趋化因子CXCL12及其受体CXCR4对卵巢上皮性癌细胞增殖、迁移和侵袭能力的影响

Effect of chemokine CXCL12 and its receptor CXCR4 on proliferation,migration and invasion of epithelial ovarian cancer cells

目的探讨趋化因子 CXCL12及其受体 CXCR4对卵巢上皮性癌(卵巢癌)细胞增殖、迁移和侵袭能力的影响。方法采用 RT-PCR 技术和免疫细胞化学染色法检测卵巢癌细胞株CAOV3细胞中 CXCL12、CXCR4 mRNA 和蛋白的表达,以及 CXCL12(100 ng/ml)作用后 CAOV3细胞中整合素β1和血管内皮生长因子 C(VEGF-C)mRNA 的表达。实验分为6组:对照组(未加药),实验组1(加100 ng/ml 的 CXCL12),实验组2(加10 ng/ml 的 CXCL12),实验组3(加100 ng/ml 的 CXCL12和10μg/ml 的 CXCR4抗体),实验组4(加100 ng/ml 的 CXCL12和1μg/ml 的 CXCR4拮抗剂——AMD3100),实验组5(加10μg/ml 的 CXCR4抗体或卵巢癌腹水)。采用四甲基偶氮唑蓝比色法检测CXCL12对 CAOV3细胞增殖的影响。以穿膜小室为模型,采用重组细胞基底膜迁移、侵袭实验检测CXCL12和卵巢癌腹水对 CAOV3细胞迁移、侵袭的影响。结果 CAOV3细胞中,CXCR4蛋白呈棕黄色强阳性表达,CXCR4 mRNA 的表达水平为0.70±0.10,经100 ng/ml 的 CXCL12作用24 h 时 CXCR4mRNA 的表达水平(1.24±0.14)显著增加(t=-7.1088,P=0.0021);CXCL12蛋白和 mRNA 均无表达。100 ng/ml 的 CXCL12作用3 h 时整合素β1 mRNA 表达水平即显著增加(作用前后分别为0.53±0.10、1.53±0.16,P<0.01),24 h 时 VEGF-C mRNA 表达水平显著增加(作用前后分别为0.52±0.09、1.11±0.15,P<0.01)。对 CAOV3细胞的增殖作用,实验组1分别与对照组和实验组2比较均明显增强(3组分别为0.428±0.051、0.325±0.045、0.328±0.039,P<0.05);实验组1分别与实验组3和实验组4比较均明显被抑制(后两组分别为0.356±0.031、0.373±0.029,P<0.05);而实验组5(0.349±0.038)与对照组比较,差异则无统计学意义(P>0.05)。对 CAOV3细胞的迁移和侵袭作用,实验组1分别与对照组和实验组2比较均明显增强[迁移细胞数分别为(523.3±25.2)、(108.0±7.2)、(211.7±24.7)个,侵袭细胞数分别为(39.3±4.0)、(4.0±1.0)、(15.7±3.1)个;P 均<0.01];实验组1分别与实验组3和实验组4比较均明显被抑制(P<0.05);但实验组5[即加入卵巢癌腹水组;迁移细胞数为(706.6±30.6)个,侵袭细胞数为(61.7±7.6)个]显著多于实验组1(P<0.05)。结论 CXCL12可促进卵巢癌细胞增殖、迁移和侵袭,并上调整合素β1和 VEGF-CmRNA 的表达,此作用可被 CXCR4抗体抑制,表明 CXCL12在卵巢癌的生长和转移中发挥一定作用。

Objective To explore the effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells. Methods CXCR4 and CXCL12 mRNA and protein expression of human ovarian cancer cell line CAOV3 was detected by RT-PCR and immunocytochemistry. Integrin 61 and vascular endothelial growth factor-C （VEGF-C） mRNA expression were detected in CAOV3 cells stimulated by CXCL12. The CAOV3 cells were divided into 6 groups： control group （un-stimulated） , experimental group 1 （stimulated by 100 ng/ml CXCL12 ）, experimental group 2 （stimulated by 10 ng/ml CXCL12） , experimental group 3 （100 ng/ml CXCL12 and 10 μg/ml neutralizing CXCR4 antibody ）, experimental group 4 （100 ng/ml CXCL12 and 1 μg/ml CXCR4 antagonist AMD3100）, experimental group 5 （10 μg/ml neutralizing CXCR4 antibody or ascites）. Methyl thiazolyl tetrazolium （MTF） was used to analyze the effects of different concentrations of CXCL12 on CAOV3 cell proliferation. Transwell invasion chamber and reconstructed basement membrane （Matrigel） were used to evaluate effect of various concentrations of CXCL12 and ascites on CAOV3 cell migration and invasion. Results CAOV3 cells expressed CXCR4 mRNA （0. 70 ±0. 10） and protein, but did not express CXCL12 mRNA or protein. Immunostaining of CXCR4 was mainly located in cytoplasm. CXCR4 mRNA was upregulated after 100 ng/ml CXCL12 stimulation （ 1.24 ± 0. 14; t = - 7. 1088, P = 0. 0021 ）. Integrin 61 mRNA was greatly increased at 3 hours by stimulation of 100 ng/ml CXCL12 （ before and after stimulation 0. 53 ± 0. 10,1.53 ± 0. 16; P 〈 0. 01 ） , and VEGF-C mRNA showed significant increase at 24 hours by treatment with CXCL12 （ before and after stimulation 0. 52 ± 0. 09,1.11 ± 0. 15 ; P 〈 0. 05 ）. Under serum- free sub-optimal culture conditions, experimental group 1 greatly enhanced cell proliferation in CAOV3 cells compared with control group and experimental group 2 （ respectively 0. 428 ± 0. 051,0. 325 ±0. 045,0. 328 ±0. 039 ;P 〈0.05 ）. Experimental group 1 was strongly inhibited compared with experimental groups 3 and 4 （the latter two groups respectively 0. 356 ±0. 031,0. 373 ±0. 029 ; P 〈 0. 05 ）. There was no significant difference between experimental group 5 （0. 349 ±0. 038 ） and control group （ P 〉 0. 05 ）. Experimental group 1 stimulated the migration and invasion of CAOV3 cells in chemotaxis assay compared with control group and experimental group 2 （ number of cell migration respectively 523.3 ± 25.2,108.0 ± 7.2,211.7 ＋24. 7, number of cell invasion respectively 39. 3 ±4.0,4.0 ± 1.0,15.7 ±3. 1 ;P 〈0. 01 ）. This enhancing effect of experimental group 1 was strongly inhibited compared with experimental groups 4 and 5 （ P 〈 0. 05 ）. The number of migrating and invading cells in experimental group 5 （migration ：706. 6 ±30. 6, invasion ：61.7 ±7. 6） was significantly higher than that of experimental group 1 （P 〈 0.05）. Conclusions This in vitro study shows CXCL12 promote proliferation, migration, invasion of ovarian cancer cell line CAOV3, and upregulate integrin β1 and VEGF-C expression, and these effects are strongly inhibited by neutralizing CXCR4 antibody. It suggests CXCL12 and its receptor CXCR4 may play important roles in ovarian cancer growth and metastasis.