CD147及其配体CypA在小鼠肝癌细胞Hepa1-6逃避T细胞免疫监视中的作用
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摘要
目的探讨CypA和CD147分子的相互作用对肿瘤细胞和T细胞生物学行为的影响。方法将pGPU6/GFP/Neo-CD147shRNA转染至Hepa1-6细胞中,用G418筛选阳性克隆,采用反转录PCR(reverse transcription PCR,RT-PCR)、Western blot法鉴定CD147的表达水平,以不同浓度CypA处理Hepa1-6和Hepa1-6-CD147shRNA的细胞24 h后,再用CCK8试剂盒检测CypA的促细胞增殖作用,利用流式细胞仪分选C57Bl/6j小鼠T细胞,Transwell小室法检测CypA在Hepa1-6和Hepa1-6-CD147shRNA细胞影响下趋化T细胞的能力,将Hepa1-6和Hepa1-6-CD147shRNA细胞接种至C57Bl/6j小鼠皮下20 d,每5 d测量肿瘤生长情况。结果 Hepa1-6-CD147shRNA细胞中CD147表达水平较Hepa1-6细胞显著下降。CypA以浓度依赖性方式促进Hepa1-6细胞的增殖,但对Hepa1-6-CD147shRNA细胞无明显作用。在加入Hepa1-6细胞后,CypA趋化T细胞的能力显著低于加入Hepa1-6-CD147shRNA细胞(P<0.01)。Hepa1-6细胞在C57Bl/6j小鼠皮下的成瘤体积显著大于Hepa1-6-CD147shRNA细胞在C57Bl/6j小鼠皮下的成瘤体积(P<0.01)。结论 Hepa1-6细胞可通过其CD147与CypA作用,促进其自身细胞增殖,并同时影响CypA对T细胞的趋化能力,进而逃避T细胞的免疫监视作用。
Objective To investigate the effects of CypA and CD147 interactions on the biological behavior of tumor cells and T cells. Methods The p GPU6/GFP/Neo-CD147 shRNA was transfected into Hepa1-6 cells and positive clone was selected by G418. The expression level of CD147 was identified by RT-PCR and Western blot, Hepa1-6 and Hepa1-6-CD147 shRNA cells were treated with different concentrations of CypA for 24 h, and then CCK8 kit was used to detect the cell proliferation. Flow cytometry was used to sort the T cells of C57Bl/6j mice, and Transwell chamber assay was employed to detect the chemotactic ability of CypA for T cells. Hepa1-6 and Hepa1-6-CD147 shRNA cells were inoculated subcutaneously into C57Bl/6j mice for 20 days, and the tumor volume was measured every five days. Results Compared with Hepa1-6 cells, the CD147 level of Hepa1-6-CD147 shRNA cells was decreased significantly. CypA promoted the proliferation of Hepa1-6 cells in a concentration-dependent manner, but there was no obvious effect on Hepa1-6-CD147 shRNA cells. Under the intervention of Hepa1-6 cells, the chemotactic ability of CypA for T cells was significantly decreased, compared with Hepa1-6-CD147 shRNA cells(P<0.01). Tumor volume of Hepa1-6 cells in C57Bl/6j mice was significantly larger than that of Hepa1-6-CD147 shRNA cells(P<0.01). Conclusion CypA stimulates the proliferation of Hepa1-6 cells, Hepa1-6 cells attenuates the chemotactic ability of CypA for T cells, and then escapes from immune surveillance of T cells, and this influence is CD147 dependent.
Objective To investigate the effects of CypA and CD147 interactions on the biological behavior of tumor cells and T cells. Methods The p GPU6/GFP/Neo-CD147 shRNA was transfected into Hepa1-6 cells and positive clone was selected by G418. The expression level of CD147 was identified by RT-PCR and Western blot, Hepa1-6 and Hepa1-6-CD147 shRNA cells were treated with different concentrations of CypA for 24 h, and then CCK8 kit was used to detect the cell proliferation. Flow cytometry was used to sort the T cells of C57Bl/6j mice, and Transwell chamber assay was employed to detect the chemotactic ability of CypA for T cells. Hepa1-6 and Hepa1-6-CD147 shRNA cells were inoculated subcutaneously into C57Bl/6j mice for 20 days, and the tumor volume was measured every five days. Results Compared with Hepa1-6 cells, the CD147 level of Hepa1-6-CD147 shRNA cells was decreased significantly. CypA promoted the proliferation of Hepa1-6 cells in a concentration-dependent manner, but there was no obvious effect on Hepa1-6-CD147 shRNA cells. Under the intervention of Hepa1-6 cells, the chemotactic ability of CypA for T cells was significantly decreased, compared with Hepa1-6-CD147 shRNA cells(P<0.01). Tumor volume of Hepa1-6 cells in C57Bl/6j mice was significantly larger than that of Hepa1-6-CD147 shRNA cells(P<0.01). Conclusion CypA stimulates the proliferation of Hepa1-6 cells, Hepa1-6 cells attenuates the chemotactic ability of CypA for T cells, and then escapes from immune surveillance of T cells, and this influence is CD147 dependent.
引文
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[15]Kim K,Kim H,Jeong K,et al.Release of overexpressed Cyp B activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress[J].Apoptosis,2012,17(8):784-96.
[16]Romano PG,Horton P,Gray JE.The Arabidopsis cyclophilin gene family[J].Plant Physiol,2004,134(4):1268-82.
[17]Yang H,Li M,Chai H,et al.Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells[J].J Surg Res,2005,123(2):312-9.
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[19]Bukrinsky MI.Cyclophilins:unexpected messengers in intercellular communications[J].Trends Immunol,2002,23(7):323-5.
[20]Sherry B,Yarlett N,Strupp A,et al.Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharideactivated macrophages[J].Proc Natl Acad Sci USA,1992,89(8):3511-5.
[21]Damsker JM,Bukrinsky MI,Constant SL.Preferential chemotaxis of activated human CD4+T cells by extracellular cyclophilin A[J].J Leukoc Biol,2007,82(3):613-8.
[22]Obchoei S,Weakley SM,Wongkham S,et al.Cyclophilin A enhances cell proliferation and tumor growth of liver flukeassociated cholangiocarcinoma[J].Mol Cancer,2011,10:102.
[2]Landskron J,Taskén K.CD147 in regulatory T cells[J].Cell Immunol,2013,282(1):17-20.
[3]Yurchenko V,Constant S,Eisenmesser E,et al.Cyclophilin-CD147interactions:a new target for anti-inflammatory therapeutics[J].Clin Exp Immunol,2010,160(3):305-17.
[4]Iacono KT,Brown AL,Greene MI,et al.CD147 immunoglobulin superfamily receptor function and role in pathology[J].Exp Mol Pathol,2007,83(3):283-95.
[5]Kanekura T,Chen X.CD147/basigin promotes progression of malignant melanoma and other cancers[J].J Dermatol Sci,2010,57(3):149-54.
[6]Kim K,Kim H,Jeong K,et al.Release of overexpressed Cyp B activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress[J].Apoptosis,2012,17(8):784-96.
[7]Yurchenko V,Constant S,Bukrinsky M.Dealing with the family:CD 147 interactions with cyclophilins[J].Immunology,2006,117(3):301-9.
[8]Liu X,Zhao Z,Liu W.Insights into the roles of cyclophilin A during influenza virus infection[J].Viruses,2013,5(1):182-91.
[9]Yurchenko V,Zybarth G,O‘Connor M,et al.Active site residues of cyclophilin A are crucial for its signaling activity via CD147[J].J Biol Chem,2002,277(25):22959-65.
[10]Malesevic M,Gutknecht D,Prell E,et al.Anti-inflammatory Effects of Extracellular Cyclosporins Are Exclusively Mediated by CD147[J].J Med Chem,2013,56(18):7302-11.
[11]Ru NY,Wu J,Chen ZN,et al.HAb18G/CD147 is involved in TGF-β-induced epithelial-mesenchymal transition and hepatocellular carcinoma invasion[J].Cell Biol Int,2015,39(1):44-51.
[12]Liang Q,Han Q,Huang W,et al.HAb18G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells[J].PLo S One,2014,9(7):e102496.
[13]Huang Q,Li J,Xing J,et al.CD147 promotes reprogramming of glucose metabolism and cell proliferation in HCC cells by inhibiting the p53-dependent signaling pathway[J].J Hepatol,2014,61(4):859-66.
[14]Tang J,Guo YS,Zhang Y,et al.CD147 induces UPR to inhibit apoptosis and chemosensitivity by increasing the transcription of Bip in hepatocellular carcinoma[J].Cell Death Differ,2012,19(11):1779-90.
[15]Kim K,Kim H,Jeong K,et al.Release of overexpressed Cyp B activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress[J].Apoptosis,2012,17(8):784-96.
[16]Romano PG,Horton P,Gray JE.The Arabidopsis cyclophilin gene family[J].Plant Physiol,2004,134(4):1268-82.
[17]Yang H,Li M,Chai H,et al.Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells[J].J Surg Res,2005,123(2):312-9.
[18]Tian-Tian Z,Jun-Feng Z,Heng G.Functions of cyclophilin A in atherosclerosis[J].Exp Clin Cardiol,2013,18(2):e118-24.
[19]Bukrinsky MI.Cyclophilins:unexpected messengers in intercellular communications[J].Trends Immunol,2002,23(7):323-5.
[20]Sherry B,Yarlett N,Strupp A,et al.Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharideactivated macrophages[J].Proc Natl Acad Sci USA,1992,89(8):3511-5.
[21]Damsker JM,Bukrinsky MI,Constant SL.Preferential chemotaxis of activated human CD4+T cells by extracellular cyclophilin A[J].J Leukoc Biol,2007,82(3):613-8.
[22]Obchoei S,Weakley SM,Wongkham S,et al.Cyclophilin A enhances cell proliferation and tumor growth of liver flukeassociated cholangiocarcinoma[J].Mol Cancer,2011,10:102.