| Background: Obesity is an important risk factor for cardiovascular disease and is closely related to the onset of diabetes,hyperlipidemia,hypertension,coronary heart disease and other diseases.With the development of social economy,the improvement of material living standards,overeating and inactivity of women of childbearing age have resulted in obesity during pregnancy and excessive weight growth,which has become a serious public health problem in many developed and developing countries including China.Obese women can cause a series of pregnancy-related complications,such as gestational diabetes,preeclampsia,etc.;mothers overweight and obesity are also considered to be important risk factors for adult diseases in offspring,leading to increased risk of metabolic disorders,cardiovascular disease and type 2 diabetes in later life.Cardiac hypertrophy is closely related to the occurrence of cardiovascular diseases.Cardiac tissue adapts to pressure or volume-overload with an enlargement of the myocardium characterized by growth of individual cardiomyocytes(cardiac hypertrophy).Pathological cardiac hypertrophy is accompanied by a series of adverse cardiovascular events,including arrhythmia,heart failure and so on.At present,the methods for preventing and treating cardiac hypertrophy are limited.MicroRNAs are small,non-coding RNAs that are approximately 20 to 22 nucleotides in length.Studies have shown that miRNAs play an important role in the development of cardiovascular diseases such as myocardial hypertrophy,atherosclerosis,and myocardial congestion.In this study,pregnant rats were divided between control and HFD(HFD-fed during gestation)groups.The effect of maternal HFD on cardiac miRNA in the male offspring was determined by miRNA sequencing method,which was verified by Real-time q PCR.The effect of miR-493-3p on cardiac hypertrophy was determined in primary cardiomyocytes.Meanwhile,cardiac miR-493-3p was overexpressed by miR-493-3p mimic,studying effect of miR-493-3p on the Ang Ⅱ-induced cardiac hypertrophy.Finally,the bioinformatics method was used to enrich and analyze the target genes of miR-493-3p,and the primary biological functions of the candidate target genes and the main biochemical metabolic and signal transduction pathways,providing a theoretical basis for the role of miR-493-3p in maternal HFD-induced cardiac hypertrophy in offspring and the sex difference of miR-493-3p.Part one The effect of maternal high-fat diet during pregnancy on cardiac miRNA in the offspringObjective: To detect the effect of HFD during pregnancy on the expression of cardiac miRNA in offspring,and explore the molecular mechanism of cardiac hypertrophy induced by maternal HFD,providing a theoretical basis for revealing the molecular mechanism of HFD-induced cardiac hypertrophy in offspring.Methods: 8-10 weeks old SD rats were mated and randomly divided into high-fat diet group(HFD)and normal control group(Con)after conception.Establish a high-fat diet(HFD)model during pregnancy and a control model of normal diet during pregnancy.All the offspring were fed standard diet.miRNA sequencing method was used to measure the miRNA expression in control and HFD male offspring;Real-time q PCR was used to verify it.Results: 1.The miRNA sequencing results showed that the HFD during pregnancy caused changes in the expression of myocardial miRNAs in offspring,including 24 up-regulated miRNAs,including miR-493-3p,miR-379-3p,miR-127-5p,miR-379-5p,miR-411-3p,miR-132-3p,miR-152-5p,miR-541-5p,miR-451-5p,miR-125a-3p.21 down-regulated expressions,including miR-6315,miR-17-2-3p,miR-96-5p,miR-73-3p,miR-384-5p,miR-136-3p,miR-61-5p,miR-10a-5p,miR-182,miR-61-5p,miR-10a-3p.The change of miR-493-3p expression was the most obvious.2.The results determined by Real-time q PCR technique was consistent with the results from miRNA sequencing.Compared with the control group,the expression of cardiac miR-493-3p in the male offspring from HFD group was the most significantly increased(p<0.05).The expressions of miR-136-3p,miR-10a-5p,miR-10a-3p,and miR-61-5p in the female offspring from HFD rats were significantly down-regulated,which was consistent with the trend of male offspring.However,compared with the control group,the expressions of miR-6315,miR-96-5p,and miR-384-5p in the female offspring from HFD rat were unchanged.Furthermore,the expressions of cardiac miR-17-2-3p and miR-182 in the female offspring of HFD group were significantly up-regulated,which was contrary to the trend of male mice.For miRNAs up-regulated in male offspring,miR379-3p,miR-411-3p,miR-132-3p,miR-152-5p,miR-451-5p are up-regulated in HFD female offspring,which was consistent with the trend of male rats.Compared with the control group,there was no significant difference in the cardiac miR-127-5p expression in HFD female offspring.However,the expression of miR-493-3p,miR-379-5p,miR-541-5p,and miR-125a-3p in HFD female offspring was significantly down-regulated,which was contrary to the trend of male offspring.Summary: 1.Maternal HFD significantly affected the expression profile of myocardial miRNAs in adult male offspring,and the differential expression of miR-493-3p was the most significant.2.Maternal HFD caused an increase miR-493-3p in male adult offspring in a sex dependent manner.Part Two Maternal high-fat diet causes cardiac hypertrophy in male offspring by upregulation of miR-493-3p and the underlying mechanismsObjective: To the role of miR-493-3p in maternal HFD-induced cardiac hypertrophy in offspring and the underlying mechanismsMethods: Isolate and culture rat primary cardiomyocytes in vitro to establish angiotensin Ⅱ(Ang Ⅱ)-induced myocardial hypertrophy model,using miR-493-3p mimic and miR-493-3p inhibitor to establish miR-493-3p overexpression model miR-493-3p expression inhibition model,and treated with estrogen.Marker of cardiomyocyte hypertrophy Nppa,Myh6,Myh7 and miR-493-3p expression was measured by Real time q PCR;cell surface area were measured by immunofluorescence;the target gene of miR-493-3p were predicted by bioinformatics,and study the signal pathway and biological process of target gene through GO,KEGG enrichment analysis;Western blotting was used to detect the expression of autophagy pathway related proteins.Results: 1.Estrogen down-regulates the expression of miR-493-3p in primary cardiomyocytes.2.Ang Ⅱ treatment established a primary cardiomyocyte hypertrophy model.Compared with the control group,the expression level of miR-493-3p was increased in the Ang II group(p <0.05)3.After the primary cardiomyocytes were transfected with miR-493-3p mimic and miR-493-3p Con mimic,miR-493-3p expression was increased significantly in miR-493-3p mimic group(p<0.05).miR-493-3p mimic increased Nppa,Myh7 expression,but decreased Myh6 expression.miR-493-3p mimic also increased cardiomyocyte surface area(p <0.05).4.The primary cardiomyocytes were transfected with miR-493-3p inhibitor and miR-493-3p Con inhibitor,and the expression level of miR-493-3p was decreased significantly in miR-493-3p inhibitor group(p<0.05).Compared with the control group,the expression of Nppa and Myh7 in the Ang Ⅱ group was increased significantly(p<0.05),and the expression of Myh6 was decreased significantly(p<0.05),and the cardiomyocyte size was increased in miR-493-3p inhibitor group(p<0.05).Compared with the Ang Ⅱ group,miR-493 inhibitor significantly reduced Nppa,Myh7 expression,increased Myh6 expression,and reduced myocardial cell surface area,indicating that miR-493 inhibitor improved Ang Ⅱ induced myocardial hypertrophy.5.Through bioinformatics target gene prediction and enrichment analysis of GO and KEGG pathways,it was found that miR-493-3p participated in organelles,cell membrane assembly and decomposition and cell regulation biological processes in rat myocardial tissue,and also was involved in PI3K-AKT Signaling pathway,human papillomavirus infection signaling pathway,estrogen signaling pathway,autophagy and other pathways.6.Compared with the control group,the expression of myocardial Beclin1 was significantly reduced in HFD offspring(p<0.05).The primary cardiomyocytes were treated with estrogen and transfected with miR-493-3p mimic.The results showed that miR-493-3p mimic significantly reduced Beclin1 compared with the control group,which was reversed by estrogen.Summary 1.The sex difference of miR-493-3p is related to estrogen,and estrogen reduced the expression of miR-493-3p in neonatal cardiomyocytes.2.The up-regulation of miR-493-3p can activate the expression of myocardial hypertrophy marker gene,causing myocardial hypertrophy.3.HFD during pregnancy increases miR-493-3p expression in offspring adult male rats,targeting down-regulation of autophagy-related protein Beclin1;estrogen reduces the expression of miR-493-3p,resulting in up-regulation of autophagy-related protein Beclin1.Conclusion: 1.Maternal HFD leads to changes in cardiac miRNA expression in the offspring;miR-493 is most up-regulated among miRNAs that are significantly differentially expressed for control versus maternal HFD in male offspring,and there are gender differences.2.Maternal HFD causes cardiac hypertrophy by upregulation of miR-493-3p in a sex dependent manner. |
