| Research BackgroundIn clinical diagnosis and treatment,the condition of unstable angina is very unstable,and it can occur during load and at rest.coronary atherosclerotic plaque rupture and thrombosis are the causes of unstable angina,and their occurrence is associated with endothelial dysfunction.At present,unstable angina lacks rapid,specific,and effective detection indicators,making early diagnosis difficult to be accurate,and can only rely on traditional indicators to make clinical suspected diagnosis.Therefore,how to quickly diagnose unstable angina remains of research significance.In the early stage of the study,we detected by RT-PCR that the expression of mi R-499a-3p in the serum of patients with unstable angina was significantly higher than that of non-coronary patients with suspected unstable angina,and then passed Target scan predicts that ADAM10 is a downstream target gene of mi R-499a-3p.At the same time,using luciferase reporter gene technology detection,it was found that mi R-499a-3p and ADAM10 have a negative targeting regulation relationship.Objective:(1)On the basis of previous studies,explore the effect of mi R-499a-3p on primary HUVEC function;(2)To verify whether mi R-499a-3p is involved in primary HUVEC regulation of ADAM10 expression.Method: Primary HUVECs were used to replace human coronary vascular endothelial cells,and primary HUVECs were divided into four groups according to the type of transfected lentivirus,ie,primary HUVECs that were not transfected with virus were blank(NC group),transfected with CO137-negative virus.The primary HUVEC was mi R-NC group(empty group),the primary HUVEC transfected with mi R-499 a was mi R-499a-3p mimics group(upward group)and the primary HUVEC transfected with mi R-499a-3p inhibitors was mi R-499a-3p inhibitors group(down-regulation group).Primary HUVEC stably transfected strains carrying high or low expression of lentiviral mi R-499a-3p were constructed.Then,real-time quantitative PCR was used to detect the expression levels of mi R-499a-3p and ADAM10 in the four primary HUVECs.Western blotting was used to detect the expression of ADAM10 in four groups of primary HUVECs.Then,using the scratch test,Transwell test,CCK8 test to determine the proliferation and migration ability of the stably transfected primary HUVEC in the blank group,mi R-NC group,mi R-499a-3p mimics group and mi R-499a-3p inhibitors group..Flow cytometry was used to determine the apoptosis of primary HUVEC in the blank group,mi R-NC group,mi R-499a-3p mimics group and mi R-499a-3p inhibitors group.Result:(1)Pre-experimental results of lentiviral transfection: The most suitable MOI for transfecting lentivirus to primary HUVEC is 10 and the most suitable condition for transfection is lentivirus + endothelial cell complete medium;(2)Screening results of stably transfected cell lines showed that the most suitable concentration of the puromycin solution for screening stably transfected cell lines was 2 ?g/m L.(3)RT-PCR results: There was no significant difference in the expression of mi R-499a-3p and ADAM10 in HUVEC between blank group and mi R-NC group(P>0.05).Compared with the blank group and the mi R-NC group,the expression of mi R-499a-3p was significantly increased in the mi R-499a-3p mimics group(P<0.01),and ADAM10 was significantly decreased(P<0.05).The expression of mi R-499a-3p was significantly decreased in the mi R-499a-3p inhibitors group(P<0.05),and ADAM10 was significantly increased(P<0.05).(4)Western blotting results: Compared with the blank group and the mi R-NC group,the expression of ADAM10 protein in the mi R-499a-3p mimics group was significantly decreased(P<0.05);the ADAM10 in the mi R-499a-3p inhibitors group The protein was significantly elevated(P<0.05).(5)Cell scratch test,Transwell test,cell proliferation test results: Compared with the blank group and the mi R-NC group,the primary HUVEC stably transfected mi R-499a-3p mimics,the number of migrated cells decreased,and the migration ability was significantly improved.Inhibition(P<0.05);while mi R-499a-3p inhibitors were stabilized,the number of migrated cells increased and the migration ability was significantly enhanced(P<0.05).(6)Flow cytometry detection of apoptosis results: Compared with the blank group and the mi R-NC group,the total number of apoptotic cells in the mi R-499a-3p mimics group was significantly higher(P<0.05).The total number of apoptotic cells in the mi R-499a-3p inhibitors group was significantly reduced(P<0.05).It was shown that mi R-499a-3p can induce apoptosis of primary HUVEC.Conclusion:(1)The lentivirus stably transfected cell line was successfully constructed.(2)Up-regulation of mi R-499a-3p expression inhibits the expression of ADAM10 in four experimental cells.mi R-499a-3p inhibits the proliferation and migration of primary HUVECs and induces apoptosis.Its mechanism may be related to the expression of the ADAM10 protein.endothelial cell dysfunction is closely related to the development of AS and thrombosis.AS rupture and thrombosis are the causes of unstable angina.Therefore,based on the results of previous studies,based on the results of this experiment,it is speculated that the role of mi R-499a-3p and ADAM10 participate in the development of UA.Detection of mi R-499a-3p expression is expected to be a new early diagnostic biomarker for UA,providing a potential intervention target for avoiding the occurrence of malignant coronary events. |
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