| Essential hypertension is one of the most common chronic diseases globally,leading to a serious public health problem.The complex in etiology of hypertension gives rise to complications,such as coronary heart disease,myocardial infarction,stroke,and renal failure.Although drugs used to treat hypertension reduce the risk of cardiovascular and cerebrovascular complications,these drugs were developed on the basis of the hypertension mechanism recognized 30 years ago,thereby affording limited efficacy and indications.Therefore,the current dilemma of hypertension prevention,control,and treatment calls for urgent insights into the deeper mechanistic details of pathogenesis of hypertension.The dysfunction of endothelial cells leads to microvascular rarefaction,further increasing peripheral vascular resistance contributing to hypertension.Micro RNAs(miRNAs)have been found to be involved in the dysfunction of vascular endothelial cells by affecting endothelial nitric oxide synthase expression,endothelial repair,angiogenesis,or the expression of inflammatory molecules.Furthermore,ascertaining miRNAs involved in hypertension holds great potential for reveling the deeper mechanistic details of pathogenesis of hypertension.Objective:To ascertain miRNAs involved in essential hypertension from differentially expressed miRNAs in essential hypertension;elucidate mechanism of the miRNAs involved in essential hypertension via biological effects of endothelial cells;investigate connectivity among the miRNAs,the miRNAs-targeted genes,clinical indices,and hypertension;and establish chain of evidence that the miRNAs contribute to the pathogenesis and treatment of hypertension.Methods:The population data and blood samples were collected from inpatients and healthy persons verified by routine medical checkup in the first hospital of Jilin University in2017,as well as persons surveyed by the "2012 Survey of Chronic Diseases and Risk Factors of Adults in Jilin Province".Differentially expressed miRNAs were screened using gene chip on the basis of six patients with hypertension and six healthy persons,and validated using RT-q PCR on the basis of 33 patients with hypertension and 33 healthy persons.Moreover,miRNAs involved in essential hypertension from differentially expressed miRNAs were confirmed using receiver operating characteristic(ROC)curve.Genes-targeting miRNAs involved in essential hypertension were predicted using Targetscan 7.2.The STRING database was utilized to construct protein–protein interaction(PPI)network.Gene ontology(GO)functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were analyzed using Metascape and Cytoscape to identify functional modules and hub genes.Levels of m RNA and protein were detected using RT-q PCR and western blot respectively.Dual-luciferase reporter assay was performed to determine miRNAtargeted regulation for genes.Effects of miRNA and miRNA-targeted genes on vascular endothelial cells were confirmed on the basis of cell proliferation,cell migration,cell apoptosis and tube formation assays.Moreover,rescue experiment was used to further confirm the biological connectivity from miRNA,via miRNA-targeted genes,to function of vascular endothelial cells.Levels of miRNA were detected using RT-q PCR,and levels of miRNA-targetedgenes-coded protein were detected using Elisa in the in the serum of 135 patients with hypertension and 135 healthy people.Spearman correlation analysis was used to clarify the nexus among miRNA,miRNA-targeted-genes,and hypertension-related clinical indicators.Results:1.Screening and validation of hypertension-related miRNAsA total of 18 differentially expressed miRNAs(six up-regulated miRNAs: miR-6125,miR-6089,miR-3656,miR-6803-5p,miR-3135 b,and miR-6068;12 downregulated miRNAs: miR-19b-3p,miR-19a-3p,miR-15a-5p,miR-106b-5p,miR-4306,miR-20b-5p,let-7i-5p,miR-107,miR-20a-5p,miR-185-5p,miR-93-5p,and miR-15b-5p)were identified using microarray in plasma from patients with hypertension and healthy control.miR-3656,miR-3135 b,and miR-107 were confirmed by RT-q PCR.miR-3656 was selected to be further investigated.ROC curve showed that area under the curve of miR-3656 was 0.875,indicating miR-3656 was a potential biomarker for hypertension.2.Connectivity between miR-3656 and vascular endothelial cell functionCompared with miR-NC mimics,miR-3656 mimics inhibited the proliferation,migration,and tube-forming ability of vascular endothelial cells,and promoted the apoptosis of vascular endothelial cells significantly(P<0.05);Compared with miR-NC inhibitor,miR-3656 inhibitor promoted the proliferation,migration,and tube-forming ability of vascular endothelial cells,and inhibited the apoptosis of vascular endothelial cells significantly(P<0.05).3.Prediction and validation of miR-3656-targeted genesA total of 332 from 1313 miR-3656-targeted genes predicted by Targetscan 7.2were screened according to screening conditions to improve confidence and specificity.We used GO enrichment analysis,finding that the 332 miR-3656-targeted genes were mainly involved in biological processes(developmental process,biological regulation,multicellular organism process,and localization),cellular components(RNA polymerase II transcriptional regulatory complex,membrane coat,endoplasmic reticulum lumen,and Golgi membrane),and molecular functions(DNA-binding transcriptional activation activity,RNA polymerase II specificity,DNA-binding transcriptional inhibitory activity,and RNA polymerase II specificity).KEGG pathway analysis showed that the 332 miR-3656-targeted genes were mainly involved in pathways,including APMK signaling pathway,insulin signaling pathway,human cytomegalovirus infection,and vascular endothelial growth factor signaling pathway.There were eight key functional modules in the interaction network of miR-3656-targeted genes,and 34 genes were included in the eight functional modules.After the34 genes were compared with the top 50 key genes screened in the interaction network,we found that 21 from the 34 genes belong to the top 50 key genes in the key modules.We surveyed literature reporting hypertension and vascular endothelium-related genes,selecting eNOS and ADAMTS13 as targeted genes for further experiments.RT-q PCR X and western blot showed that the levels of m RNA and protein expression of eNOS and ADAMTS13 in the transfected miR-3656 mimics group were down-regulated compared with those in the control group.Compared with the levels of m RNA and protein expression of eNOS and ADAMTS13 in the control group,those in the transfected miR-3656 inhibitor group were up-regulated significantly(P<0.05).In addition,dualluciferase reporter assay showed that the fluorescence value of the miR-3656mimics+eNOS/ADAMTS13 wild group was lower than that in other groups significantly(P<0.05).4.Connectivity of eNOS or ADAMTS13 with vascular endothelial cell functionCompared with si NC group,silencing eNOS or ADAMTS13 group inhibited the proliferation,migration and tube-forming ability of vascular endothelial cells,and promoted the apoptosis of vascular endothelial cells significantly(P<0.05);Compared with the vector group,the eNOS or ADAMTS13 overexpression group promoted the proliferation,migration and tube-forming ability of vascular endothelial cells,and inhibited the apoptosis of vascular endothelial cells,and the difference was statistically significantly(P<0.05).5.Connectivity of miR-3656,eNOS,and ADAMTS13 with and the function of vascular endothelial cellsRrescue experiments showed that compared with the miR-3656 mimics group,both the miR-3656 mimics+eNOS group and the miR-3656 mimics+ADAMTS13 group promoted the proliferation,migration and tube formation of vascular endothelial cells,and inhibited the apoptosis of vascular endothelial cells significantly(P<0.05),indicating that both eNOS and ADAMTS13 overexpression rescues miR-3656-injured vascular endothelial cells.6.Association of miR-3656,eNOS,and ADAMTS13 with blood pressure and clinical indicators of hypertensionCompared with levels of miR-3656 in serum of 135 healthy controls,those was high in those of 135 patients with hypertension significantly(P<0.001).Elisa showed that the protein levels of eNOS in serum of the patients with hypertension were lower than those in serum of the healthy controls significantly(P<0.05).However,there was no difference of protein levels of ADAMTS13 between the serum of the patients with hypertension and that of the healthy controls(P=0.442).Levels of miR-3656 positively correlated with systolic blood pressure,diastolic blood pressure,hypertension grade,and TG as well as with IL-6 levels(P<0.05),and negatively correlated with hs-CRP(P=0.012).The protein levels of eNOS negatively correlated with systolic blood pressure,diastolic blood pressure,and hypertension grade(P<0.05).The protein levels of ADAMTS13 positively correlated with TNF? levels(P=0.005).Conclusion:1.The blood levels of miR-3656 in patients with hypertension are higher than those in healthy controls.2.miR-3656 affects vascular endothelial cell function: high expression of miR-3656 inhibits the proliferation,migration,and tube formation of vascular endothelial cells,and promotes apoptosis;moreover,low expression of miR-3656 promotes the proliferation,migration,and tube formation of vascular endothelial cells,and inhibit s apoptosis.3.eNOS and ADAMTS13 affect vascular endothelial cell function: silencing eNOS or ADAMTS13 inhibit the proliferation,migration,and tube formation of vascular endothelial cells,and promote apoptosis;moreover,overexpression of eNOS or ADAMTS13 promote the proliferation,migration,and tube formation of vascular endothelial cells,and inhibit apoptosis.4.eNOS and ADAMTS13 are miR-3656-targeted genes,and miR-3656 leads to vascular endothelial cell dysfunction by targeting eNOS and ADAMTS13.5.miR-3656 positively correlates with systolic blood pressure,diastolic blood pressure,hypertension grade,TG,and IL-6;moreover,the levels of eNOS in patients with hypertension are lower than those in healthy controls,and it negatively correlates with systolic blood pressure,diastolic blood pressure,and hypertension grade. |
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