趋化因子CCL28在缺氧诱导肝癌细胞侵袭中的作用

Role of chemoldne CCL28 in hypoxia-induced migration of hepatocellular carcinoma

目的探讨趋化因子CCL28在缺氧诱导肝癌细胞侵袭中的作用。方法 50份肝细胞肝癌标本来自肝细胞肝癌患者，肝癌细胞株HepG2、HCCLM3为实验室冻存。用实时定量聚合酶链反应检测肝癌组织中缺氧诱导因子-1α（HIF-1α）和CCL28的mRNA水平。实时定量聚合酶链反应、Western blot和ELISA检测不同缺氧时间处理后肝癌细胞HepG2、HCCLM3中HIF-1α和CCL28的mRNA和蛋白表达水平。构建CCL28-siRNA下调HCCLM3细胞CCL28表达后，Transwell观察缺氧诱导的肝癌HCCLM3细胞侵袭改变。用X2检验分析HIF—1α和CCL28表达水平与临床资料相关性，Spearman双变量相关性分析HIF-1α和CCL28表达水平相关性，用单因素方差分析组间比较。结果HIF-1α mRNA在肝癌组织中相对表达量为0．065±0．098，CCL28mRNA水平为0．025土0．075，Spearman双变量分析显示肝癌组织中HIF-1α与CCL28表达水平呈高度相关性（r=0．595，P〈0．01），二者表达水平增高皆与术后复发相（P=0．011，P=0．019）。缺氧培养肝癌HepG2和HCCLM3细胞CCL28表达增高并呈时间依赖性，HepG2：HIF-1α F=873．5。CCL28F=151．6lHCCLM3：HIF-1α F=964．5，CCL28F=285．8，P值均〈0．01。SiRNA抑制CCL28表达后，缺氧条件下HCCLM3细胞侵袭数目为（43．2±5．4）个／室、空白对照组为（54．6±9．5）个／室、阴性对照组为（58．0±3．9）个／室，缺氧条件下HCCLM3细胞侵袭细胞数比空白对照组和阴性对照组减少，P=0．011。结论趋化因子CCL28在缺氧条件下高表达并参与缺氧诱导肝癌细胞侵袭过程。

To investigate the role of CCL28 in hypoxia-induced cell migration of hepatocellular carcinoma （HCC）. Methods Resected liver tissues from 50 HCC patients were subjected to real-time （rt）-PCR analysis to evaluate the mRNA expression levels of the hypoxia-induced factor HIF-la and the chemokine CCL28. Patient data on trealrnent and outcome were analyzed. The human HCC cell lines HepG2 and HCCLM3 were used to investigate effects of hypoxic conditions on HIF-Ia and CCL28 expressions by rt- PCR, western blotting, and enzyme-linked irnmunoassay. The CCL28-mediated effects of hypoxic conditions on cell mobility and invasion were assessed by trans-well and matrigel assays, respectively, in HCCLM3 with CCL28 expression silenced by small-interferring （si）RNA transfection. Spearman＇s rank test was used to assess the correlation between CCL28 and effects on disease- and treatment-related factors. Results The mRNA levels of CCL28 （0.025±0.075） were found to be strongly correlated with HIF-1a（0.065±0.098） in human clinical samples of HCC （r = 0.595, P 〈 0.01）, with higher expressions of both related to recurrence after surgery （P= 0.011 and 0.019, respectively）. In vitro hypoxic conditions stimulated HIF-Iu and CCL28 expression in a time-dependent manner in both bothG2 （HIF-la： F= 873.5; CCL28：ff= 151.6） and HCCLM3 （HIF-Ia： F = 964.5; CCL28： F= 285.8） （allP 〈 0.01）. siRNA inhibition of CCL28 in HCCLM3 cells led to a significant reduction in hypoxia-induced invasion and migration （allP= 0.011）. Conclusion Chemokine CCL28 expression is up-regulated in human HCC and under in vitro hypoxic conditions, and may play an important role in hypoxia-induced HCC migration and invasion.