The Role And Underlying Mechanism Of MiR-24-3p On Angiogenesis Of CoCl₂-induced Hypoxic HUVEC By Targeting SP1

Objective:A model of hypoxic human umbilical vein endothelial cell（HUVEC）induced by Co Cl₂ was established to investigate the effects of Co Cl₂ on vascular endothelial cell（VEC）morphology and mi R-24-3p and explore the effects of mi R-24-3p on the biological behavior and angiogenesis of hypoxic HUVEC induced by Co Cl₂ and its underlying molecular regulation mechanism,which may clarify the mechanism of mi R-24-3p in the occurrence and development of ischemic heart disease such as Acute Myocardial Infarction（AMI）,to provide new ideas and basis for its clinic.Methods:1.The HUVEC hypoxia model was established,and the expression of mi R-24-3p was detected in hypoxic HUVEC.To explore the appropriate concentration of Co Cl₂ to establish a HUVEC hypoxia model,the HUVEC of this study was divided into 0μM（Cont group）,100μM group,200μM group,and 400μM groups.After adding different concentrations of Co Cl₂ for 24 h,the morphology of HUVEC was observed under the microscope CCK8 detected the cell viability,and the m RNA and protein levels of HIF-1αwere detected by q RT-PCR and Western Blot.Finally,the appropriate Co Cl₂ was determined to establish the hypoxic HUVEC model in a subsequent experiment.2.Effects of mi R-24-3p on the biological behavior of hypoxic HUVEC CCK-8,clone formation assay,cell scratch test,Transwell cell invasion assay,flow cytometry,were used to detect the effects of mi R-24-3p on proliferation,migration,invasion,apoptosis,and cell cycle of hypoxic HUVEC,as well as the artificial basal gel,was used to detect the angiogenesis of hypoxic HUVEC.3.The mechanism of mi R-24-3p targeting SP1 to inhibit Co Cl₂-induced hypoxic HUVEC angiogenesis（1）Online bioinformatics software such as targetscan,pictar,mi Randa were used to analyze and predict the potential target genes of mi R-24-3p.（2）The effects of mi R-24-3p on the expression of SP1 m RNA and protein in hypoxic HUVEC were analyzed by q RT-PCR and Western blot.（3）The hypoxic model of SP1 overexpression strain was established.And CCK-8,clone formation assay,cell scratch assay,Transwell chamber invasion assay,and flow cytometry were used to detect whether SP1 affected the proliferation,migration,invasion,and apoptosis mi R-24-3p on hypoxic HUVEC and the effect of the artificial basal gel on the angiogenesis of hypoxic HUVEC.（4）q RT-PCR and Western blot were used to detect whether SP1 affected the m RNA and protein expressions of related genes such as proliferation,migration,invasion,and apoptosis of mi R-24-3p affecting hypoxic HUVEC.Results:1.The hypoxic HUVEC model was established,and the expression of mi R-24-3p in hypoxic HUVEC was detected（1）Compared with CONT group,HUVEC activity were decreased with the increment of Co Cl₂ concentration significantly（P<0.01 or P<0.001）.（2）HIF-1αm RNA levels in HUVEC were increased 4-fold and 5-fold after pretreatment with 200 or 400μM Co Cl₂ for 24h,respectively（P<0.01）.In addition,Western Blot results showed that HIF-1αwas up-regulated in hypoxic HUVEC with treatment 200μM（P<0.01）.Therefore,the HUVEC model of hypoxia was established using 200μM in the subsequent experiments.（3）After Co Cl₂ pretreatment for 24 hours,q RT-PCR results showed that mi R-24-3p level in HUVEC of 100μM Co Cl₂ group was not significantly different from that of Cont group（P<0.05）.However,mi R-24-3p levels in 200μM and 400μM HUVEC groups were increased by 2.7-fold and 3.4-fold,respectively（P<0.01）.2.Effects of mi R-24-3p on the biological function of hypoxic HUVEC（1）CCK-8 showed that there was no significant difference of absorbance between the groups on 1d and 2d（P>0.05）;On 3d,4d,and 5d,the mi R-24-3p group significantly inhibited Co Cl₂-induced hypoxic HUVEC proliferation,while mi R-24-3p inhibitor had the opposite effect and promoted Co Cl₂-induced hypoxic HUVEC proliferation significantly（P<0.05,P<0.01,P<0.001）.（2）Cloning formation unit assay showed that mi R-24-3p inhibited the clone formation ability of hypoxic HUVEC compared with mi R-NC significantly（P<0.01）,and mi R-24-3p inhibitor significantly promoted the clonogenic activity of hypoxic HUVEC（P<0.01）.（3）Compared with mi R-NC,mi R-24-3p inhibited hypoxic HUVEC migration significantly,while mi R-24-3p inhibitor promoted HUVEC migration（P<0.01）.（4）Compared with mi R-NC,mi R-24-3p inhibited the invasion of hypoxic HUVEC（P<0.01）,and mi R-24-3p inhibitor promoted the invasion of hypoxic HUVEC significantly（P<0.01）.（5）Flow cytometry detection of apoptosis revealed that mi R-24-3p promoted the total apoptosis rate and early apoptosis rate of hypoxic HUVEC significantly（P<0.01）,and mi R-24-3p inhibitor significantly inhibited the total apoptosis and early apoptosis rate of hypoxic HUVEC（P<0.001）.（6）Flow cytometry detection of cell cycle showed that both high and low expression of mi R-24-3p did not affect the G0/G1 phase of hypoxic HUVEC but had the opposite effect of S and G2/M phase.mi R-24-3p blocked hypoxic HUVEC in the S phase,and the proportion of the G2/M phase increased accordingly（P<0.01）.（7）Compared with mi R-NC,mi R-24-3p inhibited the angiogenesis ability of hypoxic HUVEC significantly.Interestingly,the mi R-24-3p inhibitor promoted the angiogenesis ability of hypoxic HUVEC（P<0.01）.3.The mechanism of mi R-24-3p targeting SP1 to inhibit Co Cl₂-induced hypoxic HUVEC angiogenesis（1）Targetscan,Pictar,and mi Randa were used to analyze and predict the potential target genes of mi R-24-3p.The results showed that there were some complementary sites between mi R-24-3p and SP1.（2）The effects of mi R-24-3p on the expression of SP1 m RNA and protein in hypoxic HUVEC were analyzed by q RT-PCR and Western Blot.The results showed that compared with mi R-NC,mi R-24-3p resulted in the down-regulation expression of SP1 m RNA and protein（P<0.01）,and its inhibitors increased the levels of SP1 m RNA and protein（P<0.01.）.（3）CCK-8 showed that there was no significant difference in absorbance of each group at 1d,2d and 3d（P>0.05）;On 4d and 5d,mi R-24-3p inhibited Co Cl₂-induced hypoxic HUVEC proliferation significantly（P<0.01,P<0.001）,SP1 significantly reversed the inhibitory effect of mi R-24-3p on the proliferation of hypoxic HUVEC（P<0.01）.Colony formation assay showed that mi R-24-3p reduced hypoxic HUVEC colony formation rate significantly,while SP1significantly reversed the inhibitory effect of mi R-24-3p on the colony formation hypoxic HUVEC（P<0.01or P<0.001）.（4）mi R-24-3p down-regulated e NOS m RNA and protein levels significantly（P<0.01）,and SP1 significantly reversed the down-regulation of mi R-24-3p on the m RNA and protein levels（P<0.01or P<0.001）.（5）The NO content in mi R-24-3p group cell was decreased significantly（P<0.01）,while SP1 could reverse the effect of mi R-24-3p on NO content of hypoxic HUVEC reduction（P<0.01or P<0.001）.（6）The m RNA and protein relative expressions of VEGF and VEGFR2 in mi R-24-3p group cell were down-regulated significantly（P<0.01）.At the same time,SP1 reversed the down-regulation effect of mi R-24-3p on the relative expression levels of VEGF and VEGFR2 m RNA in hypoxic HUVEC（P<0.01or P<0.001）.（7）mi R-24-3p inhibited the migration and invasion of hypoxic HUVEC significantly（P<0.01）;SP1 reversed the inhibitory effect of mi R-24-3p on the migration and invasion of hypoxic HUVEC（P<0.05 or P<0.01）).（8）The m RNA and protein relative expressions of MMP2 and MMP9 in the mi R-24-3p group were decreased significantly（P<0.01）,and SP1significantly reversed the down-regulation effect of mi R-24-3p on the m RNA and protein levels of MP2 and MMP9 in hypoxic HUVEC（P<0.01）.（9）mi R-24-3p promoted the rate of total apoptosis and early phase apoptosis of hypoxic HUVEC（P<0.01）.At the same time,SP1 reversed the promoting effects of mi R-24-3p on the total apoptosis rate and early phase apoptosis of hypoxic HUVEC significantly（P<0.01）.（10）mi R-24-3p increased the relative expression levels of Bax m RNA and protein.It decreased the relative expression levels of Bcl-2 m RNA significantly（P<0.01）.At the same time,SP1 reversed the effect of mi R-24-3p on the increase of the relative expression levels of Bax m RNA and protein and the reduction of the relative expression levels of Bcl-2 m RNA and protein in hypoxic HUVEC（P<0.01or P<0.01）.（11）mi R-24-3p inhibited the number,length,and nodes of mesh formation in hypoxic HUVEC tubes significantly（P<0.01）,and SP1 significantly reversed the inhibitory effects of mi R-24-3p on the number,length,and nodes of tube formation in hypoxic HUVEC tube formation（P<0.01）.Conclusions:1.The HUVEC model of hypoxia could be established at 200μm Co Cl₂.2.mi R-24-3p inhibited the proliferation,migration,and invasion of hypoxic HUVEC and its tubular ability,promoted apoptosis,and blocked it in the S phase.3.SP1 could reverse the inhibitory effect of mi R-24-3p on the proliferation,migration,invasion,and tubulation ability of hypoxic HUVEC and promote the apoptosis of hypoxic HUVEC.4.mi R-24-3p could inhibit hypoxic HUVEC angiogenesis by targeting SP1 and negatively regulating SP1,partially through the SP1/e NOS/NO signaling pathway,and affecting the expression of downstream genes,including VEGF,VEGFR2,MMP2,MMP9,Bax,and Bcl-2.