靶向CD24分子3E10增强肝癌HuH-7细胞的化疗敏感度

3E10 Tar geting CD24 Enhances Chemotherapy Sensitivity of Hepatocellular Carcinoma HuH-7 Cells

目的探讨靶向分子CD24单克隆抗体3E10调节肝癌HuH-7细胞对化疗药顺铂敏感度的作用及其分子机制。方法 Western blot和流式细胞术检测抗CD24抗体3E10的特异性;Annexin V-FITC/PI双染色检测细胞凋亡;Real-time PCR和Western blot检测耐药基因和肿瘤干细胞特性基因的表达水平;Western blot检测JAK/STAT3和PI3K/AKT信号通路的活性水平。结果 3E10可有效识别CD24蛋白及HuH-7细胞。3E10显著增强HuH-7细胞对顺铂的敏感度(P<0.05),抑制率由(10.3±3.0)%提高至(34.4±10.8)%。3E10显著降低HuH-7细胞耐药基因ABCB1、ABCB5、ABCC1和肿瘤干性基因NANOG、CD24的表达水平及干性基因β-catenin的磷酸化水平(P<0.05)。3E10显著降低STAT3和AKT的磷酸化水平(P<0.05)。结论靶向CD24分子3E10通过降低HuH-7细胞的耐药性、肿瘤干性和JAK/STAT3、PI3K/AKT信号通路活性来增强HuH-7细胞对化疗药顺铂的敏感度。

Objective To investigate the function and molecular mechanism of 3E10 molecule targeting CD24 in regulating the chemosensitivity of liver cancer HuH-7 cells to cisplatin. Methods Western blot and flow cytometry were used to verify 3E10 recognition specificity for CD24. Annexin V-FITC/PI double staining was used to detect cell apoptosis. The expression level of drug resistance gene and tumor stem cell gene were analyzed by real-time PCR and Western blot, and the activity of JAK/STAT3 and PI3K/AKT signaling pathways were measured by Western blot. Results 3E10 could effectively identify CD24 protein and HuH-7 cells. 3E10 significantly enhanced the sensitivity of HuH-7 cells to cisplatin（P〈0.05）, and the inhibition rate was increased from（10.3±3.0）% to（34.4±10.8）%. 3E10 significantly decreased the expression level of drug resistance gene ABCB1, ABCB5, ABCC1 and tumor stem gene NANOG, CD24, as well as the phosphorylation level of β-catenin in HuH-7 cells（P〈0.05）. 3E10 significantly down-regulated the phosphorylation of STAT3 and AKT（P〈0.05）. Conclusion 3E10 molecule targeting CD24 could enhance the sensitivity of HuH-7 cells to cisplatin through reducing the drug resistance, stemness and JAK/STAT3, PI3K/AKT signaling pathway activity of HuH-7 cells.