The Regulation And Mechanism Of MicroRNA-155 On Myocardial Fibrosis Induced By High Glucose

Background:MicroRNAs(miRNAs)are noncoding small RNAs that modulate gene expression at the post-transcriptional level.MicroRNA-155(miR-155)is one of the most important microRNAs(miRNAs),and is essential in many physiological and pathological processes.There is urgent need to establish a gene knock-out mouse model in order to study the function of miR-155.At present,the domestic miR-155 gene knockout mice model has not been reported.Recently,with the innovation of genetic screening and modification technology,the CRISPR/Cas 9 system has proven to be an efficient technology for gene targeting in multiplexed genome editing.In genetic studies of animal experiments,knockout models or transgenic animal models have also been widely used.Our study aims to use CRISPR/Cas 9 system to establish and breed the microRNA-155 knockout mice model.Objective:Establish the miR-155 knockout mouse model by CRISPR/Cas 9 technology.Methods:Healthy C57BL/6J mice were selected.First the amplification and sequencing of the target sequences in the mouse genome was used to confirm the sequences and loci of miR-155.Then based on the structural feature of miR-155,the Cas 9 vector plasmid and targeting construct(sgRNA)was designed,built and transcribed in vitro.And then was microinjected into the mouse zygotes cultivated in vitro.After transplanting the qualified embryos into recipients,F0 generation of mice were obtained.The genotypes of F0 mice were determined by gene detection.And the gene knockout mouse model was established after breeding the offspring.Results:A total of 112 zygotes were collected and injected,and 94 zygotes developed to the embryos.After testing sgRNA activity,81 embryos were transferred to 5 pseudo-pregnant recipients.After 21 days,18 mice were born,and only one died.After final confirmation with gene sequencing,seven mice were identified as positive gene mutants.Six of these eight mutants contained deletion mutations,and all deletions were of 1 bp.Only the 4#mice showed the deficiency of gene segments(114-bp deletion)in single-stranded DNA.After breeding,we found it had superior reproductive performance,and three heterozygous mice of F1 generation were obtained.After breeding of heterozygous mice,the homozygous mice of F2 generation were obtained and verified.The gene segments of knockout fragment were 114 bp included the miR-155 host gene sequences.Conclusions:CRISPR/Cas 9 technology can quickly and effectively establish the miR-155 knockout mice model;and the mice of miR-155 knockout can normal breed in the clean rearing environment.Background:In recent years,microRNA-155(miR-155)plays an important role in various biological processes,including immunity,inflammation,viral infections,cancer and cardiovascular disease.Especially,the homeostasis of inflammation and inflammatory cells is closely related to miR-155 expression and plays a critical role in T cell and B cell development.An increasing number of studies have demonstrated the broad potential of miR-155 in research,although few studies have characterized this miRNA in mice under normal physiological conditions.In our previous experiments,we established a miR-155 knock-out(KO)mouse model using the CRISPR/Cas 9 system and established a homozygous mouse line.Based on preliminary research,we performed a comparative analysis between miR-155 knockout(KO)mice and C57BL/6 wild type(WT)mice to characterize miR-155 under normal physiological conditions.We aimed to utilize this mouse model to identify the biological functions of miR-155.Objective:We aimed to characterize miR-155 in vivo using a comparative analysis.Methods:In our study,we performed a comparative analysis between miR-155 knockout(KO)mice and C57BL/6 wild type(WT)mice to characterize miR-155 in mice under normal physiological conditions using many evaluation methods,including a reproductive performance analysis,growth curve,ultrasonic estimation,haematology examination,and histopathological analysis.And twenty seeds(10 male and 10 female mice,aged 8 weeks)were randomly selected from both the WT and KO mice.Results:After we analyzed the data of reproductive performance analysis,growth curve,ultrasonic estimation,haematology examination,and histopathological analysis,no significant differences were found in the main evaluation indices between groups in these analyses.These phenomena were observed in all mice in the control and model groups,although some groups exhibited differences.Thus,these changes were not considered significant.The results showed that the growth and development of all mice were nearly normal and undifferentiated between the control and model groups.Using a comparative analysis and a summary of related studies published in recent years,we found that miR-155 was a non-essential factor for normal physiological processes.Conclusions:miR-155 deficiency did not affect the development and growth of naturally ageing mice during the 42 days after birth.miR-155 was a non-essential factor for normal physiological processes in mice aged 8 weeks.Thus,KO mouse modelling is necessary to study the complex biological processes of miR-155.Background:Diabetes as one of the most common diseases has played an important role in varieties of diseases,including fundus lesions,neuropathy,and fibrotic diseases.Cardiac fibrosis as one of the main fibrotic diseases often leads to cardiac dysfunction,and eventually evolves to cardiac failure.Cardiac fibrosis as a vital pathological process has played an important role in varieties of cardiac diseases.However,there are few effective treatments to prevent the cardiac fibrosis.Previous studies show that the collagens synthesis and deposition plays a vital role in the process of cardiac fibrosis.It is reported that high glucose(HG)has been shown to induce the production and multiplication of collagens,eventually leading to the cardiac dysfunction.microRNA-155(miR-155)is one of the most important miRNAs,and previous studies have reported that miR-155 as a regulatory factor in varieties of fibrotic diseases.However,the mechanism of myocardial fibrosis affected by miR-155 remains unclear.Objective:we aim to establish the biological function of miR-155 in myocardial fibrosis induced by diabetes in mice.Methods:We used normal C57BL/6 Wild Type(WT)and miR-155 knockout(KO)mice to establish the diabetic model by streptozotocin via intraperitoneal injection,and utilized echocardiography to evaluate the cardiac function at 30 and 60 days post-diabetes.And then,Hematoxylin-Eosin(HE)and Sirius-Red(SR)staining were used to evaluate the degree of myocardial lesions.Furthermore,we extracted cardiac fibroblasts(CFs)from the WT mice and transfected miR-155 inhibitors,mimics and negative control siRNAs into CFs to further research on the specific mechanism.Results:At 30 day post-diabetes,all mice were alive,and examined by echocardiography.And the result shows that the HWT group produced a slight change in the cardiac function compared to the control groups,while the HKO group did not appeared.At 60 day post-diabetes,HWT group died three mice,and HKO group one death.Alive mice were under echocardiography.The result showed that the diabetic mice at 60 day post-diabetes showed obvious cardiac dysfunction as evidenced by echocardiography data analysis.And the obvious cardiac dysfunction observed in HWT group was alleviated in HKO group at 60 day post-diabetes.The SR staining results showed that the deposition of collagen ? and ? increased significantly in the model groups as compared with the control groups.And compared to the HKO group,the HWT group exhibited more collagens deposition between the intercellular spaces of cardiomyocytes.In addition,by the HE staining,the abnormal changes of cellular structure in cardiomyocytes were easy to see in the HWT group.And these abnormal structures also decreased in HKO group.The result of the cell experiment showed that the expression of collagen ? in the normal-HG group obviously increased after stimulated by HG compared to the normal-LG(OC)group.And the mimics-miR(HG)group had obviously higher collagen ? than the other groups.Simultaneously,compared to the normal-HG group,the expression of collagen I obviously decreased in the anti-miR(HG)group.In addition,the same as TGF-?1 the Smad 2 and P-Smad 2 were significantly suppressed in anti-miR group,and increased in mimics-miR group compared to the normal group.Conclusions:miR-155 deficiency could prevent cardiac fibrosis induced by diabetes in mice.And we found that miR-155 could regulate the cardiac fibrosis via the TGF-?1-Smad 2 signaling pathway.These findings suggested that miR-155 might have a potential therapeutic target of the cardiac fibrosis induced by diabetes.