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CCL19/CCR7 Regulate the Expression of Heparanase Via Sp1 in Lung Cancer A549 Cell

Author: YinLiYing
Tutor: QiuXueShan
School: China Medical University
Course: Pathology and Pathophysiology
Keywords: Lung cancer Heparanase Sp1 Attack
CLC: R734.2
Type: Master's thesis
Year: 2010
Downloads: 63
Quote: 0
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Abstract


Preface chemokine (chemokine) CCL19 and its receptor CCR7 play an important role in lymphocyte homing. The study found that high expression of CCR7 in many solid tumors and tumor growth, angiogenesis, lymph node metastasis and invasion-related. Our group pre-trial discovery, CCR7 overexpression in non-small cell lung cancer, and its invasion is closely related with lymph node metastasis. But CCL19/CCR7 promote tumor cell invasion and metastasis exact mechanism is unclear. Join CCL19 in lung cancer cells and found that Hpa upregulation. Heparanase (heparanase, Hpa) widespread metastatic malignant cells, which can degrade the extracellular matrix and basement membrane, promote tumor invasion and metastasis. Therefore, we predict CCL19/CCR7 through regulation of the Hpa the expression, to promote the invasion of lung cancer cells, but the regulatory mechanism is unclear. The Hpa gene promoter region contains binding sites for transcription factor Sp1. This study in lung adenocarcinoma A549 cells exogenous join CCL19 activate CCR7 study Sp1 and Hpa expression and cell invasiveness. Materials and Methods 1, antibodies and reagents: polyclonal heparanase antibodies, CCR7 antibody, monoclonal antibody Sp1 antibody A Mithramycin CCL19 were purchased from peprotech. 2 cell culture: human lung adenocarcinoma A549 cells with DMEM medium containing 10% fetal bovine serum, 37 ° C, 5% CO2 incubator culture. 3, RT-PCR: take the logarithmic growth phase cells with TRIZOL (Invitrogen, USA) extraction of total RNA, reverse transcription, PCR amplification, a 1.5% agarose gel electrophoresis, expression of strength analysis using Image J software. 4, Western blot analysis: cells were collected and total protein was extracted ,60-80 micrograms of total protein after 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. Detection of heparanase, Sp1 levels. 5, chromatin immunoprecipitation of precipitate (Chip): in accordance with the ChIP kit instructions detection Sp1 and the promoter region of the binding heparanase. 6, Transwell test: inoculation of cells containing or excluding Matrigel the millipore small room, were cultured 24h, poly 4% methanol fixed, hematoxylin microscope was used to observe the changes in the cells in vitro invasion and migration ability. 7, the statistical analysis: SPSS17.0 statistical software for data processing, to P lt; 0.05 as statistically significant. Results 1 CCL19/CCR7 by Sp1 raised lung cancer A549 cell the Hpa the expression of exogenous added CCL19 different time, Sp1 and HpamRNA and protein expression levels increase. Sp1 inhibitor, MA with HpamRNA and protein inhibition of Sp1 obvious with CCR7 antibody 24h, Sp1 inhibitor Mithramycin A (MA) after adding CCL19, RT-PCR and western blot show that Sp1 expression and Hpa down . 2, CCL19 promoted the SP1 with Hpa promoter binding, we first analyzed the sequence of the promoter region of the heparanase, confirmed the Sp1 and heparanase attachment sites (5GGGGC-3) of GC-boxes. ChIP analysis showed that the promoter region of SP1 with Hpa binding increased after treatment after CCL19 decreased after blocking of CCR7, intact CCL19 impact. 3, CCL19/CCR7 through Sp1 regulation Hpa CCL19-join the invasion of A549 cells promote tumor cell invasion, compared with the control group, increased significantly. After blocking CCR7, inhibition of Sp1 decreased invasive ability. The Hpa blocking, found that cell invasion compared with the control group no significant changes in the After CCL19 role,. The conclusion CCL19 can activate CCR7 expression raised the transcription factor Sp1 to promote transcription of HpamRNA, thus contributing to the invasion of lung cancer cells.

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CLC: > Medicine, health > Oncology > Respiratory system tumors > Lung tumors
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