糖基因和N-糖链介导入肝癌细胞转移及耐药性的研究

Metastasis and drug resistance of human hepatocarcinoma cells mediated by glycogenes and N-glycans

目的通过研究糖基因、N-糖链在人肝癌高、低转移细胞株MHCC97-H和MHCC97-L中的差异表达，明确二者与肝癌转移及其耐药的相关性，确证肝癌转移诊断及抗肿瘤治疗新靶点。方法采用实时荧光定量聚合酶链反应（real-time PCR）分析糖基因，异硫氰酸荧光素（FITC）-凝集素结合分析糖链的特征；通过RNA干扰技术干预差异表达的糖基因，检测干扰前后MHCC97-H细胞的体外侵袭力及其药物敏感性。进一步修饰N-糖基化（衣霉素和糖基肽酶处理），检On,0N-糖基化修饰前后MHCC97-H细胞的体外侵袭力、体内成瘤性及药物敏感性。结果糖基因、N-糖链在人肝癌高、低转移细胞株中表达差异有统计学意义；通过特异性RNA干扰技术使MHCC97-H细胞中N-乙酰氨基葡萄糖转移酶（MGAT5）表达下调时，该细胞在体外的侵袭能力下降，药物敏感性增强（t=7．312，P〈0．05）。MHCC97．H细胞经N．糖基化修饰后在体外的侵袭能力下降，体内成瘤性受到抑制，药物敏感性增强。结论人肝癌细胞中糖基因、糖蛋白N-糖链的差异表达与肿瘤细胞的转移及其耐药密切相关，可为肿瘤化疗提供新靶点。

Objective To identify the relationship among glycogenes, N-glycans and hepatocarcinoma metastasis and drug resistance by studying the differential expressions of glycogenes and N-glycans in MHCC97-H and MHCC97-L human hepatocarcinoma cell lines, and to confirm the novel target of hepatocarcinoma metastasis and anti-tumor therapy. Methods Real-time PCR was used to quantitatively analyze glycogenes and fluorescein isothiocyanate （FITC）-lectin was used to analyze glycans characteristics. RNA interference approach was used to interfere the glycogenes, and the invasive ability in vitro and drug susceptibility of MHCC97-H cells were detected before and after interference. Modification of N-glycosylation （tunicamycin and PNGase F treatment） was done, and the invasiveness in vitro, tumorigenicity in vivo and drug susceptibility of MHCC97-H cells were detected be- fore and after modification. Results The expressions of glycogenes and glycans were different in MHCC97-H cells and MHCC97-L cells. The silence of MGAT5 in MHCC97-H cells inhibited invasion ability and increased sensitivity to 5-fluorouracil in vitro（ t = 7.312, P 〈 0.05）. Modification of N-glyeosylation decreased MHCC97-H cells invasion ability in vitro and tumorigenicity in vivo and increased sensitivity to 5-fluorouracil. Conclusion The differential expressions of glycogens and N-glycans in human hepatocarcinoma cell lines correlate with tumor invasion and drug resistance, and they are expected to be novel targets of tumor chemotherapy.