缺氧微环境中CCL28高表达促进HCCLM3细胞迁移和侵袭

 目的  探讨缺氧条件下HCCLM3细胞趋化因子CCL28表达情况及作用 。方法  实时定量PCR和ELISA方法检测缺氧条件下肝癌细胞HCCLM3 趋化因子CCL28表达水平;运用小干扰RNA(small interfering RNA,siRNA)和Lipofectamine 2 000细胞转染技术下调CCL28表达;用Transwell和 Matrigel观察肝癌细胞HCCLM3侵袭迁移情况;采用明胶酶谱法测定CCL28 siRNA干扰后细胞上清中的基质金属蛋白酶(matrix metalloproteinase enzyme,MMP)含量。应用实时定量PCR检测50例肝细胞癌组织中CCL28 mRNA表达情况,运用卡方检验和Kaplan Meier法分析CCL28表达与临床病例特征及患者总体生存时间的关系。结果  肝癌HCCLM3细胞内CCL28 mRNA在缺氧培养6 h后表达增高,蛋白水平在缺氧培养12 h后表达增高,呈时间依赖性。SiRNA抑制CCL28表达后,缺氧条件下HCCLM3细胞迁移数目(65.2±4.49)较siRNA阴性组(85±5.91)和空白组(87.4±8.44)有所减少(P<0.001);穿过Matrigel膜细胞数目(43.2±5.36)较siRNA阴性组(58±3.87)和对照组(54.6±9.53)有所减少(P=0.011)。明胶酶谱法发现CCL28表达抑制后HCCLM3分泌MMP 2减少。PCR结果提示,肝癌组织CCL28 mRNA表达量为0.0254 ±0.0755,与正常肝组织相比,23例(46%)呈高表达,CCL28表达高低与肿瘤最大径(P=0.027)、术后复发相关(P=0.019);CCL28高表达与低表达组之间总体生存无明显差异(χ2=0.008,P=0.927)。结论  缺氧条件下趋化因子CCL28高表达并参与肝癌细胞HCCLM3迁移侵袭过程。肝癌组织中CCL28表达增高与术后复发相关。

Over expression of chemokine CCL28 in hypoxia promotes HCCLM3 migration and invasion

Objective  To investigate expression and role of chemokine CCL28 in hepatic cancer cell HCCLM3 under hypoxia.Methods  Real-time PCR and ELISA were used to detect CCL28 level of HCCLM3 under hypoxia;small interfering RNA (siRNA) and Lipofectamine 2000 were used to seal up CCL28 expression;transwell and Matrigel were used to observe cellular mobility and invasion ability;gelatin zymography was used to detect MMPs activity;real-time PCR was used to detect mRNA level of CCL28 in 50 samples of hepatocellular carcinoma tissue;Kaplan Meier method and chi square test were used to analyse the relationship of clinical characteristics with CCL28 expression.Results  The mRNA levels of  CCL28 started to increase after 6 h hypoxia while the protein levels had a significant increase after 12 h hypoxia in a time dependent manner;after HCCLM3 cells were transfected with CCL28 siRNA,the number of migration cells under hypoxia (65.2±4.49) was significantly decreased as compared with negative siRNA (85±5.91) or control (87.4±8.44) (P<0.001),the number of invasion cells under hypoxia (43.2±5.36) was significantly decreased as compared with negative siRNA (58±3.87)or control (54.6±9.53) (P=0.011),and the activity of MMP2 was significantly decreased.PCR showed that CCL28 mRNA level in hepatocellular carcinoma was 0.0254±0.0755,and 23 of 50 (46%) showed high expression level as compared with control.CCL28 expression level was related with tumor size (P=0.027) and recurrence after surgery (P=0.019).Kaplan Meier showed that over survival between high and low CCL28 groups was not significant (χ2=0.008,P=0.927).Conclusions  Chemokine CCL28 is over expressed under hypoxia and plays a role in the migration and invasion of HCCLM3 cells.CCL28 expression in hepatocellular carcinoma is related with recurrence after surgery.