The Effect Of SIK2 On The Migration Ability Of Vascular Endothelial Cells And Its Mechanism

Objective:Adenovirus transfection method and CoCl₂ induction method were used to construct HUVEC models with SIK2 expression in different degrees,to explore the regulation and mechanism of SIK2 on HUVEC migration ability,and to provide theoretical basis for clinical treatment and prevention of ischemic diseases.Methods:Adenovirus transfection and CoCl₂ induction method were used to construct HUVEC models with SIK2 expression in different degrees,which were divided into Control group,CoCl₂ hypoxia induction group,CoCl₂ hypoxia induction combined with BOSUTINIB group,CoCl₂ hypoxia induction combined with adenovirus empty load Group,CoCl₂ hypoxia induction combined with adenovirus transfection SIK2overexpression h-SIK2 group,adenovirus overexpression SIK2 treatment combined with CoCl₂,BOSUTINIB group.Firstly,CCK-8,Hochest staining and LDH method were used to detect the effect of CoCl₂ on HUVEC migration under the hypoxic state of HUVEC.Secondly,the non-cytotoxic dose of BOSUTINIB was explored by CCK-8 method,and the change of SIK2 after BOSUTINIB was detected by Western blot.,Using RT-qPCR and Western blot to verify the successful transfer of SIK2 adenovirus into cells.On this basis,the scratch test and Transwell test were used to measure the migration ability of these groups of HUVEC.Use STRING database to do PPI analysis on SIK2 and its related proteins.The ELISA experiment measures the changes in the contents of MMP-2 and MMP-9 in the supernatant of HUVEC cells.Results:1.CCK-8 method to determine the best action time and concentration of CoCl₂CCK-8 results showed that low concentrations of CoCl₂（below 250μmol/L）did not affect the proliferation of HUVEC（P>0.05）;when the concentration of CoCl₂ reached250μmol/L（0.7430±0.0281%）,the cell proliferation rate was similar to that of the control group.Compared with（0.9681±0.0276%）,the inhibition began to appear（P<0.05）;the time gradient experiment results showed that when the CoCl₂ action time was extended to24 h（0.3963±0.0361%）,the inhibition effect was the most obvious（P<0.05）.2.Hochest staining and LDH method to detect the effect of CoCl₂on endothelial cell apoptosisHoechst staining fluorescence results showed that the cells in the CoCl₂ injury group did not appear a lot of apoptosis.The LDH results showed that there was no significant difference in LDH content between the control group（14.6389±0.8208%）and the CoCl₂injury group（15.3621±0.6138%）（P>0.05）,and the cells did not appear a lot of apoptosis or necrosis.3.Changes in HUVEC migration ability after CoCl₂hypoxia injuryThe results of the scratch test showed that the migration rate（14.76±3.72%）of the CoCl₂ hypoxic injury group was significantly lower than that of the control group（39.36±7.869%）（P<0.05）;Transwell’s results showed that compared with the control group（39.36±7.869%）Compared with the CoCl₂ hypoxia injury group,the migration rate（60.28±8.60%）decreased significantly,and the difference was statistically significant（P<0.05）.4.Changes of SIK2 expression level after CoCl₂ hypoxia injuryRT-qPCR results showed that when HUVEC was treated with CoCl₂ hypoxia,the expression of SIK2 mRNA decreased,and the difference was statistically significant compared with the normal group（P<0.05）;Western blot results showed that when HUVEC was treated with CoCl₂ hypoxia After the injury treatment,the expression of SIK2 protein decreased,and the difference was statistically significant compared with the normal group（P<0.05）.5.The effect of changes in the expression level of SIK2 on cell migrationThe results of the scratch test showed that compared with the control group（38.47±2.81%）,the migration rate of the Vector group（38.62±2.37%）did not change significantly,and the difference was not statistically significant（P>0.05）;the migration rate of the CoCl₂ hypoxic injury group（19.37±2.14%）significantly decreased（P<0.05）;CoCl₂ treatment SIK2 overexpression group migration rate（273.83±21.13%）significantly increased（P<0.05）;CoCl₂ treatment combined with BOSUTINIB group migration rate（37.24±9.21%）Significantly decreased（P<0.01）;the migration rate（188.73±27.16%）in the treatment group of overexpression SIK2 combined with CoCl₂ and BOSUTINIB increased significantly（P<0.01）;the results of Transwell showed that the migration rate in the no-load Vector group was（106.07±16.26%）)Compared with the blank control group,the difference was not statistically significant（P>0.05）;the migration rate（70.64±10.81%）of the CoCl₂ hypoxic injury group was significantly reduced（P<0.05）;the migration rate of the SIK2 overexpression group treated with CoCl₂ was（213.53±）15.38%)significantly increased（P<0.05）;CoCl₂ treatment combined with BOSUTINIB group migration rate（27.63±2.53%）significantly decreased（P<0.01）;overexpression SIK2 combined with CoCl₂,BOSUTINIB treatment group migration rate（162.84±24.18%）)Increased significantly（P<0.01）.The results show that SIK2 overexpression can increase the migration ability of HUVEC;after inhibiting SIK2,the migration ability decreases.6.STRING database performs PPI analysis on SIK2 and its related proteinsFind out the interaction between related proteins and SIK2 in the STRING database.Enrichment analysis of the above-mentioned protein pairs matched the GO and KEGG pathways with the first 10 significant pathways respectively.The results suggest that SIK2is highly correlated with CRTC3,CREB1,MMP-2,and MMP-9.7.The effect of the change of SIK2 expression level on the content of MMP-2 and MMP-9in the supernatantThe results of ELISA showed that,compared with the control group,MMP-2 and MMP-9 in the CoCl₂ treatment group decreased（P<0.05）;MMP-2 and MMP-9 in the CoCl₂ treatment combined with BOSUTINIB group decreased significantly（P<0.05）;CoCl₂ MMP-2 and MMP-9 in the SIK2 overexpression group increased significantly（P<0.01）;MMP-2 and MMP-9 in the overexpression SIK2 combined with CoCl₂ and BOSUTINIB treatment group increased significantly（P<0.01）.The results suggest that SIK2 may regulate the migration of HUVEC by regulating the expression of MMP-2 and MMP-9.Conclusion:SIK2 can regulate HUVEC migration,and SIK2 can regulate HUVEC migration through MMP-2 and MMP-9.