Mechanism Of RAS Gene Expression In Cardiovascular System Of Mice Conceived By Assisted Reproductive Technology

Objective: This study is to investigate the regulatory mechanism of estrogen concentration on the expression of renin-angiotensin system（Ras）related genes during the development of mouse embryonic stem cells into myocardial tissue.Explore the effect of estrogen concentration on the self-renewal ability of mouse embryonic stem cells,and to analyze the effect of ras gene expression on the formation of mouse cardiomyocytes.Material and method: 1.Establish the mouse embryonic stem cell culture,and divide them according to different estrogen concentrations.The control group with estrogen concentration of 0 and the mice embryonic stem cells cultured with estrogen concentration of 10^-10,10^-9,10^-8,10^-7,10^-6 mol/l were cultured,and the cells were collected after 3 days.2.Total RNA was extracted from the treated embryonic stem cells,and the reverse transcripted c DNA was stained with TB green.Real time PCR was used to detect the RAS related genes in different groups with GAPDH as the internal reference gene.RAS related genes include Ren1,Ace,Ace2,Agt,Agtr1 a,Agtr1b,Agtr2,Col1a1,Col1a2,Col3a1 and Ctgf.The Ras related regulatory gene expression levels in different groups were detected with α-actin as the internal reference gene.The Ras related regulatory gene included Oct4,β-Catenin,Troponin,Nanog,Sox2,Akt1,Nkx2-5,Tbx5,Srf and Myh6.The Ras related regulatory mi RNA expression levels in different groups were detected with U6 as the internal reference gene.The Ras related regulatory mi RNA included M-mi R-100-3p,M-mi R-297a-3p,M-mi R-758-5p,mmu-let-7a-5p,mmu-let-7d-5p,mmu-let-7f-2-3p,mmu-let-7c-1-3p,mmu-let-7d-3p,mmu-let-7c-2-3p.3.Ripa protein lysate containing five protease inhibitors was used to extract proteins from mouse embryonic stem cells treated with different concentrations of estrogen.GAPDH was used as the internal reference protein,and the protein concentration was determined by classical BCA method.The protein expression levels of β-Catenin,Oct-4,Sox2 and Nanog were detected.Result: RT-PCR results:1.RAS system composition and effector genes: Ren1,ACE and ACE2 expression were positively correlated with estrogen.When the concentration of estrogen reached 10^-8,the expressions of ACE,ACE2 and AGT were the highest,and then decreased.The expression of COL1A1 in different concentrations of estrogen tended to be stable,and the expression level was very high,while the expression level of COL1A2 was low.The expressions of COL3A1 and CTGF were basically positively correlated with estrogen.Estrogen could promote the expression of Agtr1 a,Agtr1b and Agtr2.2.The expression levels of myocardial marker genes: β-Catenin and Nkx2-5 were the highest at 10^-10,and then negatively correlated with the concentration of estrogen.The expression levels of Oct-4,Nanog and Sox2 were positively correlated with estrogen.It suggests that high concentration of estrogen can promote the self-renewal ability of embryonic stem cells and affect their differentiation into cardiomyocytes.3.Mi RNA expression: M-mi R-297-3p,mmu-let-7d-3p,mmu-let-7c-2-3p increased with the increase of estrogen concentration.It indicates that estrogen can also affect the role of mi RNA in the growth and differentiation of embryonic stem cells.Protein results: The expression level of β-Catenin was negatively correlated with the concentration of estrogen.The expression levels of Nanog and Sox2 were positively correlated with the concentration of estrogen,with the highest expression at 10^-9.There was no significant correlation between Oct-4 expression and estrogen concentration.It may indicate that high concentration of estrogen can promote the self-renewal of embryonic stem cells and affect their differentiation into cardiomyocytes.Conclusion: 1.High concentration of estrogen affects the differentiation from mouse embryonic stem cells into cardiomyocytes.The maintenance of self-renewal ability of embryonic stem cells may be related to Oct-4,Sox2 and Nanog gene pathways.2.High concentration of estrogen can reduce the differentiation of embryonic stem cells into cardiomyocytes,thus affecting the expression of Ras related component genes and their effector genes.Objective: This study is to investigate whether the changes of micro RNA in embryonic stage will affect the development of mouse embryonic stem cells into myocardial tissue,increase the risk of adult cardiovascular disease.To study the regulatory mechanism of Ras related gene expression changes in myocardial tissue of IVF mice,so as to provide research ideas and basis for reducing the risk of cardiovascular disease of IVF offspring.Materials and methods: 1.Lipofectamine 2000 was used to transfect mir-297 inhibitor and mimic into mouse embryonic stem cells,which were divided into four groups: in,in-nc,m m-nc（inhibitor,inhibitor control group,mimic,mimic control group）.The culture time was 3 days.2.Total RNA was extracted from the transfected embryonic stem cells,and the reverse transcripted c DNA was stained with TB green.Real time PCR was used to detect the RAS related genes in different groups with GAPDH as the internal reference gene.RAS related genes include Ren1,Ace,Ace2,Agt,Agtr1 a,Agtr1b,Agtr2,Col1a1,Col1a2,Col3a1 and Ctgf.The Ras related regulatory mi RNA expression levels in different groups were detected with U6 as the internal reference gene.The Ras related regulatory mi RNA included M-mi R-100-3p,M-mi R-297a-3p,M-mi R-758-5p,mmu-let-7a-5p,mmu-let-7d-5p,mmu-let-7f-2-3p,mmu-let-7c-1-3p,mmu-let-7d-3p,mmu-let-7c-2-3p.3.Mir-297 inhibitor and mimic were transfected into mouse embryonic stem cells.The morphological changes and the number of embryoid somatic cells were observed by embryoid formation culture test.The effects of mir-297 mimic and inhibitor on embryoid somatic cell formation were analyzed.4.Total RNA was extracted from the myocardium of 3 weeks,10 weeks,1.5 years IVF mice and control mice and the transcriptome RNA SEQ（Polya）was sequenced.A total of 18 samples were sequenced,including three 3-week IVF mice myocardial tissue samples and their control samples,three 10-week IVF mice myocardial tissue samples and their control samples,and three 1.5-year IVF mice myocardial tissue samples and their control samples.The differentially expressed genes in different age groups were screened,and the enrichment of KEGG and go was analyzed.Result（s）: 1.The mir-297 transfection was successful.PCR results showed that after transfection of mir-297 mimic for 3 days,the expression of mir-297 was significantly increased（P<0.05）,and the expression of mir-297 was significantly decreased in the inhibitor group.Overexpression of mir-297 could significantly up regulate Col1a1 and Ctgf in RAS system;inhibition of mir-297 expression could up regulate Agt,Col3a1,Agtr1 a,and Agtr1 b in RAS system.Overexpression of mir-297 could up regulate the expression of mmu-let-7f-2-3p（P<0.05）.2.Embryoid culture experiment showed that the cells transfected with mir-297 mimic group were small and irregular,which indicated that high expression of mir-297 could inhibit the growth of embryoid.And the of embryoid cells in mimic group became smaller and scattered with the extension of culture time.The cells transfected with mir-297 inhibitor were larger and regular,which indicated that the growth of embryoid bodies was promoted by low expression of mir-297.3.Among the three different age groups,there were 15 common difference genes compared with the control group,including Nppa,Myl7,Myl4 and so on.Biological enrichment showed that these genes were related to 17 KEGG signaling pathways.PPI interaction analysis showed that Comp,CD36 and TIMP genes may be related to the increase of vascular endothelial formation and atherosclerosis in IVF mice offspring.Conclusion（s）: 1.Mir-297 expression can affect the expression level of Ras related component genes and effector genes.Mir-297 may be involved in agrt1 and CTGF regulated signaling pathways.2.Mir-297 expression level can affect the process of embryonic stem cells transforming into cardiomyocytes.3.Transcriptome sequencing showed that Nppa,myl7,MYL4 and other cardiovascular disease-related genes were abnormally expressed in IVF mice in all different age groups.Through PPI interaction,we found that Comp,CD36,TIMP and other genes may be related to the increase of vascular endothelium formation and arteriosclerosis in IVF mice offspring.