缺氧增加肝癌细胞胚胎干细胞基因表达促进恶性转化

Effects of hypoxia on malignant phenotype of HCC cells

目的研究缺氧对原发性肝癌恶性表型的影响及相关机制。方法建立体内、外缺氧模型，比较缺氧对MHCC97H肝癌细胞成瘤和侵袭转移能力的影响，并通过检测细胞增殖周期，CD90、CD133标记的肝癌干细胞数量以及胚胎干细胞（embryonic stem cell，ES）样基因Oct4，Nanog，Sox2等的表达分析缺氧对肝癌细胞生物学特性的影响。结果缺氧促进MHCC97H肝癌细胞运动（t=2．792，P=0．023）、侵袭（t=7．624，P〈0．0001）、转移（χ^2=5．507，P=0．031）潜能，并增加软琼脂集落形成（t=3．292，P=0．011）和裸鼠皮下成瘤率（χ^2=8．571，P=0．015）。在缺氧环境中，MHCC97H肝癌细胞增殖能力受到抑制，G1期细胞比例显著增加，Oct4，Nanog，Sox2等基因表达明显上调。结论缺氧促进MHCC97H肝癌细胞成瘤和转移等恶性表型与获得胚胎干细胞样特征有关。

Objective To investigate the effects of hypoxia on malignant phenotype of a hepatocellular carcinoma （HCC） cell line and its molecular basis. Methods Human HCC cell line with highly metastatic potential （MHCC97H） was grown under hypoxia （induced by 100 μmol/L CoOl2 ） and normoxic conditions respectively. To observe the effects of hypoxia on MHCC97H cells was observed, the tumorigenicity was carried out by soft agar cloning method in vitro and inoculation in nude mice in vivo; Invasive and metastatic potential were measured. Experiments with and/or without Matrigel in vitro and a metastatic human HCC orthotopic nude mice model by HAL in vivo. To clarify the molecular background, the proliferation of cells were analyzed by MTT assay and the cell cycle was detected by flow cytometry; The expression of embryonic stem cell （ES） -like genes （ Oct4, Nanog and Sox2） were detected by real-time RT-PCR; CD90^ ＋ and CD133 ^＋ subpopulation from MHCC97H cells were isolated by flow cytometry and the expressions of CD90 and CD133 of MHCC97H cells were analyzed by immunofluorescence. Results Hypoxia increased tumor migration （ t = 2. 792, P = 0. 023 ）, invasiveness （ t = 7. 624, P 〈 0. 0001 ） and clone forming ability （ t = 3. 292, P = 0. 011 ） of MHCC97H cells in vitro, and promoted tumorigenesis （ χ^2 = 8. 571, P = 0. 015 ） and pulmonary metastasis （ χ^2= 5. 507, P = 0. 031 ） in vivo. The proliferation of MHCC97H cells arrest under hypoxic conditions accompanied with increased proportion of cells in G1 phase, the expressions of Oct4, Nanog, Sox2 significantly increased in response to hypoxia, but the fluorescence intensity and number of CD90 ^＋ and CD133 ^＋ MHCC97H cells decreased or unchanged. Conclusion Hypoxia increases aggressiveness of MHCC97H cells, which was correlated with the acquisition of embryonic stem cell-like characteristics.