Basic Research Of β₁ Adrenergic Receptor Gene Methylation On The Different Expression And Correlation With Blood Pressure In Spontaneously Hypertensive Rats

Objections:1. To investigate the role of DNA methylation in the control of expression ofβ1-AR gene.2. Detection the expression ofβ1-adrenergic receptor (β1-AR) between different individuals in spontaneously hypertensive rats (SHR) whose genotype is consistent.Methods:1.Forty SHRs blood pressures were measured at 14:00-16:00 every day.2.Myodardium and common carotid artery blood were gained. Using gene sequencing to check SHRβ1-AR genotype at first; extraction of myocardial tissue RNA, realtime-PCR,RT-PCR were used for myocardial mRNA expression ofβ1-AR gene; methylation specific PCR (Methylation specific PCR, MSP) and bisulfite sequencing were applied to detect methylated status inβ1-AR gene; the protein expression ofβ1-AR were identified by Western blot.3.Then they were randomly divided into 176-185mmHg group, 186-195mmHg group,196-205mmHg group according their blood pressure.Results:1.General situation of experimental animals:forty SHRs finished experimental research, systolic blood pressure, heart rate, body length, tail length, body weight, heart weight showed a normal distribution (P>0.05). There was no difference in any two groups of SHR in heart rate, body length, tail length, body weight, heart weight (P> 0.05)2. Bioinformatics analysis:no mutation was detected in SHRβ1-AR gene sequencing, demonstrates that the genotype is consistent; bioinformatics analysis showed that there are methylation modification situs inβ1-AR gene of rat. MSP and bisulfite sequencing results showed that the SHRβ1-AR gene DNA have methylation.3. Effect of methylation in the expression of SHRβ1-AR gene: Expression of myocardialβ1-AR gene mRNA in methylation group and non-methylation group are 27.8±21.7,110.2±87.5 respectively(P< 0.05). Expression of myocardialβ1-AR gene in both group are 0.2±0.1,0.4±0.1 respectively(P<0.05). But there was no significant difference in systolic blood pressure of high protein expression group and lower expression group (P> 0.05).4. The expression of three groups of experimental animals:RT-PCR shows expression of myocardialβ1-AR mRNA in the three groups are 0.21±0.04,0.27±0.02,0.33±0.03 respectively, Realtime Reverse qPCR shows expression of myocardialβ1-AR mRNA in the three groups are 25.6±6.4,67.0±15.5,149.3±10.6 respectively; Expression of myocardialβ1-AR gene in both group are 0.25±0.08,0.33±0.09,0.41±0.06 respectively, demonstrates that different tissues of SHR cardiacβ1-AR expression was different, the difference was significant (P<0.05).Conclusions:1.β1-AR gene promoter methylatied in SHR myocardium, and influence the expression ofβ1-AR gene.2.SHRβ1-AR expressed differently, the expression tended to increase with the increase of the blood pressure.