【摘要】 目的 miR-200家族与肿瘤的发生发展息息相关,可能具有调控PD-1/PD-L1的作用。文中探究肝癌组织中miR-200a、miR-200b、miR-200c与免疫检查点分子的关系。方法采用免疫组化分析2018年6月-2019年8月期间东部战区总医院秦淮医疗区60例行手术治疗的原发性肝细胞癌的癌旁组织及肝癌组织标本中的PD-L1表达。qRT-PCR检测标本中miR-200a、miR-200b、miR-200c的表达。合成miR-200a、miR-200b、miR-200c的类似物并转入肝癌细胞株HepG2中,过表达此3种microRNA。Western blot法检测过表达后PD-L1的表达;CCK-8法检测细胞增殖。使用荧光报告基因实验验证miR-200a、miR-200b、miR-200c对于PD-L1的靶向调控能力。结果肝癌患者癌组织中miR-200a(4.26±0.99 vs 30.89±4.78,P=0.0007)、miR-200b(9.59±2.04 vs 26.19±3.79,P=0.0026)、miR-200c(10.37±2.20 vs 18.70±3.35,P=0.023)含量均明显低于癌旁组织。PD-L1表达阳性的患者具有更低水平的miR-200家族表达量。结论过表达miR-200可降低PD-L1的表达,并抑制肝癌细胞的增殖。肝癌中miR-200可直接靶向PD-L1对其进行负向调控。在肝癌中PD-L1是miR-200家族的靶点,具有一定的临床治疗潜力。更多还原

【Abstract】 Objective The miR-200 family is closely related to the occurrence and development of tumors, and may have the effect of regulating PD-1/PD-L1. This research is to explore the relationship between miR-200 family(miR-200 a, miR-200 b, miR-200 c) and immune checkpoint molecules in liver cancer. Methods Immunohistochemistry was used to analyze the expression of PD-L1 in adjacent tissue and liver cancer tissue samples of 60 cases of primary hepatocellular carcinoma who underwent surgical treatment in Qinhuai Medical District of Eastern Theater General Hospital from June 2018 to August 2019. qRT-PCR was used to detect the expression of miR-200 a, miR-200 b, and miR-200 c, and the relationship between the two was statistically analyzed. Synthesize miR-200 a, miR-200 b, miR-200 c analogs and transfer them into liver cancer cell line HepG2, overexpress these three microRNAs, detect the expression of PD-L1 after overexpression by western blot method, and detect cell proliferation by CCK-8 method. Finally, aEGFP reporter gene experiment was used to detect whether miR-200 a, miR-200 b, and miR-200 c directly targeted PD-L1. Results The levels of miR-200 a, miR-200 b, and miR-200 c in cancer tissues of liver cancer patients were significantly lower than those in adjacent tissues(miR-200 a:4.26±0.99 vs 30.89±4.78,P=0.0007;miR-200 b:9.59±2.04 vs 26.19±3.79,P=0.0026;miR-200 c:10.37±2.20 vs 18.70±3.35,P=0.0230). miRNAs were inversely related to PD-L1, and patients with positive PD-L1 expression had lower miR-200 family expression level. Conclusion Overexpression of miR-200 can reduce the expression of PD-L1 and inhibit the proliferation of liver cancer cells. In liver cancer, miR-200 can directly target PD-L1 to negatively regulate it. PD-L1 is the target of miR-200 family in hepatocellular carcinoma and has certain clinical therapeutic potential.更多还原