Effects of IL-32γ on proliferation and cell cycle of rat vascular smooth muscle cells

AIM: To observe the effects of interleukin-32γ (IL-32γ)on the proliferation and cell cycle of rat vascular smooth muscle cells (VSMCs). METHODS: The VSMCs were isolated from the thoracic aorta of SD rats by the method of tissue-piece inoculation. The cells were cultured and treated with different concentrations of IL-32γ. The proliferation of the cells was examined by MTT assay. The cell cycles were analyzed by flow cytometry. The protein levels of NF-κB p65 and cyclin D1 were detected by Western blotting. The expression of proliferating cell nuclear antigen (PCNA)was examined by immunocytochemical staining. RESULTS: Administration of IL-32γ at the concentrations of 10~50 μg/L for 24~48 h significantly promoted the proliferation of VSMCs in a dose- and time-dependent manner. After stimulation with IL-32γ at the concentration of 50 μg/L for 24 h, the cell cycle transition from G₁ phase to S/G₂ phase was accelerated and the expression levels of NF-κB p65, cyclin D1 and PCNA increased as compared with those in control group. CONCLUSION: IL-32γ promotes the proliferation of rat VSMCs and accelerates the cell cycle transition via upregulating the expression of NF-κB p65 and cyclin D1.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.     

Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose

AIM: To investigate the effects of sinapic acid(SA) on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs) induced by high glucose(HG). METHODS: Cultured A7r5 cells were randomly divided and treated as indicated. The cell viability was determined by MTT assay. DNA synthesis was measured by BrdU assay. Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis. The levels of reactive oxygen species(ROS) were detected by ELISA. The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C(p-PKC), p-P38 and β-actin were evaluated by Western blot. RESULTS: Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the protein levels of cyclin D1, p-PKC and p-P38 were increased in HG group(all P<0.05). These effects were reversed by SA(0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner(all P<0.05). Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION: SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.     

Chemerin promotes proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK

AIM: To investigate the proliferation property of stable chemerin gene knockdown vascular smooth muscle cells (VSMCs) and to explore its mechanism. METHODS: The normal VSMCs, chemerin gene interfering control VSMCs and stable chemerin gene knockdown VSMCs were divided into normal group, PDGF group, control group and knockdown group. The VSMCs in PDGF group were given platelet-derived growth factor-BB (PDGF-BB) to initiate proli-feration. The cell counting and BrdU assay were employed to investigate the proliferation property of VSMCs. The mitogen-activated protein kinase (MAPK) signal pathway was determined by Western blot. RESULTS: The cell number and BrdU A value in PDGF-BB-treated VSMCs significantly increased as compared with the normal VSMCs(CONCLUSION: Chemerin promotes the proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK production.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.     

Retinoid X receptor agonists antagonize high-glucose-induced proliferation of rat vascular smooth muscle cells by inactivation of protein kinase C

AIM: To explore the effect of retinoid X receptor (RXR) agonists on high-glucose-induced proliferation of rat aortic smooth muscle cells (RASMCs). METHODS: RASMCs were cultured in DMEM containing glucose at normal concentration (5.5 mmol/L). For high glucose treatment, glucose solution was added up to a final concentration of 25 mmol/L. The proliferation of RASMCs was detected by WST-1 assay. DNA synthesis was measured by the method of BrdU incorporation. Cell cycle progression was determined by flow cytometry. Phosphorylated protein kinase C (PKC) and the expression levels of cyclin-dependent kinase 2(CDK2) and p27Kip1 were detected by immunoblotting. RESULTS: High glucose increased DNA synthesis, cell cycle progression, the expression of CDK2 and the proliferation of RASMCs. Meanwhile, the expression of p27Kip1 was decreased by high glucose. Treatment of RASMCs with RXR natural ligand 9-cis-retinoic acid (9-cis-RA) resulted in significant inhibition of high-glucose-induced proliferation, DNA synthesis, cell cycle progression and the expression of CDK2 in a concentration-dependent manner. 9-cis-RA also reversed the effect of high glucose on the expression of p27Kip1. RXR specific ligand SR11237 demonstrated the same effect as the effect of 9-cis-RA at the same concentration. PKC inhibitor showed the similar effect on high-glucose-induced proliferation and the expression of CDK2 and p27Kip1 as the RXR agonists did. Furthermore, 9-cis-RA and SR11237 rapidly inhibited high-glucose-induced activation of PKC. CONCLUSION: PKC is involved in high-glucose-induced proliferation of RASMCs. RXR agonists inhibit high-glucose-induced proliferation by depressing PKC activation in vascular smooth muscle cells.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Effects of She xiang bao xin wan (SXBXW) on phenotype transformation of vascular smooth muscle cells

AIM: To observe the effects of She xiang bao xin wan (SXBXW) of Chinese patent drug on the phenotype transformation of vascular smooth muscle cells.METHODS: The vascular smooth muscle cell line RASMC P8 was used as the cell model in this experiment. The cells were treated with SXBXW at the doses of 0.25 g/L, 0.50 g/L, 1.0 g/L and 2.0 g/L, respectively, and control cells were treated with the same volume of culture medium without SXBXW. The expression of α-smooth muscle actin (α-SMA) and the smooth muscle myosin heavy chain (SM-MHC), which served as specific molecular markers of vascular smooth muscle contraction in RASMC P8 cells, was analyzed by flow cytometry. RESULTS: The percentages of α-SMA and SM-MHC negative cells in SXBXW treated groups were lower than those in control group. Meanwhile, the percentages of α-SMA and SM-MHC positive cells were increased in RASMC P8 cells treated with SXBXW, indicating that SXBXW prompted the transformation of RASMC P8 cells from synthetic to contractile phenotype.CONCLUSION: Chinese patent drug SXBXW plays a role in transforming the cell phenotype from synthetic to the contraction in vascular smooth muscle.     

miR-17 regulates senescence of rat vascular smooth muscle cells

AIM: To investigate the effect of microRNA-17 (miR-17) on the senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanism.METHODS: The medial layer of the thoracic aorta was collected from the SD rats and isolated for primary culture. VSMCs were identified by immunofluorescence staining. The VSMCs were collected at the 4th~6th generations, and then the miR-17 mimics and miR-17 inhibitor were transfected into the VSMCs by liposome method. After 24 h, the cell senescence was induced by D-galactose. The VSMCs were divided into the following 6 groups:aging induction+miR-17 mimics (A-miR-17) group, aging induction+miR-17 inhibitor (A-anti-miR-17) group, A-control group, normal (N)+miR-17-mimics (N-miR-17) group, N-anti-miR-17 group, and N-control group. On day 3 after the addition of D-galactose, the senescence of VSMCs was observed with β-galactosidase staining. The expression of miR-17, p16 and p21 was detected by RT-qPCR and immunohistochemistry. RESULTS: miR-17 expression in the VSMCs was significantly lower in A-control group than that in N-control group (P<0.01). Compared with A-control group, the expression of miR-17 in the VSMCs was significantly increased in A-miR-17 group (P<0.01), while that was significantly decreased in A-anti-miR-17 group (P<0.01). The number of β-galactosidase positive staining cells in A-anti-miR-17 group was significantly higher than that in A-miR-17 group (P<0.01). The expression of p21 at mRNA and protein levels in the VSMCs was significantly lower in A-miR-17 group than that in A-control group (P<0.01), and the expressions of p21 at mRNA and protein levels was significantly higher in A-anti-miR-17 group than that in A-miR-17 group (P<0.01). CONCLUSION: miR-17 inhibits rat VSMCs senescence induced by D-galactose, the underlying mechanism is associated with the inhibition of p21 expression.     

Differentiation potential of human placenta derived mesenchymal stem cells into vascular endothelial cells

AIM: To investigate the differentiation potential of human placenta derived mesenchymal stem cells (PDMSCs) into endothelial cells (ECs) in vitro. METHODS: PDMSCs were isolated from human placenta tissues, characterized by flow cytometry and induced to differentiate into endothelial cells with 50 μg/L VEGF and 10 μg/L bFGF. To detect the specific markers of ECs during the process of differentiation, the method of immunocytochemistry was performed. The specific structure and function of endothelial cells were observed by transmission electron microscopy and in vitro angiogenesis assay kit, respectively. RESULTS: CD105 and CD106 were positive in PDMSCs, while CD34,CD45 and CD31 were negative.The ECs differentiated from PDMSCs showed cobblestone-like morphology, and expressed early endothelial marker of Flk-1/KDR and mature endothelial markers of CD31, vWF and CD144/VE-cadherin in a time-dependent manner during the endothelial cell differentiation (0 day, 4 days, 8 days and 12 days). The endothelial specific structure, Weibel-palade body, was observed under transmission electron microscope. The inoculation of ECs on the extra cellular matrix gel formed capillary-like structures. CONCLUSION: Plentiful PDMSCs can be isolated from placenta, and differentiate into the cells with functional characteristics of ECs in vitro, indicating that the placenta tissues will become optimal source of seed cells for vascular engineering and regenerative medicine.     

Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.     

Effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells

AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E₂) than that in VSMCs without 17β-E₂ pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.     

Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

Long noncoding RNA ZFAS1/miR-150/ROCK1 axis regulates proliferation and migration of vascular smooth muscle cells

AIM: To investigate whether long noncoding RNA ZNFX1 (zinc finger NFX1-type containing 1) antisense RNA 1 (ZFAS1) promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) by regulating microRNA-150 (miR-150)/ROCK1, and the involving mechanism of atherosclerosis. METHODS: Platelet-derived growth factor-BB (PDGF-BB) was used to induce proliferation and migration of VSMCs. Real-time PCR was used to detect the content of ZFAS1 in the VSMCs. After further down-regulating the expression of ZFAS1 by siRNA, the viability of VSMCs was detected by MTT assay, and the proliferation was measured by EdU staining. The migration ability of VSMCs was detected by Transwell method. The expression levels of miR-150 and ROCK1 were detected by RT-qPCR, and the protein level of ROCK1 was determined by Western blot. Luciferase reporter assay was used to confirm that ROCK1 was the target gene of miR-150. Finally, miR-150 expression was inhibited, and the proliferation and migration ability of VSMCs and expression of ROCK1 after down-regulation of ZFAS1 expression were examined. RESULTS: PDGF-BB up-regulated the expression of ZFAS1 in the VSMCs. After down-regulating the expression of ZFAS1, the proliferation and migration abilities of VSMCs were inhibited (P<0.05), the expression level of miR-150 was increased (P<0.05), and the expression level of ROCK1 was decreased (P<0.05). The results of luciferase reporter assay showed that miR-150 directly targeted ROCK1. Inhibition of miR-150 expression attenuated the inhibition of proliferation and migration of VSMCs by ZFAS1 expression knock-down (P<0.05) and up-regulated the expression level of ROCK1 (P<0.05). CONCLUSION: ZFAS1 promotes the proliferation and migration of VSMCs induced by PDGF-BB by regulating miR-150/ROCK1.