The Mechanism Of MiR-34a/STAT3/VAMP8 Regulating Platelet Immune Activation

Objective:To explore the mechanism of miR-34a/STAT3/VAMP8 regulating platelet immune activation.Method:（1）Human megakaryocytic leukemia cell Meg-01 was cultured and stimulated with 1mmol/L hydrogen peroxide（H₂O₂）for 2 hours to simulate the oxidative stress state of platelets.The experiment was divided into two groups:control group and H₂O₂ group.The expression level of miR-34a in each group was detected by q-RT-PCR.The protein expression levels of CD62P、CD40L、PAC-1 and VAMP8 in each group were detected by Western Blot（WB）.（2）Meg-01 cells were cultured and transfected with mimics and inhibitors of miR-34a for 48 hours,followed by H₂O₂ stimulation for 2 hours.The experiment was divided into 4 groups:control group,H₂O₂ group,miR-34a mimic+H₂O₂ group and miR-34a inhibitor+H₂O₂ group.Dense granules of the cells was observed by transmission electron microscopy.（3）Meg-01 cells were cultured and transfected with miR-34a mimic,miR-34a inhibitor and their negative control respectively.The experiment consisted of 5groups:which were the control group,miR-34a mimic group,mimic control group,miR-34a inhibitor group and the inhibitor control group.The expression levels of miR-34a and miR-96 in each group were detected by q-RT-PCR.The protein expression levels of P-STAT3、STAT3、PAC-1 and VAMP8 in each group were detected by WB.（4）The target gene prediction website Target Scan was used to predict whether STAT3 gene had binding sites with miR-34a,and dual luciferase reporter genes were used to verify whether STAT3 was the target gene of miR-34a.（5）Meg-01 cells were cultured and transfected with STAT3 overexpression plasmid（CA-STAT3）,interference fragment（si-STAT3）and their negative control,respectively.The experiment was divided into 5 groups:control group,CA-STAT3group,CA-control group,si-STAT3 group and si-control group.The expression levels of STAT3 mRNA and miR-96 in each group were detected by q-RT-PCR.The protein expression levels of P-STAT3、STAT3、PAC-1 and VAMP8 in each group were detected by WB.（6）Meg-01 cells were cultured and transfected with miR-96 mimic,miR-96inhibitor and negative control,respectively.The experiment consisted of 5 groups:namely the control group,miR-96 mimic group,mimic control group,miR-96inhibitor group,and inhibitor control group.The expression level of miR-96 in each group was detected by q-RT-PCR.The protein expression levels of CD62P、PAC-1and VAMP8 in each group were detected by WB.（7）Meg-01 cells were cultured and transfected into CA-VAMP8,si-VAMP8 and their negative controls,respectively.Five groups constituted this experiment:namely the control group,CA-VAMP8 group,CA-control group,si-VAMP8 group and si-control group.The protein expression levels of CD62P、PAC-1 and VAMP8 in each group were detected by WB.Result:（1）H₂O₂ stimulated Meg-01 cells for 2 hours.Compared with the control group,H₂O₂ could significantly increase the expression level of miR-34a,the protein expression levels of CD62P、CD40L、PAC-1 and VAMP8（P<0.05）.（2）After the cells were transfected with miR-34a mimic、miR-34a inhibitor and their respective negative controls,then stimulated with H₂O₂ for 2 hours,transmission electron microscopy results showed that the number of dense particles in the H₂O₂group and the H₂O₂+miR-34a mimic group were higher than those in the control and H₂O₂+miR-34a inhibitor groups.The number of dense particles in the H₂O₂+miR-34a mimic group is more than that in the H₂O₂ group.There is no significant change in the number of dense particles between the control group and the H₂O₂+miR-34a inhibitor group.（3）After the cells were transfected with miR-34a mimic,miR-34a inhibitor and their respective negative controls,the q-RT-PCR test results showed that the expression of miR-34a in the miR-34a mimic group was higher than that of the control group and mimic control group（P<0.05）,the expression of miR-96 was lower than the control group and mimic control group（P<0.05）;the expression of miR-34a in the miR-34a inhibitor group was lower than the control group and the inhibitor control group（P<0.05）,the expression of miR-96 was higher than that of control group and inhibitor control group（P<0.05）;while between control group,mimic control group and inhibitor control group,we don’t see remarkable difference（P>0.05）.WB results showed that the expression of P-STAT3 protein in miR-34a mimic group was significantly lower than that of control group and mimic control group（P<0.05）,while the expression of STAT3 protein had no significant difference（P>0.05）;The expression of PAC-1 and VAMP8 protein in miR-34a mimic group was obvious higher than that of the control group and mimic control group（P<0.05）.The expression of P-STAT3 protein in the miR-34a inhibitor group was significantly higher than that of the control group and inhibitor control group（P<0.05）,while the expression of STAT3 protein had no significant difference in the other two control groups（P>0.05）;the expression of PAC-1 and VAMP8 protein in miR-34a inhibitor group was significantly lower than that of control group and inhibitor control group（P<0.05）.（4）The target gene prediction website Target Scan predicts that miR-34a-5p has binding sites with the 2187-2193 base sequence of the 3’UTR region of STAT3.The dual luciferase reporter gene experiment showed that,compared with NC,after transfection of hsa-miR-34a-5p,the reporter fluorescence expression of h-STAT3-MUT was significantly higher than that of h-STAT3-WT（P<0.05）.It shows that hsa-miR-34a-5p affects the activity of luciferase through the targeted binding of STAT3 3’UTR binding site,confirming that STAT3 is the target gene of miR-34a.（5）After transfecting the cells with CA-STAT3,si-STAT3 and their respective negative controls,q-RT-PCR results showed that the expression of STAT3 mRNA in the CA-STAT3 group was obvious higher than that of the control group and the CA-control group（P<0.05）;The expression of STAT3 mRNA in the si-STAT3 group was significantly lower than that of the control group and the si-control group（P<0.05）.WB results showed that the expression of P-STAT3 protein in the CA-STAT3 group was obvious higher than that of the control group and the CA-control group（P<0.05）,but there was no significant difference in STAT3 protein expression（P>0.05）;The expression of PAC-1 and VAMP8 protein in the CA-STAT3group was significantly lower than that of the control group and CA-control group（P<0.05）;the expression of P-STAT3 protein in the si-STAT3 group was significantly lower than that of the control group and si-control group（P<0.05）,while the expression of STAT3 protein was no significantly difference（P>0.05）;the expression of PAC-1 and VAMP8 protein in si-STAT3 group was significantly higher than that of control group and si-control group（P<0.05）.（6）After transfecting the cells with miR-96 mimic,miR-96 inhibitor and their respective negative controls,q-RT-PCR results showed that the expression of miR-96in the miR-96 mimic group was significantly higher than that of the control group and mimic control group（P<0.05）,the expression of miR-96 in the miR-96 inhibitor group was significantly lower than that in the other two control groups（P<0.05）;WB results showed that the expression of CD62P,PAC-1,and VAMP8 protein in the miR-96 mimic group was significantly lower than that of the control group and the mimic control group（P<0.05）.The expression of CD62P,PAC-1,and VAMP8protein in the miR-96 inhibitor group was obvious higher than that of the control group and the inhibitor control group（P<0.05）.（7）After transfecting the cells with CA-VAMP8,si-VAMP8 and their respective negative controls.WB test results showed that the expression of CD62P、PAC-1、VAMP8 protein in CA-VAMP8 group was significantly higher than that of the other two control groups（P<0.05）,and the expression of CD62P、PAC-1、VAMP8 protein in si-VAMP8 group was obvious Lower than control group and si-control group（P<0.05）.Conclusion:（1）Under the stimulation of H₂O₂,miR-34a promoted the expression of immuneactivated protein in Meg-01 cells.（2）miR-34a/STAT3/VAMP8 may regulate the immune activation function of platelets.