Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Role of angiotensin Ⅱ in the regulation of platelet-derived growth factor receptor β subunit of vascular smooth muscle

AIM:To investigate the crosstalk between angiotensin Ⅱ (AngⅡ)-mediated and platelet-derived growth factor (PDGF)-mediated signal transduction in vascular smooth muscle proliferation.METHODS:A model of renal hypertension was made by two kidney/one-clip operation. Level of PDGF receptor β subunit of aorta was measured by Western Blot analysis. The effect of Ang Ⅱ on PDGF receptor β subunit expression was investigated in culture rat aortic vascular smooth muscle cells (VSMC).RESULTS:Systolic blood pressure obviously increased at 8th week after operation, whereas the level of PDGF receptor β subunit of aorta significantly increased by 126.6% (P＜0.05) in 2K1C rats compared with control group. The expression of PDGF receptor β subunit in cultured VSMC stimulated by AngⅡ was higher than that of control by 192.74%(P＜0.01). The effect of AngⅡ was inhibited remarkably by pretreated with losartan, a kind of specific AngⅡ receptor 1 (AT₁) subtype antagonist and U73122, a kind of phospholipase C inhibitor. The effect was partly blocked by PD98059, which inhibit the activity of mitogen-activated, ERK-activating kinase (MEK).CONCLUSION:AngⅡ-induced PDGF receptor β subunit expression is regulated by the AT₁ and its downstream signal molecule-PLC and ERK, might participate in the intracellular signal transduction pathway.     

Effects of Rho kinase on p-Smad1 nuclear translocation in rat pulmonary artery smooth muscle cells

AIM: To explore the mechanism of bone morphogenetic protein (BMP) and Rho kinase signal pathways on the proliferation of pulmonary artery smooth muscle cells. METHODS: Pulmonary smooth muscle cells were isolated from the rat distal pulmonary artery and cultured. BMP and Rho kinase pathways were activated by BMP-2 and platelet-derived growth factor BB(PDGF-BB),respectively. Rho kinase inhibitor Y-27632 and MEK inhibitor U0126 were also used. Immunofluorescent staining was applied to observe p-Smad1 distribution across the nucleus, and the cells with positive p-Smad1 nuclear accumulation were counted and the nuclear translocation rate was calculated. The total p-Smad1 and its distribution across the nucleus were quantitatively determined by Western blotting. The cell proliferation was analyzed by CCK-8 assay. RESULTS: Exposure to BMP-2 significantly increased both the total amount of p-Smad1 and its nuclear accumulation in pulmonary smooth muscle cells. Pretreatment with PDGF-BB significantly decreased the nuclear accumulation of p-Smad1 induced by BMP-2 without decrease of total p-Smad1. However, pretreatment with Y-27632 or U0126 reversed the inhibitory effect of PDGF-BB on p-Smad1 nuclear accumulation. BMP-2 significantly inhibited the cell proliferation, but PDGF-BB blocked the effect of BMP-2 and significantly increased the cell proliferation. After pretreated with Y-27632 or U0126, the PDGF-BB-activated cell proliferation was suppressed.CONCLUSION: PDGF-BB-activated Rho kinase inhibits BMP-2-induced p-Smad1 nuclear translocation via MEK/ERK1/2, and increases pulmonary artery smooth muscle cell proliferation.     

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.     

Effect of AT2 receptor on the proliferation and the NOS expression in cultured adult rat VSMC

AIM: To observe the effect of angiotensinⅡ subtype 2 receptor (AT2 receptor) on the cultured rat aortic vascular smooth muscle cells. METHODS: The plasmid contained the cDNA of AT2 receptor was transfected into cultured rat vascular smooth muscle cells. The effects of AngⅡ, Ang Ⅱ＋losartan, Ang Ⅱ+PD123319 on the expression of PCNA, the NOS activity and the cell number were observed. RESULTS: The cell number and the expression of PCNA decreased after the cells were treated with losartan. When treated with PD123319, the cell number and the expression of PCNA increased, but the expression of NOS decreased. CONCLUSIONS: These data suggest that when being activated, AT2 receptor inhibits the proliferation of vascular smooth muscle cells and antagonizes the effect of AT1 receptor, such an effect may be related to the activation of NOS.     

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.     

Effects of the antibodies against α1- adrenergic receptor on the proliferation of vascular smooth muscle cells in rats

AIM: The autoantibodies against α1-adrenergic receptor that was found in patients with malignant hypertension, primary hypertension and refractory hypertension has the agonist activity liked the NE, and may play a role in hypertension. In this paper, the effects of this antibody on vascular smooth muscle cell (VSMC) proliferation and its mechanism were to be studied. METHODS: The cultured rat VSMC proliferation induced by the antibodies against α1- adrenergic receptor that was purified by the immune affinity chromatography, was measured by the BrdU cell proliferation assay and cell cycle distribution. The expression of c-jun and c-fos were determined by RT-PCR and Western blotting. RESULTS: Compared to the normal IgG, the antibodies against α1-adrenergic receptor promoted the VSMC proliferation and increased the mRNA and protein expression of the c-jun significantly. The role was similar to the norepinephrine, and all was blocked by prazosin, while the mRNA and protein expression of c-fos were not affected by the antibodies. CONCLUSION: The antibodies against α1-adrenergic receptor promote the rat VSMC proliferation, and increase the expression of c-jun, which maybe play a role in the vascular remodeling in hypertension.     

Effects of inhibiting myosin light chain kinase on endothelin-1 induced proliferation and apoptosis of pulmonary arterial smooth muscle cells in rats

AIM: To investigate the effect of inhibiting myosin light chain kinase(MLCK) on endothelin-1(ET-1) induced proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs). METHODS: Rat PASMCs were cultured and stimulated with ET-1. The cells were randomly divided into control group, ET-1 group and ET-1+MLCK inhibitor group(ET-1+M). Western blotting, MTT assay, [³H]-TdR incorporation and flow cytometry were employed to test the expression of myosin light chain(MLC) and MLCK, cell proliferation, cell cycle and apoptotic rate of PASMCs,respectively. The phosphorylation of MLC was determined by glycerol-PAGE coupled with Western blotting. RESULTS: Compared with control group, the protein expression of MLCK and MLC phosphorylation significantly enhanced after ET-1 stimulation. ET-1 markedly induced the proliferation and decreased the percentage of apoptotic rate in the PASMCs. However, pretreatment with ML-7, a MLCK inhibitor, significantly reversed the above effects induced by ET-1. CONCLUSION: MLCK inhibitor effectively inhibits the ET-1-induced proliferation and the cell cycle progression.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Effect of CIP2A over-expression on proliferation and apoptosis of gastric epithelial cells

AIM: To investigate the effects of cancerous inhibitor of protein phosphatase 2A (CIP2A) over-expression on the proliferation and apoptosis of gastric epithelial cells.METHODS: The mRNA expression of CIP2A and cyclin D1 in the tissues of normal gastric mucosa and gastric polyps was detected by RT-qPCR. The GES-1 cells were divided into control group, Ad-emp group and Ad-CIP2A group. The cell proliferation ability was detected by MTT assay and BrdU assay, and the cell apoptosis was analyzed by flow cytometry. The expression of apoptosis-related molecules was determined by Western blot and RT-qPCR. The levels of inflammatory cytokines were measured by ELISA after GES-1 cells were infected with Ad-emp and Ad-CIP2A. Furthermore, the protein levels of p-Rb, E2F1 and cyclin D1 in the GES-1 cells was determined by Western blot after transfected with CIP2A siRNA.RESULTS: The expression of CIP2A and cyclin D1 in adenomatous gastric polyps tissues was significantly higher than that in normal gastric mucosa tissues, and no significant change of that between hyperplastic gastric polyps tissues and normal gastric mucosa was observed. After transfected with CIP2A, the proliferation ability of GES-1 cells was increased, the cell apoptosis was inhibited, the concentrations of IL-1β and IL-10 was up-regulated and the protein levels of p-Rb, E2F1 and cyclin D1 were increased, while the protein levels of p-Rb, E2F1 and cyclin D1 were significantly decreased after transfected with CIP2A siRNA.CONCLUSION: CIP2A promotes the proliferation and inhibits the apoptosis of GES-1 cells by activating Rb/E2F1.     

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.     

Yangxue qingnao-containing serum inhibits rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

AIM: To observe the effects of Yangxue qingnao-containing serum on rat vascular smooth muscle cell (VSMC) proliferation induced by lysophosphatidic acid (LPA). METHODS: The [³H]-TdR incorporation and mitogen-activated protein kinasc (MAPK) activity were measured in cultured VSMC. End product of lipid peroxidation-MDA levels were also detected. RESULTS: 1×10^－9,1×10^－8 and 1×10^－7 mol/L LPA enhanced the cultured VSMC [³H]-TdR incorporation, increased MAPK activity and MDA content in a concentration-dependent manner. 5％, 10％ and 15％ Yangxue qingnao-containing serum concentration-dependently inhibited the increase in VSMC [³H]-TdR incorporation, MAPK activity and MDA content induced by LPA. CONCLUSIONS: LPA has a stimulating effect on VSMC proliferation. The LPA-induced intracellular signal transduction may be related to MAPK activity. Yangxue-qingnao can efficiently inhibit LPA -induced VSMC proliferation,MAPK activity and lipid peroxidation.     

Quercetin inhibits endothelin-1-induced T-type Ca2+ channel expression in human umbilical arterial smooth muscle cells

AIM: To investigate the effect of quercetin on endothelin-1-induced T-type calcium channel(TCC) expression in primary cultured human umbilical arterial smooth muscle cells for exploring the protective role of quercetin in cardiovascular system. METHODS: Human umbilical arterial smooth muscle cells were verified by immunocytochemistry. The cells in 2-3 passages were used and randomly divided into control group, quercetin alone group, model group and experimental group. The cells in control group were cultured without any drugs for 24 h. The cells in quercetin alone group were cultured with 80 μmol/L quercetin for 24 h. The cells in model group were cultured with ET-1 at the concentration of 100 nmol/L for 24 h. The cells in experimental groups were pretreated with quercetin for 1 h, then coincubated with 100 nmol/L ET-1 for 24 h. The concentrations of quercetin used in this study were 20, 40and 80 μmol/L, respectively. The expression of α_1G, a TCC major subunit, was assayed at mRNA and protein levels by RT-PCR and Western blotting, respectively. The TCC currents(I_caT) were detected by the technique of whole-cell patch-clamp. RESULTS: Compared with control and experimental group, I_CaT density (P<0.01) and the expression of α_1G at mRNA (P<0.05) and protein (P<0.01) levels in model group were significantly increased. No significant difference in the results of quercetin alone group and control group was observed. CONCLUSION: The protective roles of quercetin in cardiovascular functions are related to the depressive effects of quercetin on ET-1-induced increase in both I_CaT density and the expression of α_1G at mRNA and protein levels in cultured human vascular smooth muscle cells.     

Effects of miR-130a on viability and apoptosis of rat basilar arterial smooth muscle cells

AIM: To investigate the regulatory effects of microRNA (miR)-130a on the biological characteristics of rat basilar arterial smooth muscle cells (BASMCs) and its underlying mechanisms. METHODS: The expression of miR-130a in rat BASMCs was measured by real-time PCR. After knockdown of miR-130a with inhibitor in the BASMCs, the cell viability, cell cycle distribution and apoptosis were detected by CCK-8 assay and flow cytometry. The expression of cell cycle-and apoptosis-related molecules, such as cyclin D1, cyclin-dependent kinase 2 (CDK2), p21, Bcl-2 and cleaved caspase-3/caspase-3 at protein levels was determined by Western blot. The growth arrest-specific homeobox protein (Gax) expression at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: AngiotensionⅡ (AngⅡ) up-regulated the expression of miR-130a and down-regulated the expression of Gax (P<0.05). Transfection with miR-130a inhibitor partly reversed the increase in AngⅡ-induced cell viability and promoted the Gax expression. Furthermore, the early cell apoptotic rate was significantly increased after down-regulation of miR-130a (P<0.05), and the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased, accompanied with the up-regulation of p21 and cleaved caspase-3 (P<0.05). CONCLUSION: Down-regulation of miR-130a restrains the viability and promotes the apoptosis of BASMCs by promoting Gax expression and regulating cell cycle-and apoptosis-related molecules, suggesting that miR-130a may be a potential therapeutic target of brain vascular remodeling during hypertension.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

The effect of homocysteine on airway smooth muscle cells and fibroblasts

AIM: To study the effect of homocysteine (HCY) on proliferation of airway smooth muscle cells and fibroblasts and the effect of HCY on collagen prodution of airway fibroblasts. METHODS: [³H]-TdR incorpora- tion was measured in cultured airway smooth muscle cells. The [³H]-TdR and [³H]-proline incorporation were mea- sured in cultured airway fibroblasts. RESULTS: HCY induced proliferation of airway smooth muscle cells and fibroblasts in a concentration - dependent manner. HCY also induced collagen production of airway fibroblasts in a concentration - dependent manner. The inhibitors of protein kinase C, H7 and polymyxin B, inhibited HCY - induced proliferation of airway smooth muscle cells. CONCLUSIONS: HCY induced proliferation of airway smooth muscle cells and fibroblasts, HCY also induced collagen production of airway fibroblasts. The HCY - induced proliferation of airway smooth muscle cells may be related to the pathway of PKC signal transduction.     

The regulation of peroxisome proliferator-activated receptor-γ on apoptotic related genes p53 and bcl-2in cultured vascular smooth muscle cells

AIM: We sought to evaluate the expression of apoptosis related genes, p53 and bcl-2 , in cultured vascular smooth muscle cells (VSMCs) in which apoptosis was induced by the ligands of PPARγ-ox-LDL,ciglitazone and 15-deoxy-△^12,14-PGJ₂,and then influenced by PGF_2α,an antagonist of PPARγ. To clarify whether PPARγ is involved in the regulations of p53 and bcl-2 .METHODS: Rat aorta smooth muscle cells were isolated and cultured in DMEM containing 10%FBS. ox-LDL,ciglitazone and 15 d-PGJ₂ were added into the medium for 24 h, respectively. Using PGF_2α to inhibit PPARγ activation via activation of MAP kinase. We employed the flow cytometry analysis to quantitatively analyze the apoptosis of VSMCs. Also, we measured and analyzed the discrepancies of the expression of p53 and bcl-2 between every groups. RESULTS: During the process of apoptosis induced by PPARγ,the expression of P53 protein increased (P＜0.05)and the Bcl-2 diminished. Inhibition of PPARγ through PGF_2α attenuated such influences. CONCLUSION: In the apoptosis of VSMCs, the activation of PPARγ changes the expression of p53 and bcl-2. We suppose that the apoptosis of VSMCs is partly due to changes of expression of apoptotic genes regulated by PPARγ.