Association Of CircRNAs Involved In Vascualr Endothelial Injury With Essential Hypertension

Objective: Explore the association of essential hypertension（EH）with the expression level of circRNAs involved in vascular endothelial injury,including circ₀014243、circ₀126991 and circ₀041000;Investigate the relationship between the circRNAs and environmental factors such as sex,smoking and drinking and investigate the correlated between circRNA and miRNA.Explore the differences of circRNA relative expression between cases and controls and the diagnostic value for essential hypertension.Combine the relative expression of target circRNA-miRNA to improve the accuracy in diagnosis for EH.Methods: 1.A total of 196 individuals was enrolled in the frequencing-match designed case-control study,including 98 incident EH and 98 healthy controls,which are all from Han Chinese family in Ningbo city.We collect the baseline data through the unified questionnaire,physical and laboratory examination.2.Extract the RNA of study subjects in whole blood samples,and then reverse transcribe into stable cDNA,finally detect the expression level of target circRNA（circ₀014243 、 circ₀126991 and circ₀041000）and miRNA（miR-10a-5p and miR-497-5p）by qRT-PCR.3.Compare the differences of expression level of target circRNA-miRNA in groups by univariate analysis.Apply logistic regression analysis for adjusting confounding variables,finally evaluate the diagnostic value of circRNA and combination of circRNA-miRNA for EH by receiver operating characteristic curve（ROC）analysis.Results: 1.The analysis of relative expression for target circRNA between EH and non-EH groups.（1）circ₀014243: Comparison between cases and control,circ₀014243 is highly expressed in EH（low ?Ct value）（EH VS.Control: 13.86±3.04 VS.15.27±2.06,OR（95 % CI）= 0.459（0.317-0.665）,adjusted P < 0.0001）.（2）circ₀126991: Comparison between cases and control,circ₀126991 is highly expressed in EH（low ?Ct value）（EH VS.Control: 12.28±2.16 VS.13.36±1.49,OR（95 % CI）= 0.413（0.279-0.611）,adjusted P < 0.0001）.（3）circ₀041000: Comparison between cases and control,circ₀041000 is expressed with no statistic difference（EH VS.Control: 12.72±2.26 VS.12.42±1.78,OR（95 % CI）= 1.058（0.819,1.366）,adjusted P = 0.6685）.2.The analysis of relative expression for target circRNA in different group factors:（1）circ₀014243: Compare to controls,circ₀014243 in EH is highly expressed in the subgroups of more than 60 years old,male,non-smoking,drinker,non-drinker,BMI less than 25 and miR-10a-5p ?Ct value less than 3.2（Case VS.Controls: ≥ 60 years old（13.65±3.36 VS.15.09±1.59,adjusted P<0.0001）,male（13.98±2.79 VS.15.68±1.81,adjusted P = 0.0001）、non-smoker（13.51±3.63 VS.15.10±2.10,adjusted P = 0.0022）、drinker（14.41±1.65 VS.15.69±2.25,adjusted P = 0.0158）、non-drinker（13.52±3.60 VS.15.14±2.00,adjusted P = 0.0005）、BMI ≤ 25（14.19±1.34 VS.15.02±2.09,adjusted P = 0.0033）、miR-10a-5p ?Ct > 3.2（14.19±2.66 VS.15.50±1.79,adjusted P = 0.0051））.（2）circ₀126991: Compare to controls,circ₀126991 in EH is highly expressed in the subgroups of more than 60 years old,male,female,non-smoking,non-drinking,BMI less than 25 and miR-10a-5p ?Ct value more or less than 3.2（CaseVS.Controls: ≥ 60 years old（12.27±2.19 VS.13.52±1.43,adjusted P = 0.0006）,male（12.72±1.96 VS.13.71±1.30,adjusted P = 0.0087）,female（11.66±2.28 VS.12.85±1.61,adjusted P = 0.0008）,non-smoker（11.94±2.14 VS.13.25±1.57,adjusted P = 0.0002）,non-drinker（12.09±2.02 VS.13.32±1.57,adjusted P = 0.0003）,BMI ≤ 25（12.30±2.34 VS.13.34±1.60,adjusted P = 0.0120）,miR-10a-5p ?Ct ≤ 3.2（12.00±2.73 VS.13.16±1.65,adjusted P = 0.0297）、miR-10a-5p ?Ct > 3.2（12.42±1.80 VS.13.67±1.12,adjusted P = 0.0249））.（3）circ₀041000: Compare to controls,circ₀041000 in EH is down-regulated in the subgroups of female and non-smoker（Case VS.Controls: female（12.61±2.24 VS.11.90±2.05,adjusted P = 0.0037）、non-smoker（12.63±2.11 VS.12.26±1.71,adjusted P = 0.0353））.3.The result of ROC analysis indicated that（1）There is diagnostic value for the relative expression of circ₀014243（?Ct）in the EH,the area under curve of ROC is AUC（95 % CI）:0.675（0.599-0.750）.And the sensitivity and specificity are 0.714 and 0.612,respectively.（2）There is diagnostic value for the relative expression of circ₀126991（?Ct）in the EH,the area under curve of ROC is AUC（95 % CI）: 0.699（0.624-0.774）.And the sensitivity and specificity are 0.724 and 0.673,respectively.（3）There is diagnostic value for the relative expression of miR-10a-5p（?Ct）in the EH,the area under curve of ROC is AUC（95 % CI）: 0.684（0.609-0.759）.And the sensitivity and specificity are 0.786 and 0.551,respectively.（4）There is diagnostic value for the combination of target circRNA-miRNA expression level.The AUC of combination for circ₀014243 and miR-10a-5p increase to 0.737（95 % CI: 0.667-0.807）;The AUC of combination for circ₀126991 and miR-10a-5p increase to 0.766（95 % CI: 0.699-0.832）.Conclusion: 1.Hypo-expression（higher ?Ct value）of circRNA（circ₀014243 and circ₀126991）involved in vascular endothelium injury is the protective factor for EH.However,Hypo-expression（higher ?Ct value）of miR-10a-5p,the target miRNA of circRNA,is the risk factor for EH.2.According to the logistic regression for subgroups,circRNAs’ effect for disease is changed with different factors.Compared with the effect of entire population,hypo-expression（higher ?Ct value）of circ₀014243 and circ₀126991 is more protective for EH in more than 60 years old,indicating that the expression profile of circRNA is closely related with disease as the age is growing.Similarly,the protective effect of hypo-expression of circ₀126991 was impoved when the ?Ct of miR-10a-5p is less than 3.2,which indicates that hyper-expressed miR-10a-5p can promote the protective effect of circ₀126991.3.There is diagnostic value of circ₀014243 and circ₀126991 in EH.And there is also diagnostic value for combination with the target interacting miRNA,which can improve the accuracy for diagnosing EH.