Xu Zhou-Wei, Liu Na-Na, Wang Xing-Yu, Ding Bai-Cheng, Zhang Hai-Feng, Li Ying, Sun Wu-Yi, Wei Wei
Department of Emergency Surgery, The First Affiliated Hospital of Anhui Medical University, Jixi Road, Hefei, 230022, Anhui, People's Republic of China.
Key Laboratory of Anti-Inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative Innovation Center of Anti-Inflammatory and Immune Medicine, Institute of Clinical Pharmacology, Anhui Medical University, Meishan Road, Hefei, 230032, Anhui, People's Republic of China.
Cancer Cell Int. 2021 Nov 2;21(1):587. doi: 10.1186/s12935-021-02261-8.
To study the roles of AT1R, PLC-β1, CaM and other related signal molecules in the formation and development of hepatocellular carcinoma (HCC) and their correlation.
ELISA and immunohistochemistry were used to analyze the expressions of target proteins in serum and liver tissue of HCC patients, and the correlation between AT1R, PLC-β1 and CaM and postoperative survival status of patients was followed up and determined. CCK-8 method was used to screen the doses of Ang II and candesartan sensitive to HepG2 and HCCLM3 cells. Transwell experiment was used to observe the effects of different drugs on the migration and invasion activity of HCC cells. Meanwhile, flow cytometry and Western blot were used to detect the expression levels of AT1R, PLC-β1 and CaM in the cells. Then PLC-β1 siRNA was selected to transfect HCC cells, so as to further clarify the mechanism of the above signal proteins. HepG2 cells were inoculated under the hepatic capsule of mice to induce the formation of HCC in situ. Ang II and candesartan were used to stimulate HCC mice to observe the difference in liver appearance and measure the liver index. Finally, ELISA and immunofluorescence experiments were selected to analyze the levels of target proteins in mouse serum and liver tissue.
The expression levels of target proteins in serum and liver tissue of HCC patients were significantly increased, and the postoperative survival time of patients with high expression of AT1R, PLC-β1 or CaM was obviously shortened. Ang II and candesartan could significantly promote and inhibit the motility of HCC cells, and had different effects on the levels of AT1R, PLC-β1 and CaM in cells. However, in hepatocellular carcinoma cells transfected with PLC-β1 siRNA, the intervention ability of drugs was obviously weakened. Ang II could significantly promote the formation and progression of mouse HCC, while candesartan had the opposite effect. Meanwhile, medications could affect the expressions of target proteins in mouse serum and liver tissue.
AT1R, PLC-β1 and CaM may be risk factors affecting the formation and prognosis of HCC, and the PLC-β1/CaM signaling pathway mediated by AT1R is an important way to regulate the migration and invasion activity of HCC cells.
研究1型血管紧张素Ⅱ受体（AT1R）、磷脂酶C-β1（PLC-β1）、钙调蛋白（CaM）等相关信号分子在肝细胞癌（HCC）发生发展中的作用及其相关性。
采用酶联免疫吸附测定（ELISA）和免疫组织化学法分析HCC患者血清及肝组织中靶蛋白的表达情况，并对AT1R、PLC-β1和CaM与患者术后生存状态的相关性进行随访测定。采用细胞计数试剂盒-8（CCK-8）法筛选对人肝癌细胞系HepG2和人肝癌高转移细胞系HCCLM3细胞敏感的血管紧张素Ⅱ（AngⅡ）和坎地沙坦剂量。采用Transwell实验观察不同药物对HCC细胞迁移和侵袭活性的影响。同时，采用流式细胞术和蛋白质免疫印迹法检测细胞中AT1R、PLC-β1和CaM的表达水平。然后选择PLC-β1小干扰RNA（siRNA）转染HCC细胞，以进一步阐明上述信号蛋白的作用机制。将HepG2细胞接种于小鼠肝包膜下诱导原位HCC形成。用AngⅡ和坎地沙坦刺激HCC小鼠，观察肝脏外观差异并测量肝脏指数。最后，选择ELISA和免疫荧光实验分析小鼠血清和肝组织中靶蛋白水平。
HCC患者血清和肝组织中靶蛋白表达水平显著升高，AT1R、PLC-β1或CaM高表达患者的术后生存时间明显缩短。AngⅡ和坎地沙坦可显著促进和抑制HCC细胞的运动能力，且对细胞中AT1R、PLC-β1和CaM水平有不同影响。然而，在转染PLC-β1 siRNA的肝癌细胞中，药物的干预能力明显减弱。AngⅡ可显著促进小鼠HCC的形成和进展，而坎地沙坦则有相反作用。同时，药物可影响小鼠血清和肝组织中靶蛋白的表达。
AT1R、PLC-β1和CaM可能是影响HCC发生及预后的危险因素，由AT1R介导的PLC-β1/CaM信号通路是调节HCC细胞迁移和侵袭活性的重要途径。
