Effects Of HOXC6 On The Proliferation,migration Of Rat Vascular Smooth Muscle Cells And The Apoptosis Of Vascular Endothelial Cells

BackgroundThe morbidity and mortality from atherosclerotic cardiovascular diseases(ASCVD)such as coronary heart disease(CHD),restenosis after coronary angioplasty/stenting and stroke are gradually increasing in most developing countries.ASCVD is a leading cause of death caused by noncommunicable diseases in China and even worldwide.Atherosclerosis(AS)plays a major role in the pathological basis of ASCVD.Vascular smooth muscle cells(VSMCs)located in the media are migrated to the intima and subintima and proliferate abnormally due to the functions of various pathogenic factors of AS,while the apoptosis of vascular endothelial cells(VECs)in the intima are increased,which are the main cytopathological bases for the occurrence and development of AS.However,the molecular mechanisms of above-mentioned abnormal biological behaviors of vascular wall cells and their mediating the occurrence of AS have not been fully understood.Homeobox(HOX)proteins belong to the category of homologous domain transcription factors which are highly evolutionarily conserved and affect morphogenesis in chordates.There are 39 homologous genes in HOX gene family.Recent studies have shown that the expression levels of HOXA4,HOXA5,HOXA6,HOXB1 and HOXB9 in porcine aortic wall were affected by shear stress.However,the effects of these genes on vascular biological functions and AS have not been investigated.Homeobox C6(HOXC6)gene is a member of cluster C in the HOX gene family.Previous studies have shown that HOXC6 was highly expressed in multi-organ system malignant tumor tissues which affected the biological behaviors of tumor cells,such as proliferation,migration and apoptosis.It has also been reported that HOXC6 regulated the differentiation of vascular wall pluripotent stem cells and skin fibroblasts into VSMCs and vascular wall mesenchymal cells.However,whether HOXC6 had any effect on the biological behaviors of VSMCs and VECs was not investigated.ObjectiveThe aim of this study was to investigate the differential expression of 39 Hox genes such as Hoxc6 between the aortic walls of AS rats and the normal aortic walls and to explore whether HOXC6 affected the proliferation,migration of VSMCs and the apoptosis of VECs.The mechanisms involved in above-mentioned processes will be studied.The study will establish theoretical basis for determining whether HOXC6 can become a new target for ASCVD molecular therapy.MethodsThe aortas of rats were stained with hematoxylin and eosin(H&E)to verify the establishment of AS rat model.Immunohistochemistry was used to detect in situ expressions of Hoxc6,Bcl-2,and Bax proteins in the aortas of rats.The expressions of?-smooth muscle actin(?-SMA)in VSMCs and CD31 in VECs were deteimined using immunofluorescence anslysis.Real time quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay was performed to check the expressions of mRNAs such as 39 Hox genes,p53,proliferating cell nuclear antigen(PCNA),phospholipase C beta(PLC ?)and platelet-activating factor receptor(PTAFR).Western blot analysis was applied to measure the expressions of proteins such as Hoxc6,p53 and PCNA in the aortas,hearts and VSMCs of rats,and to detect the expressions of HOXC6,BCL-2,BAX,Caspase-3,Cleaved caspase-3,Caspase-9,PTAFR,PLC ? and IL-18 proteins in VECs,and to evaluate the levels of phosphorylation of protein kinase C zeta(PKC?)and NF-?Bp65 and membrane translocation of PKC?.Cell counting kit-8(CCK-8)and 5-ethyl-2'-deoxyuridine(Ed U)assays were used to detect the proliferation of VSMCs.The migration of VSMCs was determined by cell scratch and transwell assays.The apoptosis of VECs was measured using the TUNEL and the flow cytometry assays.Bioinformatics analysis methods were used to collect and analyze the expressions of genes related to CHD and their possible signal pathways in the GEO database.Dual luciferase reporter gene assay was applied to detect the binding of HOXC6 to the DNA sequence of PTAFR promoter.Chromatin immunoprecipitation(Ch IP)combined with qPCR(Ch IP-qPCR)was used to validated the specific target sequences where HOXC6 binded to PTAFR promoter.Results1.Abnormal expression of Hox genes in the aortic walls of AS ratsThe results of H&E staining showed that the average ratio of aortic intima/media in the aortas of AS rats was significantly higher than that in the aortas of normal rats(P<0.01).qRT-PCR detection results of 39 Hox genes mRNAs showed that 17 Hox genes were up-regulated or down-regulated(the mean absolute value of the multiple increased or decreased was?2)in aortic walls of AS rats,among them,Hoxa1,Hoxa3,Hoxa9,Hoxb7,hoxc5,Hoxc6,Hoxc8,Hoxc9 and Hoxc10 were up-regulated(all P<0.01),while Hoxa2,Hoxa4,Hoxa5,Hoxa6,Hoxb2,Hoxb3,Hoxb4 and Hoxb6 were down-regulated(all P<0.01).The expression of Hoxc6 was increased by more than 10 times among the 9 up-regulated Hox genes.Compared with Hoxc9 or Hoxc10 which was increased by higher multiples,Hoxc6 had the highest basic expression abundance in rat aortic wall,and had the best repeatability,hence Hoxc6 was our first choice for further verification.2.Abnormal expressions of Hoxc6 and proliferation-apoptosis related molecules in the aortic walls of AS ratsThe results of qRT-PCR and western blot assays showed that the expressions of Hoxc6 mRNA and protein in the aortic walls of AS rats were significantly higher than those in the aortic walls of normal rats(all P<0.01),accompanied by a significant decrease in the expressions of p53 mRNA and protein and a significant increase in the expression of PCNA mRNA and protein(all P < 0.01).The results of immunohistochemical staining showed that the expression of Hoxc6 protein was significantly increased in the atherosclerotic plaque lesions of aortas and VSMCs migrating to intima of AS rats(P<0.01),accompanied by a significant decrease in the expression of anti-apoptotic protein Bcl-2 and a significant increase in the expression of pro-apoptotic protein Bax(all P<0.01).3.The proliferation and migration of rat VSMCs(RVSMCs)were inhibited by knocking down Hoxc6 geneThe immunofluorescence staining results showed that after RVSMCs were stained using anti-?-SMA antibody,the positive rate of the expression of?-SMA in the cytoplasm was decreased in oxidized low density lipoprotein(ox-LDL)treated cells(P<0.05).The results from the Ed U,CCK-8,cell scratch and transwell assays showed that after RVSMCs were treated with ox-LDL,the cell proliferation was significantly increased,and the migration ability was siginficantly increased as well(all P<0.01).Meanwhile,the expressions of p53 mRNA and protein were decreased while the expressions of PCNA mRNA and protein were increased(all P<0.01).However,after the expression of Hoxc6 was down-regulatied in RVSMCs treated with ox-LDL or cultured normally,it was found that the cell proliferation and migration were significantly decreased(all P<0.01),accompanied by an increase of p53 protein expression and a decrease of PCNA protein expression(all P<0.01).Furthermore,after the expressions of Hoxc6 and p53 in the group where were knocked down in RVSMCs,the cell proliferation and migration were increased compared with those in the group that knocked down Hoxc6 alone(all P<0.05),but decreased compared with those in normal control group(all P<0.05).4.The apoptosis of VECs was inhibited by down regulating HOXC6expressionThe results from the TUNEL,flow cytometry,qRT-PCR and western blot showed that the apoptosis rate of rat aortic endothelial cells(RAECs)or human umbilical vein endothelial cells(HUVECs)was significantly increased compared with the normal control group after ox-LDL treatment(all P<0.05),accompanied by a significant decrease in the expression of anti-apoptotic protein BCL-2(all P<0.01),and an increase in the expressions of pro-apoptotic proteins such as BAX,Caspase-3,Cleaved caspase-3 and Caspase-9(all P < 0.05).However,after inhibiting the expression of HOXC6 in RAECs or HUVECs cultured normally or treated with ox-LDL,the apoptosis rate of RAECs or HUVECs was significantly decreased compared with the normal control group or simple ox-LDL treatment group(all P<0.05).Meanwhile,the expression of anti-apoptotic protein BCL-2 was significantly increased(all P<0.01)and the expressions of pro-apoptotic proteins such as BAX,Caspase-3,Cleaved caspase-3 and Caspase-9 were decreased(all P<0.05).On the other hand,over-expressing HOXC6 in HUVECs,the trend of HUVECs apoptosis rate and apoptosis-related proteins changes were opposite to that of knocking down of HOXC6 expression(all P<0.05).5.HOXC6 targeting PTAFR affected the apoptosis of HUVECs via PLC?/PKC?/NF-?Bp65/IL-18 pathwayAfter analyzing the GSE20681,GSE40231 and GSE46560 data sets in GEO database related CHD,68 genes including PTAFR were found to have intersections.KEGG pathway analysis showed that PTAFR could affect apoptosis through PLC/PKC pathway.The predictive results using the JASPAR database showed that there were several specific binding sites where HOXC6 protein could bind to the promoter sequence of PTAFR.In view of the consistency of HOXC6's influence on the apoptosis of RAECs and HUVECs,HUVECs were selected as the target cells to explore the mechanism that HOXC6 would affect the apoptosis of VECs.qRT-PCR and western blot results showed that the expressions of PTAFR mRNA and protein were increased after treating with ox-LDL or over-expressing HOXC6 in HUVECs(all P<0.05),with an increase of the downstream signal molecule PLC? expression(all P<0.05),PKC? phosphorylation and membrane translocation levels were enhanced(all P < 0.05),the levels of NF-? Bp65 phosphorylation and IL-18 expression were raised as well(all P<0.05).However,after inhibiting the expression of HOXC6 in HUVECs treated with ox-LDL or cultured normally,the changes of the above-mentioned molecules were opposite to that of promoting the expression of HOXC6(all P<0.05).In addition,PTAFR agonist platelet-activating factor and PKC?agonist lysophosphatidylcholine could reverse the inhibition effect of HUVECs apoptosis and the activity of PLC?/PKC?/NF-?Bp65/IL-18 signaling pathway caused by down-regulating HOXC6 expression(all P<0.05).Furthermore,the results of dual luciferase reporter gene assay showed that after over-expressing HOXC6 in HUVECs,the binding activity of HOXC6 protein to PTAFR promoter was significantly increased(P<0.01).The results of Ch IP-qPCR assay showed that HOXC6 could bind to PTAFR promoter at specific sites AGAGGAATAGATTAAA and(or)GAAGCAATCAACCAAG(P<0.01).Conclusions1.Seventeen of the 39 Hox genes were abnormally expressed in the aortic walls of AS rats,among which Hoxc6 expression was increased,accompanied by a decreased expression of p53 and Bcl-2,while an increased expression of PCNA and Bax.2.HOXC6 promoted the proliferation and migration of VSMCs via negative regulating the expression of p53,on the other hand facilitated the apoptosis of VECs via targeted up-regulating the expression of PTAFR which increased the activity of PLC?/PKC?/NF-?Bp65/IL-18 pathway.These results suggested that there was the heterogeneity that HOXC6 influenced the proliferation and migration of VSMCs and the apoptosis of VECs.