The Regulation And Mechanism Of MiRNA-145on Phenotype Of Arterial Vascular Smooth Muscle Cells In Spontaneously Hypertensive Rats

Part1:The expression of miR-145and determination of cell phenotypes in arterial vascular smooth muscle cells of SHRObjectives:(1)To observe the expression of miR-145in the thoracic aortic VSMC of SHR at8、12、16、20weeks old, and compared with the expression of Wistar rats at same weeks old respectively;(2) To detect the expression of cell phenotypic markers of a-SMA and SMemb in the thoracic aortic VSMC of SHR at8、12、16、20weeks old, and compared with the expression of Wistar rat’s at same weeks old respectively; Systolic arterial pressure (SAP)、 diastolic arterial pressure (DAP) and the average arterial pressure (MAP) of SHR and Wistar rats were measured respectively at the end of8、12、16、20weeks old, and compared with each other; Thoracic aortic intima-media thickness (MT) and the percentage of membrane area (WA)/lumen area (LA) of SHR and Wistar rats were measured respectively at the end of8、12、16、20weeks old, and compared with each other; Correlational analysis were conducted between the relative expression of miR-145and MT and SAP of the corresponding weeks old in SHR. Methods:Real-time PCR technique was used to detect the expression of miR-145in thoracic aortic VSMC of SHR and Wistar rat at different weeks old;Real-time PCR and Western blot methods were used to detect the expression of a-SMA and SMemb mRNA and protein of VSMC phenotypic marker in the thoracic aortic of SHR and Wistar rats; ALC-NIBP noninvasive blood pressure measurement analysis system were used to measure the blood pressure of SHR and Wistar rats in different weeks old. HE staining slices were used to observe the morphology、 the membrane thickness (MT) and the percentage of membrane area (WA)/lumen area (LA) of thoracic aortic VSMC in SHR and Wistar rat.Results:(1)The relative expression of miR-145in SHR is lower than that in Wistar rats (p<0.05); the relative expression of miR-145was gradually decreased with the increase of weeks old in SHR (p<0.05),but there was no statistical significant in the SHR group after16weeks;The relative expression of miR-145has no obvious difference in Wistar rat with different weeks old (p>0.05);(2) The relative expression of mRNA and protein of a-SMA in SHR is significantly lower than that of the Wistar rats (p<0.05);while The SMemb is obviously higher than that of Wistar rats (p<0.05);The relative expression of mRNA and protein of a-SMA decrease gradually with the increase of weeks old in SHR (p<0.05);while the mRNA and protein of SMemb increase gradually, but there is no statistically significant difference after16weeks in the same SHR group(p>0.05);There are no significant difference in the relative expression of mRNA and protein of a-SMA and SMemb between Wistar rats with different weeks old(p>0.05);(3) SAP、 DAP and MAP of SHR are obviously higher than that of Wistar rats (p<0.05),and increased with the weeks old(p<0.05); Blood pressure increases up to peak after16weeks, and then maintain this high level; Blood pressure of Wistar rats do not increase with weeks and maintain the normal range.(4) Aortic wall in SHR is thicker than that in Wistar rats obviously, and the smooth muscle cell layer having increased、 disordered arrangement and elastic fibers having distorted significantly. The layers of smooth muscle cells also increased gradually with the increase of weeks, but the elastic fiber layers are arranged orderly in Wistar rats; The thickness of MT and percentage of WA/LA increase in SHR when compared with Wistar rats (p<0.05)and tend to increase with weeks old. there is no statistically significant difference (p>0.05).MT and WA/LA have no statistically significant difference in different weeks of Wistar rats (p>0.05);(5)Both blood pressure and MT have been showed Significant negative correlation relationship with relative expression of miR-145in SHR(p<0.01).Conclusions:(1)miR-145is expressed in thoracic aortic VSMC of both SHR and Wistar rats. High level of miR-145is expressed in Wistar rats but low level is found in SHR, and the expression level of miR-145is descend by the increase of blood pressure in SHR;(2) The thoracic aortic VSMC of SHR and Wistar rat have different cell phenotypes; The phenotype of VSMC is synthetic phenotype in SHR and is contractive phenotype in Wistar rats;(3)The relative expression level of miR-145is descend by the increase of blood pressure increase and thickness of MT in SHR. Part2:The impacts of drug intervention on the expression of miR-145and cell phenotype in arterial VSMC of SHRObjectives:Research is to investigate these questions:whether the expression of miR-145and cell phenotype will be affected in arterial VSMC of SHR after the blood pressure has been reduced by drug intervention, meanwhile to observe the expression changes on proliferation-related gene of VSMC.Methods:16weeks old SHR male30were randomly divided into2groups:the control group (group SHRc) and Valsartan (Valsartan, generation) intervention group (SHRv group).30males16weeks old SHR were randomly divided into2groups:the control group (SHRc group) and Valsartan intervention group (SHRv group). SHRv group was given30mg/kg/d valsartan, but SHRc group was given the same volume of physiological saline. To determinate the SAP, DAP and MAP of each group at the end of Ow,6w,12w after drug interventionand compared with each other. At the same time, Real-time PCR technique was used to detect the expression differences of miR-145in thoracic aortic VSMC between SHRv group and SHRc group; Real-time PCR and immune-histochemical methods were used to detect the expression of phenotypic marker of VSMC of a-SMA and SMemb mRNA and protein in the thoracic aorta between two groups; So do the proliferation-related gene p38MAPK and KLF-5.HE staining slices were used to observe the morphology、 the membrane thickness (MT) and the percentage of membrane area (WA)/lumen area (LA) of thoracic aortic VSMC between two groups.Results:(1)SAP, DAP and MAP decreased gradually with the increase of intervention time in SHRv group.One-way ANOVA showed that the difference have statistical significant between each other at Ow,6w and12w after drug intervention(F=37.46,p=0.0000); SAP, DAP and MAP did not decreased gradually with the increase of intervention time in SHRc group, and the difference have no statistical significant between each other(p>0.05); Blood pressure was obviously lower in SHRv group compared with in the SHRc group (p<0.05);(2) The relative expression of thoracic aortic VSMC of miR-145increased gradually with the increase of intervention time in SHRv group. The statistical analysis showed that there was statistical significant between each other at Ow,6w and12w intervention time(F=25.13,p=0.0000). The relative expression did not increased with the increase of intervention time in SHRc group.The relative expression of miR-145was obviously higher in SHRv group compared with in the SHRc group at the end of Ow,6w and12w, and the difference had statistical significant(p<0.05);The relative expression of miR-145increased gradually with the decrease of blood pressure in SHRv group, And correlation analysis showed that have significant negative correlation between them(r=0.8176);(3)The relative expression of mRNA of alpha SMA, SMemb, p38MAPK and KLF-5had no statistical significant compared with each other at different intervention time in SHRv group(F=0.78, p=0.69;F=0.89,p=0.53;F=1.21,p=0.27; F=0.67, p=0.91);One-way ANOVA showed that the difference have statistical significant among the relative expression of mRNA of alpha SMA,SMemb, p38MAPK and KLF-5at different intervention time in SHRv group(F=9.8l,p=0.00; F=7.04, p=0.00;F=4.28, p=0.03;F=5.88, p=0.006); The relative expression of mRNA of alpha SMA increased gradually with the increase of intervention time of valsartan in SHRv group, but the relative expression of mRNA of other factors all decreased gradually with the increase of intervention time of valsartan in SHRv group; So do the relative expression of protein between two groups;(4) The aorta was been seen obvious thickening and elastic fiber hyperplasia in the membrane, disordered arrangement, distorted obviously in HE slice in SHRv group at OW; The aortic thickening and elastic fibrous hyperplasia reduced significantly with the drop in blood pressure;And during the whole observation the phenomena of aorta obviously thickening and membrane elastic fiber hyperplasia have no improvement in SHRc group; MT and WA/LA reduced when compared between two groups after intervention(p<0.05). Conclusions:(1)Angiotensin Ⅱ (AT1) receptor antagonist valsartan can reduce the SAP, DAP and MAP of SHR;(2) The expression of thoracic aortic VSMC of miR-145increased gradually with the decrease of blood pressure in SHR. The phenotype of VSMC cell was being transformed from synthetic phenotype to contractile phenotype;(3)The proliferation of VSMC lessened and the expression of proliferation-related factors decreased with the decrease of blood pressure in SHR. Part3:Overexpression and suppression-expression of miR-145can modulate phenotypic transformation of VSMC in SHRObjectives:To further explore the role of miR-145on the phenotypic transformation, and to observe the effects of transfected with different concentrations of miR-145mimics and miR-145inhibitors on the phenotypic transformation of arterial VSMC in SHR.Methods:VSMC from thoracic aorta in8weeks old male SHR were cultured in vitro using tissue piece anchorage primary culture and were identified by immunohistochemical method.6-8generation of VSMC were used to do intervention trial. VSMC were transfected with various concentration (3nM、50nM、100nM) of miR-145mimics and miR-145inhibitors by lipofectamineTM2000.After24h-incubation, Real-time PCR was used to detect the expression of a-SMA and SMemb mRNA of VSMC and MTT method was used to detect cell proliferation after transfected with various concentration (3nM、50nM、100nM) of miR-145mimics and miR-145inhibitors.Results:(1)Tissue piece anchorage primary culture successfully cultivates VSMC; Immunocytochemistry detection shows that the positive VSMC is good in quality with purity over99%;(2)The relative expression of mRNA of a-SMA is up-regulated in a concentration-dependent manner after transfected with various concentration of miR-145mimics;One-way ANOVA shows that the difference of the relative expression of mRNA of a-SMA have statistical significant (F=36.63,P=0.00); The relative expression of mRNA of a-SMA is4.71times of that in Mock group by overexpression of miR-145mimics in100nM group;(3)The relative expression of mRNA of SMemb is down-regulated in a concentration-dependent manner after transfected with various concentration of miR-145mimics; One-way ANOVA shows that the difference of the relative expression of mRNA of SMemb have statistical significant (F=14.84, p=0.02); The relative expression of mRNA of SMemb is only30.47%of that in Mock group by overexpression of miR-145mimics in100nM group;(4) The relative expression of mRNA of a-SMA is down-regulated in a concentration-dependent manner after transfected with various concentration of miR-145inhibitors; One-way ANOVA shows that the difference of the relative expression of mRNA of a-SMA have statistical significant (F=27.06, p=0.00); The relative expression of mRNA of a-SMA is only26.48%of that in Mock group by overexpression of miR-145mimics in100nM group;(5)The relative expression of mRNA of SMemb is up-regulated in a concentration-dependent manner after transfected with various concentration of miR-145inhibitors; One-way ANOVA shows that the difference of the relative expression of mRNA of SMemb have statistical significant (F=17.29, p=0.03);The relative expression of mRNA of SMemb is2.08times of that in Mock group by overexpression of miR-145inhibitors in100nM group;(6) MTT method shows that VSMC proliferation activity is reduced to27.6%±7.8%and67.4±9.3%respectively by miR-145mimics with concentrations of50nM and100nM, and the difference has statistical significant(p<0.05)when compared with each other; while VSMC proliferation activity is increased to145.2%±11.2%and205.7±15.6%respectively by miR-145inhibitors with concentrations of50nM and100nM, and the difference has statistical significant(p<0.05).Conclusions:(1)miR-145could modulate cell phenotype transformation of VSMC;VSMC phenotype changes to the synthetic phenotype by inhibition of the expression of miR-145;VSMC phenotype changes to contractive phenotype by overexpression of the expression of miR-145;(2) Overexpression of miR-145can inhibit VSMC proliferation; suppression-expression of miR-145can promote VSMC proliferation. Part4:miR-145regulat phenotypic transformation of arterial VSMC by p38MAPK signaling pathwaysObjectives:To investigate whether the miR-145regulate the phenotypic transformation of arterial VSMC by p38MAPK signaling pathways.Methods:(1)Real-time PCR was used to detect the expression of p38MAPK and KLF-5mRNA of VSMC after overexpression and suppression-expression of miR-145;Western-blot was used to detect the expression of protein of Myocardin and KLF-5;(2) The relationship between p38MAPK and KLF-5in phenotypic transformation of VSMC: VSMC were divided into three groups according to the experiment needs: normal control group; and SB203580treatment group.VSMC in normal control group are the6-8passages cell;Ang Ⅱ induced group are induced VSMC in which Ang Ⅱ is added with the final concentration of10-7M; SB203580treatment group are cells in which SB203580of p38MAPK inhibitor is added firstly with the final concentration of10-6M, after4h Ang Ⅱ is added with the final concentration of10-7M; Real-time PCR was used to detect the effects of p38MAPK inhibitor SB203580on the expression of a-SMA and SMemb mRNA of Ang II induced VSMC;At the same time,Western-blot was used to detect the expression of protein of p-p38MAPK and KLF-5;and MTT was used test the proliferation in VSMC; Results:(1)The expression of mRNA of p38MAPK and KLF-5are down-regulated in a concentration-dependent manner after transfected with various concentration of miR-145mimics; One-way ANOVA shows that the difference of the relative expression of mRNA of p38MAPK and KLF-5all have statistical significant (F=53.19, p=0.00; F=38.70, p=0.00); And correlation analysis showed that have significant highly positive correlation between them;(2) The relative expression of mRNA of p38MAPK and KLF-5are up-regulated in a concentration-dependent manner after transfected with various concentration of miR-145inhibitors; One-way ANOVA shows that the difference of the relative expression of mRNA of p38MAPK and KLF-5have statistical significant (F=13.77,p=0.00; F=7.16,p=0.02); And correlation analysis showed that have significant highly positive correlation between them;(3)MTT method shows that VSMC proliferation activity is increased to236.3%±13.6%in AngⅡ induced group, VSMC proliferation activity in SB203580treatment group is increased to120.7±5.4%compared with control group; and the difference has statistical significant(p<0.05)when compared among SB203580treatment group, normal control group and AngⅡ induced group; but the difference has no statistical significant(p<0.05)when compared normal control group with SB203580treatment group(p>0.05); (4) The expression of mRNA of a-SMA in Ang Ⅱ induced group decreased obviously to0.37±0.04of that in control group, while the expression of mRNA of SMemb increased obviously to3.27±0.68times of that in control group, and the difference all have statistical significant(p<0.05).The expression of mRNA of a-SMA and SMemb mRNA in SB203580treatment group didn’t appear to be much different and only1.12±0.06times and1.23±0.12times of that in control group respectively(p>0.05);(5) Western blot show that the expression of protein of p38MAPK and KLF-5can be induced by Ang Ⅱ,and the difference all has statistical significance when compared with control group(p<0.05);The expression of protein of p38MAPK and KLF-5decreased significantly After giving p-38MAPK inhibitors, and the difference all has statistical significance when compared with control group and Ang Ⅱ induced group (p<0.05);(6) Overexpression and suppression-expression of miR-145can affect the protein expression of Myocardin and KLF-5;Western blot show that he protein expression of Myocardin, p-p38MAPK and KLF-5has statistical significance when compared with each group(p<0.05);And correlation analysis showed that have significant highly positive correlation between p-p38MAPK and KLF-5;but highly negative correlation between KLF-5and Myocardin.Conclusions:(1)miR-145regulat phenotypic transformation of arterial VSMC by p38MAPK signaling pathways;(2) p38MAPK positively regulats the expression of KLF-5and KLF-5negatively regulats the expression of Myocardin in p38MAPK signaling pathways;(3)VSMC phenotype can be changed from the contractive phenotype to the synthetic phenotype by induced with AngⅡ; After p38MAPK being inhibited, it can prevent Ang Ⅱ induced VSMC phenotypic transformation.