降脂药奥利司他通过靶向脂代谢途径对肝细胞癌细胞HepG2增殖和凋亡的影响

Effects of lipid-lowering drug Orlistat on proliferation and apoptosis of hepatocellularcarcinoma cellline HepG2 through targeted lipid metabolism pathway

目的探究脂肪酸合成酶(fatty acid synthase,FASN)在肝细胞癌(hepatocellular carcinoma,HCC)进展中的功能,以期为HCC治疗提供有利的理论依据。方法生信分析FASN在HCC肿瘤组织中的表达及富集通路;细胞计数盒8(cell counting kit-8,CCK-8)、克隆形成、流式细胞术分别检测细胞活力、增殖和凋亡;实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测FASN的水平;蛋白质免疫印迹法(Western blot)检测BCL2相关X蛋白(BCL2-associated X,Bax)和B细胞淋巴瘤2(B-cell lymphoma-2,Bcl-2)的表达;亲脂性荧光染料BODIPY 493/503检测中性脂质的积累情况;试剂盒检测游离脂肪酸和甘油的水平。结果FASN在HCC肿瘤组织中的表达高于邻近癌旁组织。在体外细胞模型中进行验证,结果显示,与MIHA细胞相比,HCC细胞系(Huh-7、HepG2、Hep3B)中FASN的表达显著上调(P<0.05)。与对照组相比,si-FASN组细胞中FASN的表达降低,而oe-FASN组细胞中FASN的表达提高(P<0.05)。与对照组相比,si-FASN组HepG2细胞活力和克隆数降低,而oe-FASN组Hep3B细胞活力和克隆数提高(P<0.05)。与对照组相比,si-FASN组HepG2细胞凋亡率提高,而oe-FASN组HepG2细胞凋亡率降低(P<0.05)。与对照组相比,si-FASN组细胞中Bax的表达提高,Bcl-2的表达降低;oe-FASN组细胞中Bax的表达降低,Bcl-2的表达提高(P<0.05)。与对照组相比,si-FASN组细胞的中性脂质含量降低,而oe-FASN组细胞中的中性脂质含量升高(P<0.05)。与对照组相比,si-FASN组细胞中游离脂肪酸水平和甘油水平降低,而oe-FASN组细胞中游离脂肪酸水平和甘油水平提高(P<0.05)。与oe-NC+DMSO组相比,oe-NC+Orlistat组细胞中FASN的表达降低,而与oe-NC+Orlistat组相比,oe-FASN+Orlistat组细胞中FASN的表达提高(P<0.05)。与oe-NC+DMSO组相比,oe-NC+Orlistat组细胞的增殖水平降低,并诱导细胞凋亡;而与oe-NC+Orlistat组相比,oe-FASN+Orlistat组细胞的增殖水著提高,并抑制细胞凋亡(P<0.05)。与oe-NC+DMSO组相比,oe-NC+Orlistat组细胞中促凋亡因子Bax的表达提高,降低抗凋亡因子Bcl-2的表达;而与oe-NC+Orlistat组相比,oe-FASN+Orlistat组细胞中促凋亡因子Bax的表达显著降低,提高抗凋亡因子Bcl-2的表达(P<0.05)。与oe-NC+DMSO组相比,oe-NC+Orlistat组细胞中的中性脂质含量、游离脂肪酸含量以及甘油水平降低;与oe-NC+Orlistat组相比,oe-FASN+Orlistat组细胞中的中性脂质含量、游离脂肪酸含量以及甘油水平提高(P<0.05)。结论本研究强调FASN在HCC进展及脂质代谢中的促进作用。脂肪酸合酶抑制剂奥利司他可以抑制FASN的表达,通过调控脂肪酸代谢重编程抑制HCC的进展。

Objective To explore the function of fatty acid synthase(FASN)in the progression of hepatocellular carcinoma(HCC),in order to provide a favorable theoretical basis for the treatment of HCC.Methods The expression and enrichment pathway of FASN in HCC tumor tissues were analyzed by bioinformatics.Cell Counting Kit-8(CCK-8),colony formation and flow cytometry were used to detect cell viability,proliferation and apoptosis,respectively.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the level of FASN.The expression of BCL2-associated X(Bax)and B-cell lymphoma-2(Bcl-2)was detected by Western blot.Lipophilic fluorescent dye BODIPY 493/503 was used to detect the accumulation of neutral lipids,and the levels of free fatty acids and glycerol were detected by reagent kit.Results The expression of FASN in HCC tumor tissue was higher than that in paracancerous tissues.Based on verification in an in vitro cell model,the results showed that compared with MIHA cells,the expression of FASN was significantly upregulated in HCC cell lines(Huh-7,HepG2,Hep3B)(P<0.05).Compared with the control group,the expression of FASN decreased in cells in the si-FASN group,but increased in the oe-FASN group(P<0.05).Compared with the control group,the si-FASN group showed a decrease in HepG2 cell viability and clone count,while the oe-FASN group showed an increase in Hep3B cell viability and clone count(P<0.05).Compared with the control group,the apoptosis rate of HepG2 cells increased in the si-FASN group,but decreased in the oe-FASN group(P<0.05).Compared with the control group,the si-FASN group showed an increase in Bax expression and a decrease in Bcl-2 expression in cells.The expression of Bax decreased and the expression of Bcl-2 increased in the oe-FASN group cells(P<0.05).Compared with the control group,the neutral lipid content decreased in the si-FASN group,but increased in the oe-FASN group(P<0.05).Compared with the control group,the levels of free fatty acids and glycerol decreased in the si-FASN group but increased in the oe-FASN group(P<0.05).Compared with the oe-NC+DMSO group,the expression of FASN decreased in the oe-NC+Orlistat group cells decreased,while compared with the oe-NC+Orlistat group,the expression of FASN in the oe-FASN+Orlistat group cells increased(P<0.05).Compared with the oe-NC+DMSO group,the proliferation level of cells in the oe-NC+Orlistat group decreased and apoptosis was induced.Compared with the oe-NC+Orlistat group,the oe-FASN+Orlistat group showed increased cell proliferation and inhibited cell apoptosis(P<0.05).Compared with the oe-NC+DMSO group,the expression of pro-apoptotic factor Bax increased in cells of the oe-NC+Orlistat group,while the expression of anti apoptotic factor Bcl-2 was reduced.Compared with the oe-NC+Orlistat group,the expression of pro-apoptotic factor Bax in the Orlistat+oe FASN group cells significantly reduced,while the expression of anti-apoptotic factor Bcl-2 increased(P<0.05).Compared with the oe-NC+DMSO group,the neutral lipid content,free fatty acid level,and glycerol level in the oe-NC+Orlistat group cells reduced.Compared with the oe-NC+Orlistat group,the neutral lipid content,free fatty acid level,and glycerol level in the oe-FASN+Orlistat group cells increased(P<0.05).Conclusion The results of this study emphasize the role of FASN in the promotion of HCC progression and lipid metabolism.The fatty acid synthase inhibitor Orlistat can inhibit FASN expression,which can inhibit HCC progression by regulating reprogramming in fatty acid metabolism.