ZNF300基因在肝癌耐药细胞HepG2/VCR中的表达及功能初探

目的建立人肝癌HepG2/VCR耐药细胞株,检测ZNF300基因在HepG2/VCR中的表达并初步分析其在肝癌多药耐药(MDR)中发挥的功能。方法采用体外低浓度梯度递增的诱导方法建立长春新碱(VCR)获得性HepG2/VCR耐药细胞株。MTT法检测确定HepG2/VCR耐药细胞株对VCR的耐药性,用Western blot方法检测人锌指蛋白ZNF300基因编码的ZNF300及多药耐药基因编码的P糖蛋白(P-gp)在HepG2和HepG2/VCR细胞中的表达差异;在HepG2/VCR细胞中转染ZNF300基因正向或反向cDNA质粒后,MTT法检测VCR对耐药细胞IC50值的变化,Westernblot方法检测细胞内P-gp表达的影响。结果 MTT检测确认HepG2/VCR耐药细胞构建成功,Western blot检测发现耐药细胞中ZNF300及P-gp的表达相对于HepG2细胞明显增高。在HepG2/VCR细胞中转染正向ZNF300 cDNA质粒后,MTT和Western blot检测发现ZNF300过表达可使VCR对耐药细胞的IC50值增高,并使细胞内P-gp表达上调;在转染反向cDNA质粒Knockdown ZNF300基因表达后得到相反的结果。结论 ZNF300基因在HepG2/VCR耐药细胞中表达明显增高,并能通过上调耐药蛋白P-gp的表达促进肝癌细胞耐药性,可以作为逆转肝癌多药耐药的分子作用靶点。

The expression and primary functional analysis of ZNF300 gene in hepatic cancer drug resistant cell line HepG2/VCR

Aim To establish human hepatic cancer drug resistant cell line HepG2/VCR,to detect the expression of human ZNF300 gene in HepG2/VCR cells,and to primarily analyze the possible roles of ZNF300 gene in hepatic cancer multi-drug resistance(MDR).Methods HepG2/VCR cell line was established by using treatment with vincristine(VCR) of low concentration gradually increased.MTT analysis was performed to confirm the VCR resistance feature of HepG2/VCR.The expression variances of ZNF300 and multi-drug resistant protein P-glycoprotein(P-gp) between HepG2 and HepG2/VCR were detected by Western blot.Then a transient transfection to HepG2/VCR cells was performed using plasmids containing ZNF300 sense or antisense cDNA.After plasmid transfection,MTT analysis was used to detect the effect of ZNF300 gene overexpression or knockdown on VCR-IC50 value alternation of HepG2/VCR,and Western blot analysis was used to detect the expression alternation of P-gp.Results MTT analysis showed the successful construction of HepG2/VCR.Western blot analysis showed that the expression of ZNF300 and P-gp was significantly elevated in HepG2/VCR cells,compared with HepG2 cells.After plasmid transient transfection,MTT and Western blot analysis showed that ZNF300 overexpression increased the VCR-IC50 value of HepG2/VCR cells,and promoted the expression of P-gp,whereas ZNF300 gene knockdown had an opposite effect.Conclusions ZNF300 gene expression is evaluated in hepatic cancer drug resistant cell line HepG2/VCR,and it can promote the drug resistance feature of HepG2/VCR through up-regulating the expression of P-gp.It is suggested that ZNF300 may be a molecular target of reversing the hepatic cancer MDR.