Alteration In The Expression Of The Renin-Angiotensin System In The Myocardium Of Mice Conceived By In Vitro Fertilization

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Alteration in the expression of the renin-angiotensin system in the myocardium of mice conceived by in vitro fertilizationObjective:This study aimed to assess the effects of IVF on the expression levels of genes in the RAS pathway and related effect genes in the myocardial tissue of the newborn,adult and elderly mice conceived by IVF.Materials and methods:1.IVF and in vivo fertilization models were established using C57BL/6J mice.The mice were euthanized to obtain the heart tissues at 3 weeks,10 weeks and 1.5 years after birth respectively.2.For mRNAs(Renl,Ren2,Ace,Ace2,Agt,Agtr1a,Agtr1b,Agtr2a,Col1al,Col1a2,Col3,and Ctgf),RNA samples were reverse-transcribed and performed using the real-time quantitative polymerase chain reaction(PCR)analysis.3.The protein of myocardium was extracted with RIPA protein lysis solution contained protease inhibitor.The protein concentration was determined by BCA method.The relative expression of AGTR1,AGTR2,CTGF and COL3 protein was detected by Western blot.Result:1.Compared with those in the in vivo group,the expression levels of Renl,Ace,Agtrla,Col3,and Ctgf were found to be significantly increased in the IVF group at 3 weeks of age.At 10 weeks of age,the expression levels of Ace,Agt,Agtr1b,Agtr2,Col1al,and Col3 were found to be significantly increased in the IVF group.When the mice reached the elderly age of 1.5 years,the expression levels of Ren1,Ace,Agtr1b,Col3,and Ctgf were obviously increased in the IVF group.2.At 3 weeks of age,the expression levels of AGTR1 and CTGF were significantly increased in the IVF group compared with the in vivo group.The protein expression levels of AGTR1,AGTR2,and CTGF were not statistically different between the two groups at 10 weeks of age.In old age,the protein expression of AGTR1 was obviously upregulated in the IVF group.Conclusion:The expression levels of partial genes in RAS system and related effector genes were increased in the myocardium of IVF-conceived mice from 3 weeks old to the old age.Altered expression of the RAS in the myocardial tissue might be one of the causes of cardiovascular malformations or dysfunction in the IVF offspring.Part ? The mechanisms of the alteration in the expression of the renin-angiotensin system in the myocardium of mice conceived by in vitro fertilizationObjective:This study aimed to understand the regulatory mechanism of RAS changes in myocardial tissue of IVF offspring and to further explore the interactions between miRNA and differentially expressed gene.Materials and methods:1.The genomic DNA of myocardial tissue was extracted using the DNeasy Blood and Tissue Kit.Genome bisulfite modification was performed using the EpiTect Bisulfite Kit.Bisulfite-converted DNA template was performed using the targeted bisulfite sequencing.2.Total RNA was extracted from myocardium of mice.After reverse transcription,the miRNA expression profiles of two groups of mice in old age were detected by miRNA microarray.3.MiR-100,miR-297,and miR-758 were chosen as the detection genes because they interacted with the mRNA of Col3a1,Agtr1a,and Col1a2,respectively,after miRNA database prediction.The expression of miR-100,miR-297,and miR-758 in both groups was detected by quantitative PCR.4.The luciferase reporter gene vector carrying AGTR1 3'-UTR or AGTR1 3'-UTR mutation was transfected miR-297a-3p mimic or negative control(NC)into 293T cells.Fluorescence intensity of luciferase reporter gene vector was measured respectively after transfection.5.MiR-297 was overexpressed in H9c2 cardiomyocytes.6.The protein in cardiomyocytes was extracted after transfection.The protein concentration was determined by BCA method.The relative expressions of AGTR1,CTGF and COL3 were detected by Western blot.7.The 5-ethynyl-20-deoxyuridine(EdU)(10 ?,M)was added to the culture medium after transfection and the cells were cultured for another 2 hours.Flow cytometry and confocal microscopy were used to observe the proliferation of H9c2 cardiomyocytes.Result:1.Compared with in vivo group,the mean DNA methylation level of Ctgf was decreased significantly in the IVF group at 3 weeks.The methylation levels of the CpG site 18 of Ctgf at 10 weeks and the CpG sites 2 and 14 of Ctgf at old age significantly reduced in the IVF group.2.At 3 weeks of age,the methylation level of the CpG site 4 of Col1al was significantly reduced in the IVF group compared with in vivo group.The methylation levels of CpG sites 6 and 8 of Col1al at 10 weeks and of CpG sites 1,2,and 3 of Collal at an old age were significantly reduced in the IVF group.3.At 3 weeks,the expression levels of miR-100,miR-297,and miR-758 were not found to be significantly altered between the two groups.Whereas at 10 weeks,a significant increase in miR-100 and miR-758 was observed in the IVF group compared with the in vivo group.A similar trend was found during old age.4.The results of the luciferase activity assay showed that miR-297a-3p directly targeted AGTR1 and regulated its expression.5.The protein expression of AGTR1 and CTGF in the miR-297 overexpression group significantly increased after 48 hours.6.Compared with in vivo group,miR-297 overexpression could significantly promote the proliferation of cardiomyocytes.7.The mean DNA methylation level of Igf2r in the IVF group was significantly reduced at 1.5 years.The mean DNA methylation level of Mest in the IVF group was significantly increased at 10 weeks.The mean DNA methylation level of Snrpn in the IVF group was reduced at 3 weeks of age.Conclusion:1.Elevated Ctgf and Col1al gene expression levels might be due to decreased methylation levels at the CpG sites in their promoter regions in IVF mice.2.MiR-100,miR-297,and miR-758 might be involved in the regulation of COL3,AGTR1,and COL1 expression in this process.Phenotypic changes of cardiomyocyte proliferation were observed after miR-297 overexpression.3.IVF treatment could regulate RAS and related genes in the myocardium through epigenetic regulatory networks in IVF mice from 3 week to old age.