CCL19/CCR7 Regulate The Expression Of Heparanase Via Sp1 In Lung Cancer A549 Cell

chemokine CCL19 and its receptor CCR7 play an important role in the homing of lymphocytes. CCR7 overexpression in many solid tumors, was found to be associated with lymph node metastasis,tumor growth,angiogenesis and invision.Our previous study found that CCR7 overexpressed in non-small cell lung cancer and have closely relationship with lymph node metastasis and invasion.But the mechanism that CCR7 promote tumor cells invision and metastasis is unclear.We added CCL19 exogenously and found the upregulation of Hpa. Heparanase(heparanase, Hpa) is common,which can degrade the extracellular matrix and basement membrane,promoting the invasion and metastasis of tumor. Therefore we predict CCL19/CCR7 can promote the invasion of lung cancer cells by regulating the expression of Hpa,but the regulatory mechanism remains unclear. Hpa gene promoter region contains transcription factor Sp1 binding sites. In this study,we added CCL19 to A549 cell exogenously and activated CCR7 to detect the expression of Sp1 and Hpa and cell invasiveness.Materials and Methods1. Antibodyies and ReagentPolyclonal anti-CCR7,heparanase and monoclonal anti-spl antibodies, Mithramycin A, Recombinant human cc119 was purchased from Peprotech..2. Cell cultureHuman lung cancer cell lines A549 (adenocarcinoma) was cultured in DMEM supplemented with 10% foetal bovine serum,at 37℃in a 5% CO2 incubator. 3. RT-PCRTotal RNA was isolated using TRIZOL (Invitrogen, USA), reverse transcription, polymerase chain reaction,1.5% agarose gel electrophoresis, using Image J sofeware analysis of eexpression intensity.4.Western Blot analysisTotal protein was extracted with lysis buffer, and 60-80ug of protein lysates were separated on a 10% SDS-polyacrylamide electrophoresis gel, transferred to Polyvinylidene Fluoride (PVDF) membranes,detect the expression of heparanase and Sp1.5.Chromatin immunoprecipitation (ChIP):According to the instructions of the ChIP assay kit,detect the Sp1 binding to the heparanase promoter.6.Cell migration and invasion assays100μl cells were added to dhe upper chamber of an 8μm pore size coated with Matrigel or not.600μl DMEM/10%/2%FBS was added to the lower chamber and incubated for 24h.Cells were fixed in 4% Paraformaldehyde and stained with hematoxylin.,using Microscope count the migrated cells.7.Statistical analysisThe SPSS 17.0 software was employed to analyze the data.P<0.05 was considered as statistical significance.Results1.CCL19/CCR7 upregulate the expression of heparanase via Sp1Treatment human lung adenocarcinoma A549 cells with recombinant human CCL19 obviously increased the expression of Sp1 and heparanase mRNA and protein. Sp1 inhibitor(MA) inhibite the expression of Sp1 and heparanase mRNA and protein significantly.After blocking CCR7 and the expression of Sp1 respectively, RT-PCR and western blot analyses showed that the expressions of Spl and heparanase mRNA and protein decreased, while decreased expression of heparanase was not affected by CCL19.2.CCL19 enhance the DNA binding of Spl to the promoter of heparanaseWe first analysed the promoter sequence of heparanase and indentified spl binding sites(consensus sequence 5GGGGC3),also known as GC box.Then we performed ChIP assay.ChIP analysis demonstrated that spl could bind to the heparanase promoter.3.CCL19/CCR7 regulate the expression of heparanase via Spl to enhance the invasion ability of the lung cancer cellsAfter incubation with CCL19, the invasion ability of A549 cell increased in comparison the control group.After blocking CCR7 and Spl respectively, the invasion ability decreased. Migration assay showed that the invasion ability of A549 cell did not change.ConclusionsCCL19 can activate CCR7 to up-regulate the expression of Spl and promote the transcription of heparanase mRNA and cell invasiveness in lung adenocarcinoma A549 cell.