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@#Objective To study the effect of ankyrin repeat domain 49(ANKRD49)on the migration of human lung adenocarcinoma cell line NCI-H1299 and its mechanism.Methods NCI-H1299 cells were infected with lentivirus vector carrying ANKRD49 gene and shRNA targeting ANKRD49 to construct the cell models stably overexpressing and knocking down ANKRD49. Meanwhile,the control cell models infected with empty lentivirus vector and lentivirus vector with scramble sequences were constructed respectively. The expression levels of ANKRD49 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot. The effect of ANKRD49 on cell migration was measured by scratch test. The mRNA and protein levels of matrix metalloproteinase(MMP)-2/9 and tissue inhibitor of metalloproteinase(TIMP)-1/2 were detected by real-time fluorescence quantitative PCR and Western blot. The protein expression levels of p65,p-p65,IκBα and p-IκBα were detected by Western blot.Results The levels of ANKRD49 mRNA and protein in the ANKRD49 overexpression group were significantly higher than those in the control group(t = 70. 02 and 45. 68,respectively,each P < 0. 001). Compared with the control group,the migration ability of cells in the ANKRD49 overexpression group significantly increased at 24 h and 48 h(t = 5. 343 and 3. 282,P = 0. 005 9 and 0. 030 4,respectively);The mRNA transcription levels and protein expression levels of MMP-2 and MMP-9 significantly increased(t = 9. 304 and 6. 193,P =0. 000 7 and 0. 003 5,respectively),while the mRNA and protein expression of TIMP-1 and TIMP-2 decreased significantly(t = 3. 858 and 3. 517,P = 0. 018 2 and 0. 024 5,respectively),and the values of MMP-2/TIMP-1 and MMP-9/TIMP-2 significantly increased(t = 17. 7 and 9. 682,P < 0. 001 and < 0. 01,respectively);The expression of p-p65 and pIκBα significantly increased,the total protein levels of p65 and IκBα showed no obvious change,and the values of p-p65/p65 and p-IκBα/IκBα significantly increased(t = 3. 962 and 5. 370,P = 0. 016 7 and 0. 005 8,respectively). However,knocking down of ANKRD49 presented the opposite results.Conclusion ANKRD49 promotes the migration of NCI-H1299cells by enhan-cing the expression of MMP-2/9,the values of MMP-9/TIMP-1 and MMP-2/TIMP-2 via activating NF-κB/p65 signa-ling pathway.
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Objective To investigate the correlation between moderate hypoxia and tumor invasiveness in colon carcinoma cell line HT‐29 and detect the expression of NF‐κB protein in nucleus and activities of MMP‐2/9 ,so as to explore its latent mecha‐nism of malignant transformation .Methods The moderate hypoxia model was established according to the pressure of O2 of tumor in vivo .After different period in moderate hypoxia ,activities of MMP‐2/9 secreted by HT‐29 cells were measured by gelatin zymog‐raphy ,NF‐κB levels in nucleus were assessed by Western blot after extraction of nucleic protein .Results MMP‐2/9 activities under moderate hypoxia gradually up‐regulated in 12 h and reached the peak at 24 h .The expression of NF‐κB protein in nucleus was simi‐lar to that of MMP‐2/9 .Conclusion Moderate hypoxia could induce malignant phenotype in HT‐29 cells through up‐regulating MMP‐2/9 activities ,in which NF‐κB may involve in this progression .
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BACKGROUND: CM1 (centrocyte/-blast marker 1) was defined by a mAb against concanavalin A (Con A) activated PBMC. It is expressed in germinal center of human tonsil and on the surface of activated PBMC as well as cancer cells. Recently, increased productions of pro-inflammatory mediators were detected from activated PBMC by CM1 ligation. METHODS: However, there is a limitation to explain the exact role of CM1 on inflammation and its related mechanisms, since the identity of CM1 is still not clarified. In our previous study, we have already confirmed that soluble form of CM1 was produced by Raji. Therefore, we performed Q-TOF analysis after immunoprecipitation of concentrated Raji culture supernatant using anti-CM1 mAbs. RESULTS: As a result, we found that CM1 is identical to enolase-1(ENO1), a glycolytic enzyme, and we confirmed that results by silencing ENO1 using siRNA. It was also confirmed through competition assay between anti-CM1 and anti-ENO1 mAbs. Finally, we investigated the possible role of CM1 in inflammatory response and cancer. The ligation of CM1 on Raji cells with anti-CM1 mAbs induces the extensive production of prostaglandin E2(PGE2). In addition, the increased activity of matrix metalloproteinase (MMP)-2/9 was shown in NCI-N87, stomach cancer cell line by CM1 stimulation. CONCLUSION: CM1 is identical to ENO1 and it might be an important role in the regulation of inflammatory responses.
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Humans , Cell Line , Concanavalin A , Dinoprostone , Germinal Center , Immunoprecipitation , Inflammation , Ligation , Palatine Tonsil , RNA, Small Interfering , Stomach NeoplasmsABSTRACT
AIM:Xilei Powder,a traditional Chinese prescription,has been used to treat wounds for hundreds of years,but the mechanism has not been fully understood.METHODS:The effects of Xilei Powder on fibroblast proliferation,collagen accumulation,matrix metalloproteinases-2,9 ( MMP-2,9 ) activities and tissue inhibitor of metalloproteinase 1 (TIMP-1) production were investigated by MTT,chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays ( ELISA),respectively.RESULTS: The aqueous extract of Xilei Powder significantly promoted fibroblasts proliferation in a time and concentration manner,the population doubling time (125 μg/mL) was 33.8 h,it also significantly (P ＜0.05 ) promoted collagen production.Both of the aqueous and alcoholic extracts could significantly ( P ＜ 0.05 ) increase MMPo2 activity,and also very significantly ( P ＜ 0.01 )promote TIMP-1 production.CONCLUSION: Xilei Powder could promote fibroblasts proliferation,collagen and TIMP-1 production,this might be parts of mechanism to promote wound healing.
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Objective:To explore the effect of Naoshuning on the protein expression of Matrix metalloproteinases(MMPs) in experimental injuried brain tissue of rats. Methods:Immunohistochemistry was used to determine the changes of protein expression of MMPs. Brain tissue water content,permeability and ultramicrostructure of blood-brain barrier(BBB) were also observed. Results:Compared with the sham group,the brain tissue water and EB content of injured side and the level of MMP-2 and MMP-9 protein expression in brain tissue around contusion in model group increased obviously(all P
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PURPOSE: The purpose of this study is to clarify the degree of corneal neovascularization and its expression of MMP-2. 9(matrix metalloproteinase) and TIMP-1, 2(tissue inhibitor of metalloproteinases) according to the different concentrations of VEGF(vascular endothelial growth factor). METHODS: After the pellets with different amounts of VEGF(VEGF 125, 250 ng) were inserted into the corneal stroma of rat model, their degree of corneal neovascularization and the expression of MMP-2, 9 and TIMP-1, 2 were compared with those of control group where pellets were filled with phosphate-buffered saline. RESULTS: At the 7th day after the pellet insertion, degree of neovascularization was most highly scored in the group with pellet which contained the largest amount of VEGF, 250 ng, and there was statistically noticeable increase of eovascularization with the increase of VEGF amount(P<0.05). On immunohistochemical staining, as the amount of VEGF increases, not only MMP-2, 9 and flk-1, but also TIMP-1, 2 were expressed more. CONCLUSIONS: In conclusion, when it comes to neovascularization, MMP-2, 9, which induces angiogenesis, as well as its inhibitor TIMP-1, 2 are increased to maintain the homeostasis of the cornea.
Subject(s)
Cornea , Corneal Neovascularization , Corneal Stroma , Homeostasis , Models, Animal , Tissue Inhibitor of Metalloproteinase-1 , Vascular Endothelial Growth Factor AABSTRACT
AIM:Xilei Powder,a traditional Chinese prescription,has been used to treat wounds for hundreds of years,but the mechanism has not been fully understood.METHODS:The effects of Xilei Powder on fibroblast proliferation,collagen accumulation,matrix metalloproteinases-2,9(MMP-2,9)activities and tissue inhibitor of metalloproteinase 1(TIMP-1)production were investigated by MTT,chloramine T method,gelatin zymography and enzyme-linked immunosorbent assays(ELISA),respectively.RESULTS:The aqueous extract of Xilei Powder significantly promoted fibroblasts proliferation in a time and concentration manner,the population doubling time(125 ?g/mL)was 33.8 h,it also significantly(P
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Objective To examine the changes of matrix metalloproteinase-2,9 (MMP-2,9) in gingival crevicular fluid(GCF) after phase 1 periodontal treatment of adult patients with periodontitis. Methods GCF was sampled with filter paper strips by intra-pocket method to determine MMP-2,9 levels. Forty teeth of forty adult patients with periodontitis and forty teeth of forty periodontally healthy persons were included in this study. Assays for MMP-2,9 in GCF were performd by ELISA. Results Contents of MMP-2,9 were higher in AT group than those in controls(P