Lin28 对肝癌HepG2 细胞5-Fu 敏感性的影响及其分子机制

目的：探讨RNA结合蛋白Lin28 对肝癌HepG2细胞5-氟尿嘧啶（5-Fu）敏感性的影响及其机制。方法：应用pcDNA3.1-Lin28 或si-Lin28 转染HepG2 细胞，采用qPCR及WB法检测转染后HepG2 细胞Lin28 的表达水平，CCK-8 法检测5-Fu 作用后转染细胞增殖活性变化并计算5-Fu 对细胞作用的IC50，流式细胞术检测5-Fu 处理后细胞凋亡情况、WB法检测凋亡相关蛋白的表达变化，qPCR法检测转染后耐药相关miRNA（let-7a 和let-7b）及肿瘤干细胞标记物（Oct4、Nanog和Sox2）的mRNA表达水平。结果：与空载对照组（HepG2/Vector）相比，HepG2/Lin28 组细胞中Lin28 mRNA和蛋白的表达水平均显著升高（P<0.05 或P<0.01），过表达Lin28 可显著抑制HepG2 细胞对5-Fu 作用的敏感性（IC50明显升高，P<0.05）和增强细胞增殖活性、使细胞凋亡率和凋亡相关蛋白caspase-3 表达明显降低（均P<0.01）。与阴性对照组（si-control）相比，si-Lin28 细胞中Lin28 表达水平显著下降（P<0.01）；敲减Lin28 可使细胞增殖活性及5-Fu 的IC50显著降低（均P<0.01），凋亡率及凋亡蛋白表达明显增加（P<0.01）。与HepG2/Vector 组比较，HepG2/Lin28 细胞中let-7a、let-7b 及肿瘤干细胞标记物（Oct4、Nanog 和Sox2）的表达水平明显上调（均P<0.01），而si-Lin28 细胞中let-7a、let-7b 及Oct4、Nanog、Sox2 的表达水平较si-control 组明显下调（均P<0.01）。结论：Lin28 可通过调节miRNAs的表达及肿瘤干细胞的形成从而调节肝癌HepG2细胞对化疗的敏感性，靶向调节Lin28 表达有望成为提高肝癌化疗疗效的手段。

Effect and molecular mechanism of Lin28 on 5-Fu sensitivity of hepatocellular carcinoma HepG2 cells

Objective: To investigate the effect and mechanism of RNA binding protein Lin28 on the 5-fluorouracil (5-Fu) sensitivity of HepG2 cells. Methods: HepG2 cells were transfected with plasmid pcDNA3.1-Lin28 or si-Lin28 (small interfering RNA of Lin28).qPCR and Western blotting were used to detect the expression of Lin28 in HepG2 cells after transfection. Changes of cell proliferation in transfected cells after 5-Fu treatment was detected by CCK8 assay and the 50% inhibitory concentration (IC50) was calculated. Flow cytometry was used to detect apoptotic rate after 5-Fu treatment and the expression of apoptosis-related protein was assayed by Western blotting. The mRNA expressions of drug-resistant miRNAs (let-7a and let-7b), as well as cancer stem cell markers (Oct4, Nanog and Sox2) after transfection were detected by qPCR. Results: As compared to the HepG2/Vector cells, the mRNA and protein expressions of Lin28 were significantly up-regulated in HepG2/Lin28 cells (P<0.05 or P<0.01). Over-expression of Lin28 significantly suppressed the sensitivity of HepG2 cells to 5-Fu (IC50 elevated obviously, P<0.05) and significantly increased cell proliferation while decreased apoptotic rate and expression of apoptotic-related protein caspase-3 (all P<0.01). As compared to si-control group, expression of Lin28 in HepG2/si-Lin28 cells was significantly down-regulated (P<0.01). Lin28 knockdown significantly reduced cell proliferation and IC50 of 5-Fu (all P<0.01) but increased apoptotic rate and expression of apoptosis-related protein (P<0.01). Compared with HepG2/Vector group, expressions of let-7a and let-7b, as well as cancer stem cell markers (Oct4, Nanog and Sox2) were significantly increased in HepG2/Lin28 cells (all P<0.01); while these molecules were significantly decreased in HepG2/si-Lin28 cells as comparing to si-control group (all P<0.01). Conclusion: Lin28 can modulate the chemosensitivity of HepG2 cells by regulating the expression of miRNAs and the formation of cancer stem cells. Targeting Lin28 might be a promising approach to improve the chemotherapy efficacy in HCC.