| Purpose:Ischemic stroke due to chronic carotid artery occlusion is not common but very important reason.Its short-term re-stroke and mortality are high,gradually get attention.But its treatment has not been unified.In recent years,hybrid operation treatment has appeared,but few cases have been reported.This study summarizes the safety and effectiveness of hybrid operation for patients with chronic carotid artery occlusion,and analyzes the related factors that affect the success or failure of surgery.Atherosclerosis is the most common reason of chronic carotid artery occlusion,and its molecular mechanism remains to be further elucidated.Increasing evidences show that long non-coding RNA(lncRNA)plays an important role in various biological processes and physiological and pathological progress of human diseases.In this study,carotid atherosclerotic plaques from patients with chronic carotid artery occlusion were taken to detect the correlation between the expression of MEG3 and the expression of inflammatory factors.Further in vitro experiments were conducted to determine the function and molecular mechanism of lncRNA MEG3 in Raw264.7 cells treated with oxidized low density lipoprotein(ox-LDL).Methods:(1)Patients with chronic carotid artery occlusion who were hospitalized in the department of neurovascular surgery of the first hospital of jilin university from January2016 to December 2018 and received hybrid operation were collected for statistical analysis of their basic data,clinical data,postoperative complications,postoperative clinical follow-up and imaging follow-up.(2)Collect clinical samples of chronic carotid occlusion cases(atherosclerotic plaque),and then detect the expression of MEG3 in the samples by real-time quantitative PCR;Detection of TNFαexpression in the samples by Elisa;Then do a correlation analysis.(3)Raw 264.7 cells were treated with ox-LDL,and cell viability and proliferation were detected by CCK8 and EdU,expressions of TNFα and IL1β were measured by Elisa and Western blotting,cell apoptosis was tested flow cytometry and Western blotting.(4)Raw 264.7 cells were treated with x-LDL,real-time quantitative PCR was used to detect the expression of MEG3;Raw 264.7 cells were transfected with small interfering RNA of MEG3,and then the expression of MEG3,miR-204 and CDKN2 A was detected by real-time quantitative PCR and Western blotting assay;CCK8 and EdU were used to detect cell viability and proliferation;Elisa and Western blotting were used to detect TNFα in cells;Flow cytometry and Western blotting werw used to measure the cell apoptosis.(5)Dual fluorescein plum reporter gene was employed to detecte the binding between MEG3 and miR-204,miR-204 and CDKN2 A.(6)Raw 264.7 cells were transfected with miR-204 mimics and inhibitors,and drug intervention was performed,and then CDKN2 A expression was detected by real-time quantitative PCR and Western blotting.(7)Raw 264.7 cells were transfected with small interfering RNA against CDKN2 A,and then drug intervention was performed.The expression of CDKN2 A was detected by real-time quantitative PCR and Western blotting assay.(8)Raw 264.7 cells were subjected to drug intervention and then performed the modulation of MEG3/miR-204/CDKN2 A pathway;the expression of CDKN2 A was detected by real-time quantitative PCR and Western blotting;cell viability and proliferation were detected by CCK8 and EdU;expressions of TNFα and IL1β were measured by Elisa and Western blotting,cell apoptosis was tested flow cytometry and Western blotting.Results:(1)The success rate of combined surgery for chronic carotid artery occlusion is high.(2)There was a clear correlation between the postoperative reflux and the position of the distal reflux at the level below the level of the clinoidal segment with the success of the operation.(3)In clinical samples(atherosclerotic plaque),the expression of MEG3 was positively correlated with the expression of inflammatory factors TNFα.(4)Ox-LDL inhibits the activity and proliferation of RAW264.7 cells,induces apoptosis and promotes the expression of inflammatory factors(5)Ox-LDL plays a role in the RAW264.7 cells by regulating the expression of MEG3.(6)MEG3 played a role via regulation of the MEG3/miR-204/CDKN2 A regulatory axis inRaw264.7 cells treated with ox-LDL.Conclusion:In summary,hybrid operation can be used as a safe and effective surgical method for recanalization of chronic carotid artery occlusion.After successful recanalization,the perfusion of the affected side was significantly improved and the neurological function recovered well.Our study found that the expression of MEG3 was positively correlated with the expression of inflammatory factors TNFα in clinical samples of chronic carotid occlusion cases(atherosclerotic plaque)and further,identified the role of the MEG3/miR-204/CDKN2 A pathway in Raw264.7 cells treated with ox-LDL,revealed a new regulatory pathway in AS and indicated potential new characteristic biomarkers and therapeutic targets for AS. |
