刺芒柄花素通过COX-2/cyclin D1轴调控肝癌的发生和发展

Formononetin affects the pathogenesis of liver cancer by inhibiting the COX-2/cyclin D1 axis

目的:分析环氧合酶2(cyclooxygenase-2,COX-2)的表达与肝癌患者临床病理特征及预后的关系,探讨刺芒柄花素(formononetin,FOR)在肝癌发生和发展中的作用及其机制。方法:收集2021年1~5月河北医科大学第四医院20例手术治疗肝癌患者的癌和相应的癌旁组织、60例手术治疗肝癌患者的临床资料,以及人肝癌细胞系HepG2和Bel-7402。用qPCR法和免疫组织化学染色法检测肝癌组织中COX-2的表达,分析其表达与患者临床病理特征和预后的关系。构建小鼠二乙基亚硝胺诱导肝癌模型,验证FOR对肝癌发病的影响。用0.003、0.006μmol/ml的FOR分别处理肝癌细胞HepG2和Bel-740248 h后,用CCK-8法和流式细胞术检测FOR对肝癌细胞增殖和周期的影响,qPCR和WB法检测细胞中COX-2、CDK4、CDK6和cyclin D1表达的变化。结果:肝癌组织中COX-2 mRNA表达水平显著高于癌旁组织(P<0.01),COX-2蛋白表达阳性率为68.3%(41/60);COX-2表达阳性与患者TNM分期晚、肿瘤大、生存期短有关(均P<0.05)。诱导小鼠肝癌模型中,FOR处理组小鼠肝癌的发生率显著降低、肝癌组织中COX-2表达显著降低(均P<0.05)。FOR处理可显著抑制HepG2和Bel-7402细胞的增殖能力,增加G0/G1期细胞比例、降低S和G2期细胞比例(均P<0.05),抑制细胞中COX-2和cyclin D1的表达(均P<0.01)。结论:COX-2表达阳性肝癌患者临床分期较晚且预后较差,FOR可通过抑制COX-2和cyclin D1表达来降低肝癌的发生和发展。

Objective:To analyze the relationship between the expression of cyclooxygenase-2(COX-2)and clinicopathological features and prognosis of patients with liver cancer,and to explore the role and mechanism of formononetin(FOR)in the pathogenesis of liver cancer.Methods:The clinical data of 60 patients with liver cancer and 20 pairs of surgically resected cancer tissues and corresponding para-cancerous tissues from liver cancer patients that treated in the Fourth Hospital of Hebei Medical University during January 2021 and May 2021 were collected for this study;in addition,human liver cancer cell lines HepG2 and Bel-7402 were also collected.The expression of COX-2 in liver cancer tissues was detected by qPCR and immunohistochemical staining,and the relationship between COX-2 expression and clinicopathological characteristics and prognosis of patients was analyzed.Diethylnitrosamine-induced mouse model of liver cancer was established to verify the effect of FOR on the incidence of liver cancer.After being treated with 0.003 and 0.006μmol/ml FOR 48 h,the effects of FOR on proliferation and cell cycle of HepG2 and Bel-7402 cells were detected by CCK-8 and Flow cytometry.The changes in the expression of COX-2,CDK4,CDK6 and cyclin D1 were detected by qPCR and WB.Results:The mRNA expression of COX-2 in liver cancer tissues was significantly higher than that in paracancerous tissues(P<0.01).The positive expression rate of COX-2 in liver cancer tissues was 68.3%(41/60),and the positive expression of COX-2 was correlated with advanced TNM stage,large tumor and short survival(all P<0.05).In the xenograft mouse model of induced liver cancer,the incidence of liver cancer in mice treated with FOR was significantly reduced,and the expression of COX-2 in liver tissues was significantly decreased(all P<0.05).FOR significantly inhibited the proliferation,increased the proportion of cells at G0/G1 phase,decreased the proportion of cells at S and G2 phase(all P<0.05),and inhibited the expression of COX-2 and cyclin D1 in HepG2 and Bel-7402 cells(all P<0.01).Conclusion:Liver cancer patients with positive COX-2 expression have an advanced clinical stage and a poor prognosis.FOR can prevent the occurrence and development of liver cancer by inhibiting the expression of COX-2 and cyclin D1.