| ObjectiveResistin(RE)plays an important role in the formation of thrombus and participates in the occurrence and development of thrombotic diseases.We predicted that mir-631 and re have complementary binding sites through pre experiment and online software,which is an upstream regulator of re.This study aims to explore the mechanism of mir-631 regulating re involved in deep vein thrombosis from three aspects of endothelial injury,inflammatory response and coagulation.Methods1.80 DVT inpatients in our hospital from 2017/12 to 2018/12 were enrolled in the study.The expression levels of miR-631 and RE in the patients’ plasma was detected to explore the meaning of the two expressions to DVT.2.Human umbilical vein endothelial cells were cultured in vitro and luciferase reporter gene system combined with quantitative PCR was used to verify the interaction between miR-631 and RE.Cells were transfected with miR-631 and RE exogenous expression vector or interference sequence first,then ELISA was to detect endothelial injury-related indicators(eNOS,ET-1,ICAM-1,PGI2,vWF),inflammation-related indicators(CRP,IL-6,IL-1,TNF-a,IL-12),as well as coagulation-related indicators(PAI-1,T-PA),and flow cytometry was to detect endothelial cell apoptosis.3.SD rats were taken to establish a DVT model,miR-631 mimic/inhibitor and oeRETN/siRNA-RETN was injetced through tail vein to observe thrombosis formation.ELISA was to detect the endothelial damage,inflammation,and coagulation-related factors of plasma.Results1.Expression levels of miR-631 and RE in clinical cases:(1)The relative expression level of plasma miR-631 in DVT group was(0.76±0.12),which was lower than that in normal control group(P<0.05);Plasma RE concentration was(0.37±0.09)ng/mL and plasma D-dimer concentration was(1.08±0.08)ng/mL,both of which were higher than those in normal control group(P<0.05).The relative expression level of miR-631 was negatively correlated with RE concentration(r=-0.611,P<0.05),the level of miR-631 was negatively correlated with D-dimer(r=-0.439,P<0.05),and RE concentration was positively correlated with D-dimer(r=0.377,P<0.05).2.In vitro experimental results of human umbilical vein endothelial cells(HUVECs):(1).After transfection of wild-type RE reporter gene and miR-631 mimic with HUVECs,the enzyme activity was significantly lower than that of the control group(P<0.01).After co-transfection with mutant RE reporter gene and miR-631 mimic,there was no difference in enzyme activity compared with control group(P>0.05).(2).miR-631 The expression levels of eNOS,ET-1,PGI2 and vWF in HUVECS cells of mimic group were(59.63±2.36)μmol/L,(12.24±1.02)pg/ml,(11.67±0.53)pg/ml and(17.21±1.15)ng/ml,respectively.ENOS and PGI2 were increased compared with blank control group(P<0.05),ET-1 and vWF were decreased compared with blank control group(P<0.05).The expression levels of eNOS,ET-1,ICAM-1 and VWF in HUVECs cells of OE-RE group were(34.22±2.72)μmol/L,(8.98±0.67)pg/ml,(29.10±2.25)ng/ml and(23.21±1.76)ng/ml,respectively.Compared with blank control group,eNOS was decreased(P<0.05),and other indexes were increased(P<0.05).miR-631 The expression levels of eNOS,ET-1,ICAM-1,PGI2 and vWF in HUVECS cells treated with mimic+OERE were(41.31±2.72)μmol/L,(10.55±1.12)pg/mL,(24.50±2.04)ng/mL,(7.33±0.52)P,respectively G/ml and(21.38±1.43)ng/ml,eNOS and PGI2 were lower than those of miR-631 mimic+NC-Re group(P<0.05),and the others were higher than those of miR-631 mimic+NC-Re group(P<0.05).(3).miR-631 The expression levels of CRP,IL-6,IL-1,TNF-α and IL-12 in HUVECS cells of mimic group were(15.12±1.11)mg/L,(65.20±3.47)mg/L,(88.3 5±4.23)pg/ml,(112.33±8.32)pg/ml and(3)5.56±2.71 pg/ml,lower than that of blank control group(P<0.05).The expression levels of CRP,IL-6,IL-1 and TNF-a in HUVECs cells of OE-RE group were(19.64±1.23)mg/L,(72.23±2.55)mg/L,(84.41±3.61)pg/ml and(119.31±5.14)pg/ml,respectively.Were increased compared with blank control group(P<0.05).miR-631 The expression levels of CRP,IL-6,IL-1,TNF-A and IL-12 in HUVECs cells treated with mimic+OE-RE were(26.34±1.56)mg/L,(63.20±2.54)mg/L,(69.24±2.43)pg/mL,and(97.33±4.12)pg/mL,respectively Ml and(34.64±2.22)pg/ml were increased compared with miR-631 mimic+NC-RE group(P<0.05).(4)The expression levels of PAI-1 and t-PA in HUVECs cells with miR-631 mimic were(9.13±1.00)ng/ml and(7.38±0.65)ng/mL,respectively,which were lower than those in blank control group(P<0.05).The expression levels of PAI-1 and t-PA in HUVECs cells of OE-RE group were(13.37±0.78)ng/mL and(10.55±0.89)ng/mL,respectively,which were higher than those of blank control group(P<0.05).The expression levels of PAI-1 and t-PA in HUVECs endothelial cells of miR-631 mimic+OE-RE group were(17.22±1.21)ng/mL and(14.67±1.04)ng/mL,respectively,which were higher than those of miR-631 mimic+NC-Re group(P<0.05).(5).The apoptosis rate of HUVECs cells in miR-631 mimic group was(8.60±0.68)%,which was lower than that in blank control group(P<0.05).The apoptosis rate of HUVECs cells in miR-631 inhibitor group was(13.62±1.11)%,which was higher than that in blank control group(P<0.05).The apoptosis rate of HUVECs cells in OE-RE group was(16.23±1.23)%,which was higher than that in blank control group(P<0.05).The apoptosis rate of HUVECs cells in siRNA-RE group was(10.62±0.98)%,which was lower than that in blank control group(P<0.05).The apoptosis rate of HUVECs cells in miR-631 mimic+OERE group was(19.55±1.18)%,which was higher than that in miR-631 mimic+NC-RE group(P<0.05).The apoptosis rate of endothelial cells in miR-631 inhibitor+siRNA RE group was(12.73±1.45)%,which was lower than that in miR-631 inhibitor+NC-RE group(P<0.05).3.In vivo experimental results of SD rats:(1)The average length and mass of thrombus in miR-631 mimic group were(6.23±1.02)mm and(17.11±0.87)mg,both of which were lower than those in sham group(P<0.05).The mean thrombus length and mass of miR-631 mimic+OE-RETN group were(7.77±0.78)mm and(19.59±0.98)mg,which were higher than those of miR-631 mimic+NC-RETN group(P<0.05).(2).MiR-631 The expression levels of eNOS,ET-1,ICAM-1,PGI2 and vWF in plasma of rats in mimic group were(68.23±2.96)μmol/L,(14.41±1.34)pg/ml,(32.78±2.13)ng/ml,(17.33±1.60)pg/ml and(23)respectively.95±1.65)ng/mL,eNOS and PGI2 were increased,ET-1 and ICAM-1 were decreased compared with sham group(P<0.05).miR631 mimic+ The expression levels of eNOS,ET-1,ICAM-1,PGI2 and vWF in plasma of OE-RETN group were(43.11±2.78)μmol/L,(21.23±1.24)pg/ml,(42.74±2.62)ng/ml,(10.61±1.02)pg/ml and(1),respectively 9.44±1.12)ng/mL,eNOS and PGI2 were decreased compared with sham operation group,while ET-1,ICAM-1 and vWF were increased compared with sham operation group(P<0.05).(3)The expression levels of CRP,IL-6 and IL-1 in plasma of rats with miR-631 mimic were(14.21±1.00)mg/L,(62.31±3.77)mg/L and(82.87±5.19)pg/mL,respectively,which were lower than those in sham group(P<0.05).miR-631 mimic+The expression levels of CRP,IL-6,IL-1,TNF-A and IL-12 in plasma of OE-RETN group were(23.32±1.23)mg/L,(77.32±2.42)mg/L,(94.45±3.78)pg/ml,(118.51±5.56)pg/ml and(39.89±2.54)pg/ml was higher than that of miR-631 mimic+NC-RENT group(P<0.05).(4)The expression levels of PAI-1 and t-PA in plasma of rats with miR-631 mimic were(11.16±1.04)ng/mL and(9.56±0.56)ng/mL,respectively,which were lower than those in sham group(P<0.05).The expression levels of PAI-1 and t-PA in plasma of rats with miR-631 mimic+OE-Re group were(19.22±0.98)ng/mL and(13.45±1.02)ng/mL,respectively,which were higher than those of rats with miR-631 mimic+NC-Re group(P<0.05).ConclusionsThe plasma miR-631 level was low and RE level was high in patients with acute DVT,and the two were negatively correlated.The plasma miR-631 was negatively correlated with D dimer,and RE were positively correlated with D dimer.MiR-631 is the upstream regulator of RE,which can negatively regulate the expression of RE.miR-631 can reduce vascular endothelial damage,inhibit inflammation,correct coagulation-fibrinolytic disorders,and have a protective effect on thrombosis during DVT,which may be achieved by downregulating the level of RE. |
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