The Research On Circulating MicroRNA As Vascular Endothelial Injury Biomarkers

Backgroud: Vascular endothelial injury is the initial pathology and pathway ofatherosclerosis (AS) and many other cardiovascular diseases. Multiple risk factors such ashyperlipidemia, hyperglycemia, hypertension may cause endothelial injury which couldstart the pathological process of AS and is involved in all the process. Therefore, the earlydiagnosis of vascular endothelial injury and the early interventions are important toprevent or delay the development of cardiovascular diseases.MicroRNA (miRNA) are agroup of endogenous small non-coding RNA, which play important gene regulatory rolesat the post-transcriptional level by binding to the3′untranslated region of the targetedmRNA. Recent reports have demonstrated that miRNA was stably present in plasma andhave been proven to be potential biomarkers for diagnosis and prognosis of manydiseases including cardiovascular diseases.Objective: The aim of our research is to determine circulating miRNA levels afterendothelial cell injury, and to look for the sensitive circulating miRNA which could beserved as potential biomarker diagnostic of vascular endothelial injury.Methods: MiRNA microarray is used to detect the expression profile of miRNA invascular endothelial cells. We determine the candidate miRNA according to the normalplasma miRNA in healthy people. The rat model of vascular endothelial injury could beconstructed by hyperlipidemic and hyperglycemic rats. Hyperlipidemic rats group (n=20)are intraperitoneally injected with a single dose of vitamin D3combined with high-fat dietand the rats of control group (n=5) were intraperitoneally injected with same volume ofsaline combined with standard diet. Hyperglycemic rats group (n=20) are given a singleintraperitoneal injection of freshly prepared solution of streptozocin (STZ) and the rats ofcontrol group (n=5) are intraperitoneally injected with the same volume of citric buffer.Specimens of blood are collected regularly (once every10days in hyperlipidemic rats;once every5days in hyperglycemic rats).Hyperlipidemic rats are confirmed by the level of blood total cholesterol (TC),triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) withenzyme-linked immunosorbent assay (ELISA) and hyperglycemic rats were identificated by level of blood glucose. The rats that are failed to meet the level of hyperlipidemia orhyperglycosemia are excluded. Hematoxylin-eosin staining (HE) was used for themorphologic changes of vascular endothelial cell. Oil red O staining was used for the lipiddeposition under the vascular endothelium. The level of plasma souble intercellularadhesion molecule-1(sICAM-1) was detected by ELISA. Plasma miRNA was measuredby quantitative PCR.Results:(1) We found27miRNA was highly expressed in vascular endothelial cellaccording to the miRNA expression profile of human vascular endothelial cell identified bymiRNA microarray.We selected11miRNA (miR-21, miR-126, let-7a, miR-222, miR-23a,miR-221, miR-125b, miR-26a, miR-29a, miR-16and miR-100) as candidate miRNAaccording to the normal plasma.(2) Survival rate of hyperlipidemic rats was70%. Tendays after intraperitoneal injection of vitamin D3combined with high fat diet, the levels ofTC and LDL were significantly increased (TC:4.00±1.14mmol/L vs1.81±1.03mmol/L;LDL:2.72±1.13mmol/L vs0.63±0.32mmol/L). HE staining showed no significantmorphologic changes in endothelial cell and in oil red O staining confirm that there wasnot lipid deposition under the endothelium. Twenty days after intraperitoneal injection ofvitamin D3combined with high fat diet, the levels of plasma lipid remained high (TC:2.65±0.94mmol/L, LDL:1.17±0.43mmol/L). There was no changes in endothelial cellsby HE staining and there was no lipid deposition under the endothelium. Thirty days afterintraperitoneal injection of vitamin D3combined with high fat diet, the level of plasmalipid remained high (TC:2.80±1.03mmol/L；LDL:1.71±0.63mmol/L), while HE stainingdiscovered typical plagues characterized by hclewglnouo vaccine of thickening of theintima and lipid was detected under the endothelium by oil red O staining. The level ofsICAM-1was increased (2628.78±188.35pg/mL vs1352.43±101.52pg/mL) and andquantitative PCR found that the levels of miR-126、let-7a、miR-21�'�miR-26a wereelevated and miR-29a was detected decreased while miR-125b、miR-23a、miR-221、miR-222、miR-100and miR-16were detected no significant change.(3) The successfulrate of hyperglycosemic rat model was about80%. Five days after intraperitoneal injectionof STZ solution, blood glucose was raised significantly compared to the control group(24.61±6.96mmol/L vs7.91±0.37mmol/L). There were no notable changes aboutendothelium by HE staining. Fifteen days after intraperitoneal injection of STZ solution,blood glucose was still higher (26.28±6.34mmol/L) and HE staining discovered thethicking intima. Then the level of plasma sICAM-1was detected increased significantly compared with control group, and quantitative PCR found that the levels of miR-126、miR-21and miR-221were detected increased, the level of miR-16and miR-23a weredecresed and miR-125b、miR-29a、miR-222、miR-100、let-7a and miR-26a were detectedno significant change.Conclusion:(1) The levels of plasma miR-126and miR-21were increasedsignificantly when endothelium was injuried.(2) The levels of plasma miR-126andmiR-21could have the potential diagnostic biomarker for vascular endothelial injury.