探讨β-谷甾醇对肝癌细胞增殖的影响,凋亡,迁移,入侵,和上皮-间质转化(EMT),并利用网络药理学研究其潜在机制。将人肝癌细胞系(Huh-7和HCCLM3)暴露于梯度浓度的β-谷甾醇(5μg/mL,10μg/mL,和20μg/mL)。使用MTT评估细胞活力和增殖,CCK-8,集落形成,和EdU测定。流式细胞术用于评估细胞周期和凋亡。进行划痕和Transwell测定,分别,检测细胞迁移和侵袭。凋亡相关蛋白的水平(BAX,BCL2和裂解的caspase3)以及EMT相关蛋白(E-cadherin,N-钙黏着蛋白,蜗牛,和波形蛋白)使用Western印迹分析在Huh-7和HCCLM3细胞系中检测到。通过PubChem筛选β-谷甾醇的药物靶基因,随后评估GSE112790数据集中的表达。此外,分析了癌症基因组图谱-肝细胞癌(TCGA-LIHC)数据库中糖原合酶激酶3β(GSK3B)的表达水平,以及它与肝细胞癌患者生存结局的相关性。通过分析ROC曲线评估GSK3B的诊断效率。随后,用GSK3B的过表达载体转染Huh-7和HCCLM3细胞系,然后用β-谷甾醇处理以进一步验证GSK3B和β-谷甾醇之间的关联。GSK3B在肝细胞癌患者中表达显著升高,基于GEO数据集和TCGA数据库,可以预测肝细胞癌患者预后受损。GSK3B抑制剂(CHIR-98014)显著抑制细胞增殖和侵袭,促进肝癌细胞G0/G1期细胞凋亡和细胞周期阻滞。β-谷甾醇处理进一步促进了GSK3B抑制剂对肝癌细胞的作用。已发现GSK3B过表达可增强肝细胞癌细胞的增殖和侵袭能力。此外,已经观察到GSK3B过表达,它可以部分逆转β-谷甾醇对肝细胞的抑制作用。β-谷甾醇抑制肝癌细胞增殖和侵袭,并通过抑制GSK3B表达增强细胞凋亡。
To explore the effects of β-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of β-Sitosterol (5 μg/mL, 10 μg/mL, and 20 μg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for β-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with β-Sitosterol to further validate the association between GSK3B and β-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients\' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. β-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of β-Sitosterol upon hepatocellular. β-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.