Effect And Mechanism Of CCL28 On The Hepato Carcinoma In The Hypoxia Microenvironment

Objectives:To make clear that whether hypoxia promote the increase of CCL28 in hepatoma cells through HIF1? and whether CCL28 affect the biological characteristics of hepatoma cells and has the ability to recruit regulatory T cells(Tregs),so as to provide more theoretical support on the discuss of hypoxia may have important role in the process of tumor.Methods:1.To a variety of human hepatoma cell lines,cells were cultured in hypoxia(1%O2,5%CO2)or normoxic(21%O2,5%CO2)condition,and supernatants were collected at 0,8,24,48 h.Q-PCR and ELISA were used to detect the mRNA and protein expression of CCL28.2.Collect hepatocarcinoma tissue samples from cancer patients at III-IV stage and make paraffin sections to detect the expression of CCL28 and hypoxia marker protein CA IX(carbonic anhydrase IX)by immunohisto chemistry and correlation analysis.3.Knockdown the expression level of HIFla in human hepatoma SK-Hep-1 cell by the mediate of lentivirus and detect the effect of HIFla gene silencing on the expression of CCL28 in hypoxia condition by Q-PCR and ELISA method.4.Overexpress CCL28 in human hepatoma SK-HEP-1 cell by the mediate of lentivirus and analyze the effect of CCL28 gene overexpression on the biological behavior,including cell proliferation,invasion,migration,apoptosis and cell adhesion,etc.5.Prepare mouse hepatoma Hepal-6 cells which could express CCL28 and luciferase gene stably and control cells.Establish the subcutaneous tumor model for fluorescence imaging by using C57BL mice.Detect tumor growth by fluorescence imaging and sampling.6.Isolate tumor-infiltrating lymphocytes from tumor sample of step 5.Detect the expression of Treg cell-specific marker molecule Foxp3 by Q-PCR,so as to reflects the number levels of Treg cells in hepatocarcinoma indirectly.7.Isolate human peripheral blood mononuclear cells(PBMC).Add into the transwell chamber for chemotaxis experiment and add hypoxia/normoxic conditioned medium or the recombinant of CCL28/isotype control antibody into lower chamber,blocked with the neutralizing antibodies of CCL28 or CCR10(CCL28 receptor).Analyze the CD4+CD25+Foxp3+/CD4+ cell ratio in the lower chamber by flow cytometry.Make clear that whether hypoxia could recruit Treg cells in PBMC through CCL28 and whether Treg cell type is CCR10 positive.8.Obtained high purity human CD4+CD25+Foxp3+ Treg cells using immunomagnetic separation kit.Add into the transwell chamber for chemotaxis experiment and add hypoxia/normoxic conditioned medium or the recombinant of CCL28/isotype control antibody into lower chamber,blocked with the neutralizing antibodies of CCL28 or CCR10.Count the cell number in the lower chamber directly by flow cytometry.Make clear that whether hypoxia could recruit Treg cells directly through CCL28 and whether Treg cell type is CCR10 positive.Results:1.Compared with normoxia condition,hypoxia condition could induce the level increase of CCL28 mRNA and protein in the supernatant significantly in a variety of hepatoma cell line(SK-Hep-1,HepG2,Hep3B)and the increase level is hypoxia time depended.2.Immunohistochemistry result of human hepatocarcinoma clinical samples showed that the expression level of CCL28 is correlated with hypoxia marker protein CA?.3.Use lentivirus-mediated RNA interference technology to knock down the expression level of HIF1? in human hepatoma cells.CCL28 mRNA and protein levels corresponding decrease under hypoxic conditions,compared with the control group,P<0.05.4.CCL28 overexpression in human hepatoma SK-Hep-1 cells promotes the ability of cell proliferation,migration and adhesion,but does not affect cell apoptosis and invasion.5.Successfully prepare mouse hepatoma Hepal-6 cells that express CCL28 and luciferase gene stably and control cells and establish the subcutaneous tumor model for fluorescence imaging by using C57BL mice.Fluorescence imaging system and final sampling measurement both show that tumor from CCL28 overexpression group is significantly greater than the control group,P<0.05.6.Isolate tumor-infiltrating lymphocytes from tumor sample of step 5.Result of Q-PCR show that Foxp3 mRNA expression level of CCL28 overexpression group is significantly higher than the control group.7.In transwell chamber chemotaxis experiment,human PBMC cells were added into the upper chamber,hypoxic cell supernatants recruit more Treg cells than oxic cell supernatants and Treg cell migration towards hypoxic medium is attenuated by blockad of CCL28.Human recombinant CCL28 recruits Treg cell and this recruitment is abrogated by blockade of CCR10.8.In transwell chamber chemotaxis experiment,high purity human Treg cells the chamber was added into the upper chamber.hypoxic cell supernatants recruit more Treg cells than oxic cell supernatants.Treg cell migration towards hypoxic medium is attenuated by blockade of CCL28 and CCR10.Conclusions:Hypoxia mediates the CCL28 level increasing in hepatoma cells through HIFla.CCL28 have a variety of biological functions.On one hand,it could promote the ability of hepatoma cells,including proliferation,migration and adhesion.On the other hand,it could induce the chemotaxis of Treg cells through the interaction with CCL28 receptor CCR10 on the surface of Treg cell.CCL28 level increasing induced by hypoxia may play an important role in the hepatocarcinoma process.