The Effect And Mechanism Of Down-regulating BOP1 In Vascular Smooth Muscle Cells On Neointimal Hyperplasia

Part?The expression of BOP1 is up-regulated in the neointimal hyperplasia tissuesResearch significance Neointimal hyperplasia is a common pathological change of coronary heart disease,restenosis after stenting,graft vasculopathy and many other vascular proliferative diseases.However,there is still a lack of effective clinical intervention targets for it.The increase of Blocking of Proliferation 1(BOP1)has been confirmed in a variety of proliferative diseases.The purpose of this part was to explore the expression of BOP1 in neointimal hyperplasia tissues and determine whether BOP1 is involved in neointimal hyperplasia.Methods The atherosclerotic coronary artery,in-stent restenosis and normal coronary artery specimens were collected from patients undergoing heart transplantation in our hospital.The m RNA expression levels of BOP1 and Ki-67 were detected by RT-PCR and Western blot(WB)was used to detect the protein expression of BOP1 in the coronary artery tissues.The localization of BOP1 in the coronary arteries were observed by immunofluorescence(IF)staining.Rat carotid artery balloon injury model was established,and the carotid arteries were collected for IF staining to verify the expression changes of BOP1 in neointimal hyperplasia.Mice carotid artery ligation model was also established,HE staining was used to evaluate the efficiency and IF staining was used to observe the expression of BOP1 over time of ligation.Results Compared with normal coronary arteries,the m RNA and protein levels of BOP1 in atherosclerotic and in-stent restenosis coronary arteries were significantly increased(P<0.05),and the m RNA levels of Ki-67 were also significantly increased(P<0.05).The results of IF staining showed that BOP1 was highly expressed on the inside edge of the neointimal and was co-localized in vascular smooth muscle cells(VSMCs)with Ki-67.HE staining of mice carotid arteries showed that neointimal hyperplasia was successfully induced.IF staining showed that the expression of BOP1was increased with the time of ligation.Conclusions BOP1 is highly expressed in VSMCs on the inside edge of the neointimal and the expression increased with the intensification of neointimal hyperplasia.Up-regulation of BOP1 may be one of the factors that drive the proliferation and migration of VSMCs and thus induce neointimal hyperplasia.Part?Down-regulation of BOP1 inhibited the proliferation and migration of VSMCs and alleviated neointimal hyperplasiaResearch significance Although it has been confirmed that BOP1 expression is upregulated in VSMCs in the process of neointimal hyperplasia,the relationship between BOP1 expression and functional changes of VSMCs remains unclear.This part aims to investigate whether the change of BOP1 expression affects the proliferation and migration ability of VSMCs and whether the down-regulation of BOP1 has the effect on neointimal hyperplasia.Methods The neointimal hyperplasia model of VSMCs was established by platelet-derived growth factor-BB(PDGF-BB)treatment.VSMCs were treated with BMH-21 to detect the inhibition efficiency of BOP1.The m RNA and protein expression levels of BOP1 in VSMCs were detected by RT-PCR and WB.The lentivirus loaded with BOP1 specific RNAi(LV-BOP1)and scrambled sequences(LV-SCR)was used to construct VSMC lines.WB was used to detect the knockdown efficiency of BOP1,the proliferation ability was tested by CCK8 and Ki-67 IF staining and the migration ability was compared by Wound Healing Assays and the Transwell Assay.VSMCs were transfected with adenovirus to overexpress BOP1(Ad-BOP1),and co-treated with BMH-21 to detect the migration and proliferation ability.Topical transfection with LV-BOP1 or Ad-BOP1 of the carotid arteries was used and the severity of neointimal hyperplasia was evaluated by HE staining or EVG staining.The proteins expression levels of BOP1were detected by IF or immunohistochemical(IHC)staining.Results PDGF-BB significantly increased the m RNA and protein expression of BOP1in VSMCs,in a dose-and time-dependent manner(P<0.05).The BOP1 protein level of LV-BOP1 group was significantly decreased,as well as the proliferation and migration ability(P<0.05),compared to LV-SCR group.The m RNA and protein levels of BOP1 were decreased in a time-and dose-dependent way after BMH-21treatment(P<0.05).The proliferation and migration of VSMCs were significantly inhibited by 1?M BMH-21,and was partially reversed by transfection with Ad-BOP1(P<0.05).Topical transfection of LV-BOP1 of mice carotid artery significantly decreased the expression level of BOP1,inhibited cells proliferation,and alleviated the neointimal hyperplasia(P<0.05).BMH-21 inhibited neointimal hyperplasia and was partially reversed by local transfection of Ad-BOP1(P<0.05).Conclusion The change of BOP1 expression affects the function of VSMCs.BMH-21treatment or LV-BOP1 transfection can inhibit the proliferation and migration of VSMCs thus alleviate the neointimal hyperplasia.Part?BOP1 knockdown regulates the function of VSMCs by activating the RPL11/MDM2/p53 nucleolar stress response pathway and inhibiting nascent protein synthesisResearch significance Although it has been clear that BOP1 knockdown can regulate the function of VSMCs and inhibit neointimal hyperplasia,the specific mechanism of action remains mysterious.The purpose of this section is to explore the mechanism through which BOP1 knockdown regulates the function of VSMCs.Methods In order to verify whether ribosomal protein L11(RPL11)is the downstream of BOP1 knockdown,we transfected VSMCs with LV-BOP1 or LV-SCR and conducted synchronous treatment.According to whether RPL11 was knocked down by si RNA,the cells were divided into four groups:LV-SCR+NC si RNA group,LV-SCR+RPL11 si RNA group,LV-BOP1+NC si RNA group and LV-BOP1+RPL11si RNA group.The proliferation ability of VSMCs in each group was compared by CCK8 detection and Ki-67 IF staining,the migration ability was measured by the Transwell Assay and the Wound Healing Assays.Flow cytometry was used to compared the reactive oxygen species(ROS)level,cell cycle and apoptosis ratio of each group,the protein levels of RPL11,murine double minute 2(MDM2),p53 and p21 were determined by WB.In order to certify whether p53 is downstream of BOP1knockdown,we used a similar manner to divide VSMCs into four groups:LV-SCR group,PFT-?group,LV-BOP1 group and LV-BOP1+PFT-?group.The corresponding indexes of cells in each group were detected by the same method.The co-localization of RPL11 and MDM2 after BOP1 knockdown was observed by laser confocal microscopy.The stability of p53 was detected by actinomycin treatment and the synthesis of nascent protein was detected by puromycin treatment.The carotid artery neointimal hyperplasia model of p53 knockout mice was constructed and was topically transfected with LV-BOP1(p53^-/-+LV-BOP1 group),the severity of neointimal hyperplasia and the proportion of proliferating cells were compared with the wild-type mice(p53^+/++LV-BOP1 group).Results Compared to LV-SCR+NC si RNA group,VSMCs in LV-BOP1+NC si RNA group have lower proliferation and migration ability,while the ROS level,cells arrested in G0/G1 phase as well as the proportion of apoptotic cells were higher.MDM2,p21 and p53 protein levels were also significantly increased as detected by WB.However,all these effects can be partly reserved by RPL11 si RNA transfection(LV-BOP1+RPL11 si RNA group)(P<0.05).The results of inhibiting p53 by PFT-?treatment were similar to RPL11 si RNA transfection.After BOP1 knockdown,the co-localization of RPL11 and MDM2 was increased as detected by IF staining,the degradation rate of p53 was decreased,the synthesis of nascent proteins was also decreased whereas could not be reversed by PFT-?treatment.Compared with the p53^+/++LV-BOP1 group,the neointima hyperplasia and the proportion of proliferating cells in p53^-/-+LV-BOP1 group were increased(P<0.05).Conclusion BOP1 knockdown activated the RPL11/MDM2/p53 nucleolar stress response pathway and inhibited the nascent proteins synthesis,thereby regulating the function of VSMCs and relieving the neointimal hyperplasia.