基于外泌体miR-602介导的RASSF1A/p38 MAPK通路对乙肝相关性肝癌术后复发调控机制及健脾化瘀方干预研究

Hepatitis B is one of the leading causes of liver cancer in China. Surgery is the main treatment of liver cancer. According to our previous researches, we suspected that the expression of miR-602 in exosomes can inhibit the expression of ROSF1A gene, which regulate the p38 MAPK signaling pathway. Then, the miR-602 plays a role in promoting the tumor cells apoptosis and affect the postoperative dormancy/recurrence of liver cancer. We intend to investigate the effect of RTLF1A/p38 MAPK pathway mediated by miR-602 on the postoperative dormancy/ recurrence on HBV-transgenic mice model of hepatitis B-related hepatocellular carcinoma. Then, we observed the cytotoxic activity of spleen CTL in different states to normal expression and overexpression of miR-602 hepatoma cells and the changes of miR-602/RTLF1A/p38 MAPK pathway. Finally, clarify the mechanism of Jianpi Huayu Decoction on the prevention and treatment of hepatitis B-related hepatocellular carcinoma. The results of this research are useful in clinical prevention and treatment of hepatitis B-related hepatocellular carcinoma.

乙肝是我国诱发肝癌的主要因素之一，手术是肝癌的主要治疗手段。本课题基于前期研究，提出外泌体中miR-602表达的变化，可通过抑制RASSF1A基因介导p38 MAPK信号通路的促进乙肝相关性肝癌细胞凋亡，影响肝癌术后休眠/复发。本项目拟先在雄性乙肝病毒转基因小鼠上建立乙肝相关性肝癌术后休眠/复发小鼠模型，探讨外泌体miR-602介导的RASSF1A/p38 MAPK通路对促进乙肝相关性肝癌术后休眠，预防乙肝相关性肝癌术后复发的作用。再运用细胞转染等技术，得到稳定表达RASSF1A、过表达达miR-602的HepG2.2.15细胞，并观察不同状态小鼠脾CTL分别对正常表达和过表达miR-602的肝癌细胞的杀伤活性以及对miR-602介导的RASSF1A/p38 MAPK通路的影响。最终阐明健脾化瘀方预防乙肝相关性肝癌的作用及机制，为中医药干预乙肝相关性肝癌术后复发提供了新的思路。

目的：阐明外泌体miR-602介导的RASSF1A/p38 MAPK通路对乙肝相关性肝癌术后复发调控机制及健脾化瘀方干预.方法：（1）体外实验：a. 提取HepG2.2.15 细胞外泌体并检测miR-602和RASSF1A的表达情况；b. 选择SMCC-7721细胞稳定转染RASSF1A，在此基础上加入mimic/inhibitor miRNA-602，检测各组miR-602，RASSF1A/p38 MAPK表达，细胞的增殖、迁移、凋亡情况以及健脾化瘀方作用（2）体内实验：a. 建立转基因乙肝相关性原发肝癌动物模型，提取鉴定小鼠外周血中外泌体并比较各组miR-602表达；b. 构建过表达RASSF1A的SMCC-7721细胞肝癌原位复发裸鼠模型，检测miR-602，RASSF1A/p38 MAPK表达水平; c.以SMCC-7721细胞建立原位肝癌裸鼠模型，并观察健脾化瘀方对肝癌生长的影响，同时检测miR-602，RASSF1A/p38 MAPK的表达水平。.结果：（1）体外实验：a. HepG2.2.15与LO2共培养后，其外泌体miR-602表达升高，RASSF1A表达降低；b.与NC组比较，RASSF1A组的RASSF1A表达升高、miR-602表达下降。在此基础上，与 mimic NC 组相比，miR-602 mimic 组中miR-602和下游 ATF-2 和 p-C-JUN，C-JUN表达升高， RASSF1A 的表达降低， inhibitor 和 inhibitor NC 组的结果相反。健脾化瘀方可以使SMCC-7721,HepG2,Huh7细胞的增殖侵袭能力降低、凋亡增多（2）体内实验：a. 肝癌复发组外泌体中miR-602 的表达显著高于原位肝癌组和肝癌术后休眠组，与正常对照组相差较小;b. 过表达RASSF1A组原发肿瘤组织较小，RASSF1A表达升高，miR-602， ATF-2 和 p-C-JUN，C-JUN表达下降，复发组结果相反；c. 与NC组相比，JPHY组miR-602和Bcl-2表达降低， RASSF1A，Caspase-8和-9以及BAX的表达升高，明显抑制肿瘤生长.结论：健脾化瘀方通过下调miR-602而促进RASSF1A/p38 MAPK的表达，从而抑制肝癌的增殖迁移，促进凋亡，防止复发。