The Effect Of Long Non-coding RNA MALAT1 On Platelet Activation And Mechanism

Objective:Platelets are an important component of blood,and platelets play an important role in the formation of thrombus and physiological hemostasis.When the platelet activation function is abnormal,it will promote the formation of pathological thrombus which is an important cause of cardiovascular disease in myocardial infarction,hypertension and stroke.Currently,one of the core strategies for the treatment of cardiovascular disease is the timely treatment of antiplatelets.Therefore,exploring the mechanism by which platelets are thrombus in vivo provides a new therapeutic approach for the clinical treatment of cardiovascular diseases associated with thrombosis.The long non-coding RNA in vivo has abundant biological functions,especially The long non-coding RNA MALAT1,which is of great significance with the development of various tumors.Studies have now found that some long-chain non-coding RNAs are closely related to the occurrence and development of cardiovascular diseases.However,the study of long-chain non-coding RNA in platelets is still in its infancy.We have found that the expression of IncRNA MALAT1 in platelets is very high and the difference in expression between the two groups is through sequencing of people with high platelet aggregation rate and low aggregation rate.very large.A large number of literature studies have shown that lncRNA MALAT1 is mainly expressed in the nucleus of cells and platelets are non-nuclear cells.This differential expression has attracted our attention.Let us make a bold assumption:lncRNA MALAT1 can affect the activation of platelets,thus affecting the formation of thrombosis in the body.Methods:(1)We established the IncRNA MALAT1 knockdown MEG-01 cell membrane model by si-RNA and the adhesion and spreading experiments of MEG-01 cells and platelet like particle platelet like particle surface antibody detection experiments and Experiments on the size and number of platelet like particle particles in vitro explored the effect of IncRNA MALAT1 knockdown on MEG-01 and its resulting PLP function and its mechanism of action.(2)Using lncRNA MALAT1 lentivirus in the tail vein to establish a mouse model of lncRNA MALAT1 knockdown by analyzing blood parameters of mice,spreading experiments of mouse platelets,mice tail bleeding,Fecl3 induced mice Mesenteric artery thrombosis experiments,microfluidic shear force experiments and flow cytometry on the platelet active markers ??b?3 and P-Selectin were used to investigate the effect of lncRNA MALAT1 on platelet activation in mice.(3)The localization of lncRNA MALAT1 on MEG-01 cells and platelets was determined by nuclear separation and FISH(fluorescence in situ hybridization)of MEG-01 cells.The expression of Akt,P-Akt,PDK1,GSK-3? in PI3K/Akt pathway associated with platelet activation was detected by Western blot.Results:The experimental results showed that(1)lncRNA MALAT1 had significant expression differences in the two groups by analyzing the sequencing results of people with high platelet aggregation rate and low aggregation rate.Detection of lncRNA by Quantitative PCR MALAT1 is highly expressed in both mouse platelets and human platelets.When thrombin was added to stimulate MEG-01 cells,the expression of lncRNA MALAT1 decreased with increasing time of thrombin stimulation,which was time-dependent.The adhesion and spreading ability of cells and platelet like particle in the MALAT1 KD group were significantly increased by thrombin stimulation of MEG-01 cells and platelet like particle secreted by cells.The volume and activity of the platelet like particle secreted by the MALAT1 KD group were significantly increased by flow cytometry.(2)In animal experiments,we analyzed the blood parameters of mice,and all the indexes of the mice were within the normal range,indicating that the lncRNA MALAT1 deletion did not affect the normal hematopoietic function of the mice.However,the time of tail bleeding in the MALAT1 KD group was significantly shortened.Under the induction of Fecl3,the mesenteric artery of the mouse was more likely to form a thrombus.The whole blood was more easily combined with fibrinogen to form a thrombus under the action of 5dyn/cm2 shear force.It is easier to spread on fibrinogen under the action of thrombin,and the activity of integrin ??b?3 on platelets is higher.(3)In terms of mechanism of action,we determined that lncRNA MALAT1 is mainly expressed in the nucleus of MEG-01 by fluorescence in situ hybridization.The distribution of lncRNA MALAT1 on platelets is relatively wide.Western blot analysis revealed that lncRNA MALAT1 knockdown increased the phosphorylation level of Akt in MEG-01 cells and platelets,and decreased the phosphorylation levels of PTEN and GSK-3?.Conclusion:The above data indicate that lncRNA MALAT1 plays a very important role in platelet activation.lncRNA MALAT1 can promote Akt phosphorylation by regulating the PI3K/Akt pathway,inhibiting its downstream target protein GSK-3?,thereby changing the conformation of integrin ??b?3,promoting platelet activation,and thus affecting thrombus formation.This provides a theoretical basis for the treatment of pathological thrombosis in vivo by lncRNA MALAT1.