabbits’ age,there was a gradual decline in heart rate,especially 6 months rabbits’average heart rate compared with 3 years rabbits’and 4 years rabbits’,the average heart rate were reduced with increasing age,There is a significant differrence.And the expression of miR-1976 increased gradually with the increase of rabbits’ age.Then,we check for the proteins of different age groups rabbits’pace-making gene Cav1.2 and Cav1.3 relate to miR-1976,we found that with the increase of rabbits’ age,Cav1.2 and Cav1.3 expression was decreased.We made a correlation analysis on miR-1976 and pace-making gene expression,there is a negative correlation between miR-1976 and pace-making gene Cav1.2,Cav1.3.Their correlation coefficients respectively are R2=0.7739 and R2=0.8333,which showed significant difference and P<0.05.2.In miR-1976 over-expression transgenic mice,homozygous mice were grew slower than wild type mice,and their sizes are smaller.The average heart rate in each experimental group,the statistical analysis found that wild type mice,heterozygous mice and homozygous mice showed the average heart rate,gradually decreased.Conclusion:miR-1976 may down regulate the transcription and expression of target gene Cav1.2,Cav1.3,resulting in sinus node dysfunction,cause animals’developmental retardation and the average heart rate decreased.