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Tissue Inhibitor of Matrix Metalloproteinases-1 Knockdown Suppresses the Proliferation of Human Adipose-Derived Stem Cells.

Zhang P, Li J, Qi Y, Tang X, Duan J, Liu L, Wu Z, Liang J, Li J, Wang X, Zeng G, Liu H - Stem Cells Int (2016)

Bottom Line: Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA.The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting.The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plastic Surgery, Stem Cell Research and Clinical Translation Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China.

ABSTRACT
Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional matrix metalloproteinase, and it is involved in the regulation of cell proliferation and apoptosis in various cell types. However, little is known about the effect of TIMP-1 expression on the proliferation of adipose-derived stem cells (ADSCs). Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA. Cell proliferation was then evaluated by MTT assay and Ki67 staining, respectively. Cell cycle progression was determined by flow cytometry. The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting. The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels. These findings suggest that TIMP-1 works as a positive regulator of cell proliferation in ADSCs.

No MeSH data available.


Related in: MedlinePlus

TIMP-1 expression was suppressed by shRNA-TIMP-1. TIMP-1 expression was analyzed by real-time RT-PCR and western blotting, respectively. (a) The levels of TIMP-1 mRNA in parental ADSCs, mock, and shTIMP-1 ADSCs were measured by real-time RT-PCR 72 h after shRNA infection. TIMP-1 mRNA expression in parental ADSCs is defined as 1. The y-axis represents the normalized TIMP-1 mRNA expression relative to parental ADSCs. (b) Representative western blot images for the detection of TIMP-1 protein expression in the ADSCs infected with shRNA against TIMP-1. (c) The relative expression level of TIMP-1 protein was quantified by the ratio of optical density of TIMP-1 and β-actin. The data are denoted as mean ± SD (n = 3). ∗∗P < 0.01 versus mock-shRNA group. Parental ADSCs: uninfected ADSCs; mock ADSCS: control lentivirus infected ADSCs; shTIMP-1 ADSCs: shRNA-TIMP-1 lentivirus infected ADSCs.
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fig2: TIMP-1 expression was suppressed by shRNA-TIMP-1. TIMP-1 expression was analyzed by real-time RT-PCR and western blotting, respectively. (a) The levels of TIMP-1 mRNA in parental ADSCs, mock, and shTIMP-1 ADSCs were measured by real-time RT-PCR 72 h after shRNA infection. TIMP-1 mRNA expression in parental ADSCs is defined as 1. The y-axis represents the normalized TIMP-1 mRNA expression relative to parental ADSCs. (b) Representative western blot images for the detection of TIMP-1 protein expression in the ADSCs infected with shRNA against TIMP-1. (c) The relative expression level of TIMP-1 protein was quantified by the ratio of optical density of TIMP-1 and β-actin. The data are denoted as mean ± SD (n = 3). ∗∗P < 0.01 versus mock-shRNA group. Parental ADSCs: uninfected ADSCs; mock ADSCS: control lentivirus infected ADSCs; shTIMP-1 ADSCs: shRNA-TIMP-1 lentivirus infected ADSCs.

Mentions: To elucidate whether TIMP-1 expression has any effect on the proliferation of ADSCs, RNAi was used to generate TIMP-1 knockdown cell lines. In this study, lentivirus-mediated infection was performed in ADSCs. The results of RT-PCR confirmed that TIMP-1 mRNA expression in the shTIMP-1 ADSCs was suppressed by about 80% compared to the cells infected with mock-shRNA (Figure 2(a)). Additionally, western blot analysis demonstrated that TIMP-1 protein expressed in the shTIMP-1 ADSCs was also markedly downregulated (Figures 2(b) and 2(c)), whereas TIMP-1 protein bands were clearly observed in the mock-shRNA cells and parental ADSCs.


Tissue Inhibitor of Matrix Metalloproteinases-1 Knockdown Suppresses the Proliferation of Human Adipose-Derived Stem Cells.

Zhang P, Li J, Qi Y, Tang X, Duan J, Liu L, Wu Z, Liang J, Li J, Wang X, Zeng G, Liu H - Stem Cells Int (2016)

TIMP-1 expression was suppressed by shRNA-TIMP-1. TIMP-1 expression was analyzed by real-time RT-PCR and western blotting, respectively. (a) The levels of TIMP-1 mRNA in parental ADSCs, mock, and shTIMP-1 ADSCs were measured by real-time RT-PCR 72 h after shRNA infection. TIMP-1 mRNA expression in parental ADSCs is defined as 1. The y-axis represents the normalized TIMP-1 mRNA expression relative to parental ADSCs. (b) Representative western blot images for the detection of TIMP-1 protein expression in the ADSCs infected with shRNA against TIMP-1. (c) The relative expression level of TIMP-1 protein was quantified by the ratio of optical density of TIMP-1 and β-actin. The data are denoted as mean ± SD (n = 3). ∗∗P < 0.01 versus mock-shRNA group. Parental ADSCs: uninfected ADSCs; mock ADSCS: control lentivirus infected ADSCs; shTIMP-1 ADSCs: shRNA-TIMP-1 lentivirus infected ADSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4863124&req=5

fig2: TIMP-1 expression was suppressed by shRNA-TIMP-1. TIMP-1 expression was analyzed by real-time RT-PCR and western blotting, respectively. (a) The levels of TIMP-1 mRNA in parental ADSCs, mock, and shTIMP-1 ADSCs were measured by real-time RT-PCR 72 h after shRNA infection. TIMP-1 mRNA expression in parental ADSCs is defined as 1. The y-axis represents the normalized TIMP-1 mRNA expression relative to parental ADSCs. (b) Representative western blot images for the detection of TIMP-1 protein expression in the ADSCs infected with shRNA against TIMP-1. (c) The relative expression level of TIMP-1 protein was quantified by the ratio of optical density of TIMP-1 and β-actin. The data are denoted as mean ± SD (n = 3). ∗∗P < 0.01 versus mock-shRNA group. Parental ADSCs: uninfected ADSCs; mock ADSCS: control lentivirus infected ADSCs; shTIMP-1 ADSCs: shRNA-TIMP-1 lentivirus infected ADSCs.
Mentions: To elucidate whether TIMP-1 expression has any effect on the proliferation of ADSCs, RNAi was used to generate TIMP-1 knockdown cell lines. In this study, lentivirus-mediated infection was performed in ADSCs. The results of RT-PCR confirmed that TIMP-1 mRNA expression in the shTIMP-1 ADSCs was suppressed by about 80% compared to the cells infected with mock-shRNA (Figure 2(a)). Additionally, western blot analysis demonstrated that TIMP-1 protein expressed in the shTIMP-1 ADSCs was also markedly downregulated (Figures 2(b) and 2(c)), whereas TIMP-1 protein bands were clearly observed in the mock-shRNA cells and parental ADSCs.

Bottom Line: Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA.The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting.The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plastic Surgery, Stem Cell Research and Clinical Translation Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China.

ABSTRACT
Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional matrix metalloproteinase, and it is involved in the regulation of cell proliferation and apoptosis in various cell types. However, little is known about the effect of TIMP-1 expression on the proliferation of adipose-derived stem cells (ADSCs). Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA. Cell proliferation was then evaluated by MTT assay and Ki67 staining, respectively. Cell cycle progression was determined by flow cytometry. The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting. The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels. These findings suggest that TIMP-1 works as a positive regulator of cell proliferation in ADSCs.

No MeSH data available.


Related in: MedlinePlus