益母草碱调节Hippo-YAP信号通路对结肠癌细胞增殖、凋亡和化疗耐药性的影响

Effect of Leonurine on proliferation,apoptosis,and chemotherapy resistance of colon cancer cells by regu⁃lating the Hippo⁃YAP signaling pathway

目的探讨益母草碱(Leo)调节Hippo-Yes相关蛋白(YAP)信号通路对结肠癌细胞增殖、凋亡和化疗耐药性的影响。方法分别用0、1.875、3.75、7.5、15、30、60μg/mL Leo处理人结肠癌LOVO细胞48 h,筛选适宜的Leo浓度用于实验。将LOVO细胞分为空白组、Leo低剂量组、Leo中剂量组、Leo高剂量组、Leo高剂量+pcDNA组、Leo高剂量+pcDNA-YAP组,CCK-8法、5-乙炔基-2’-脱氧尿苷(EdU)染色检测细胞增殖;流式细胞术检测细胞凋亡;qRT-PCR检测LOVO细胞中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、Bcl2相关X蛋白(Bax)、多药耐药关联蛋白1(MRP1)、P-糖蛋白(PGP)mRNA表达;Western blot检测LOVO细胞中YAP、结缔组织生长因子(CTGF)、富半胱氨酸蛋白61(CYR61)蛋白表达。以LOVO/五氟尿嘧啶(5-FU)细胞为研究对象,按照上述分组进行处理,并分为A-空白组、A-Leo低剂量组、A-Leo中剂量组、A-Leo高剂量组、A-Leo高剂量+pcDNA组、A-Leo高剂量+pcDNA-YAP组,CCK-8检测LOVO/5-FU细胞的耐药性。结果与空白组相比,Leo低剂量组、Leo中剂量组、Leo高剂量组LOVO细胞A_(450)值(分别为0.86±0.08、0.75±0.06、0.43±0.04比0.97±0.08)、EdU阳性细胞率[分别为(50.96±2.17)%、(43.37±2.05)%、(28.84±1.19)%比(57.75±2.36)%]、PCNA、MRP1、PGP mRNA表达及YAP(分别为2.05±0.17、1.78±0.14、1.23±0.11比2.36±0.19)、CTGF、CYR61蛋白表达降低,细胞凋亡率[分别为(15.54±0.65)%、(23.38±1.01)%、(36.69±1.78)%比(8.34±0.31)%]、Bax mRNA表达升高,且呈剂量依赖性(P<0.05);与Leo高剂量组、Leo高剂量+pcDNA组相比,Leo高剂量+pcDNA-YAP组LOVO细胞A_(450)值(分别为0.77±0.05比0.43±0.04、0.45±0.04)、EdU阳性细胞率[分别为(46.69±1.95)%比(28.84±1.19)%、(27.67±1.25)%]、PCNA、MRP1、PGP mRNA表达及YAP、CTGF、CYR61蛋白表达升高,细胞凋亡率、Bax mRNA表达降低(P<0.05);与A-空白组相比,A-Leo低剂量组、A-Leo中剂量组、A-Leo高剂量组LOVO细胞活力降低,且呈剂量依赖性(P<0.05);与A-Leo高剂量组、A-Leo高剂量+pcDNA组相比,A-Leo高剂量+pcDNA-YAP组LOVO细胞活力升高(P<0.05)。结论Leo可能通过抑制Hippo-YAP信号通路抑制结肠癌细胞增殖和化疗耐药性,促进细胞凋亡。

Objective To investigate the effects of Leonurine(Leo)on the proliferation,apoptosis,and chemotherapy resistance of colon cancer cells by regulating the Hippo-Yes associated protein(YAP)signaling path-way.Methods Human colon cancer LOVO cells were treated with Leo at concentrations of 0,1.875,3.75,7.5,15,30,and 60μg/mL for 48 hours,respectively,and suitable Leo concentrations were selected for the experiment.LOVO cells were separated into blank group,Leo low-dose group,Leo medium-dose group,Leo high-dose group,Leo high-dose+pcDNA group,and Leo high-dose+pcDNA-YAP group,CCK-8 method and 5-ethynyl-2'-deoxyuridine(EdU)staining were applied to detect cell proliferation;flow cytometry was applied to detect cell apoptosis;qRT-PCR was applied to detect the mRNA expression of proliferating cell nuclear antigen(PCNA),Bcl2 associated X protein(Bax),multidrug resistance associated protein 1(MRP1),and P-glycoprotein(PGP)in LOVO cells;Western blot was applied to detect the expression of YAP,connective tissue growth factor(CTGF),and cysteine rich protein 61(CYR61)in LOVO cells.LOVO/pentafluorouracil(5-FU)cells were studied and treated according to the above groups.They were divided into A-blank group,A-Leo low-dose group,A-Leo medium dose group,A-Leo high-dose group,A-Leo high-dose+pcDNA group,A-Leo high-dose+pcDNA-YAP group.CCK-8 was applied to de-tect the drug resistance of LOVO/5-FU cells.Results Compared with the blank group,the A_(450) value of LOVO cells(0.86±0.08,0.75±0.06,0.43±0.04 vs 0.97±0.08),EdU positive cell rate(50.96%±2.17%,43.37%±2.05%,28.84%±1.19%vs 57.75%±2.36%),the expression of PCNA,MRP1,PGP mRNA,and the expression of YAP(2.05±0.17,1.78±0.14,1.23±0.11 vs 2.36±0.19),CTGF,CYR61 proteins in the Leo low dose group,Leo medium dose group,and Leo high dose group decreased,while the apoptosis rate(15.54%±0.65%,23.38%±1.01%,36.69%±1.78%vs 8.34%±0.31%)and the expression of Bax mRNA increased,in a dose-dependent manner(P<0.05);compared with the Leo high-dose group and Leo high-dose+pcDNA group,the A_(450) value of LOVO cells(0.77±0.05 vs 0.43±0.04,0.45±0.04),EdU positive cell rate(46.69%±1.95%vs 28.84%±1.19%,27.67%±1.25%),the expression of PCNA,MRP1,PGP mRNA,and the expression of YAP,CTGF,CYR61 proteins in the Leo high-dose+pcDNA-YAP group increased,while the apoptosis rate and the expression of Bax mRNA de-creased(P<0.05);compared with the A-blank group,the LOVO cell viability of the A-Leo low-dose group,A-Leo medium dose group,and A-Leo high-dose group decreased,in a dose-dependent manner(P<0.05);compared with the A-Leo high-dose group and the A-Leo high-dose+pcDNA group,the LOVO cell viability of the A-Leo high-dose+pcDNA-YAP group increased(P<0.05).Conclusion Leo may inhibit proliferation and chemotherapy resistance of colon cancer cells and promote cell apoptosis by inhibiting the Hippo-YAP signaling pathway.