抑制肾癌细胞自噬可增强癌细胞对舒尼替尼的敏感性

Knockdown of ATG5 enhances the sensitivity of human renal carcinoma cells to sunitinib

目的检测肾癌中自噬的表达水平及其对舒尼替尼耐药性的影响。方法采用免疫组织化学法检测自噬相关基因5(ATG5)与微管相关蛋白1轻链3(LC3)在99例肾透明细胞癌组织中的表达,并进行临床病理及生存分析;通过慢病毒感染技术构建ATG5低表达的A-498肾癌细胞株,Western blot法检测干扰效果;流式细胞术检测ATG5正常表达与低表达的A-498细胞增殖的变化;经舒尼替尼刺激后,MTT法检测A-498细胞存活率。结果 ATG5与LC3在肾癌组织中的表达显著高于癌旁组织,且ATG5与LC3的表达与肾癌的分型、分化程度、TNM分期、术后生存率具有相关性,而与性别、年龄、部位、大小无关;与对照组相比,ATG5低表达的A-498细胞ATG5与LC3蛋白表达明显降低、细胞增殖率(G2/M期百分比)明显降低;且对舒尼替尼敏感性增加,与舒尼替尼呈剂量依赖性。结论肾癌组织中自噬活跃,抑制自噬ATG5表达后,明显增强人肾癌细胞对舒尼替尼的敏感性。

Objective To investigate the expression levels of autophagy-related gene 5（ATG5） and microtubuleassociated protein 1 light chain 3（LC3） and their effects on sunitinib resistance in human renal carcinoma cells. Methods After clinic-pathologic feature and survival analysis,99 renal clear cell carcinoma tissues with different histological grades were used to detect the expression of ATG5 and LC3 by immunohistochemistry. Renal carcinoma cell line A-498 was infected with lentivirus-mediated ATG5 shRNA. Western blot analysis was performed to confirm the efficiency of ATG5 knockdown.Proliferation rate of A-498 cells in control group and ATG5 low expression group was determined by flow cytometry. Finally,the survival rate was detected by MTT assay after A-498 cells were treated with different concentrations of sunitinib. Results The expression levels of ATG5 and LC3 in renal clear cell carcinoma tissues were significantly higher than those in para-tumor tissues. The expression levels of ATG5 and LC3 were associated with classification,histological grade,TNM stage and survival rate,rather than gender,age,location,tumor size. Compared with the control group,the protein expressions of ATG5 and LC3 significantly decreased in A-498 cel s with ATG5 low expression. The cel proliferation rate in ATG5 downregulation group was lower than that in the control group. Compared with control group,the survival rate in ATG5 low expression group were significantly reduced in a dose-dependent manner after sunitinib treatment. Conclusion Autophagy is active in renal clear cell carcinoma,and the drug sensitivity to sunitinib in renal cancer cells can be enhanced by the downregulation of ATG5.