CRISPR/Cas9系统靶向敲除多溴蛋白1基因对肝癌细胞功能的影响

Effect of PBRM1 gene knock-out via CRISPR/Cas9 on hepatocellular carcinoma cell

目的探讨应用规律成簇的间隔短回文重复(CRISPR)/CRISPR相关蛋白9(Cas9)系统靶向敲除多溴蛋白1(PBRM1)基因对肝癌细胞功能的影响。方法应用CRISPR/Cas9系统靶向敲除Huh7细胞的PBRM1基因。采用Sanger测序和蛋白质印迹法(Western blot)检测基因敲除情况。基于转录本测序(RNA-seq)结果,对Huh7敲除(KO)细胞株与Huh7野生(WT)细胞系差异表达基因进行功能富集。采用流式细胞术分析细胞周期的改变。通过CCK-8法和克隆形成实验研究细胞增殖情况。采用不同浓度的γ-干扰素(IFN-γ)处理Huh7-KO细胞株和Huh7-WT细胞系,验证GO富集中IFN-γ应答通路的改变。结果成功构建PBRM1基因敲除的Huh7细胞株。Huh7-KO细胞株的生长曲线和克隆形成率均显著慢于Huh7-WT细胞系(P<0.0001)。Huh7-KO细胞株中下调基因主要与细胞周期相关,上调基因主要与免疫应答相关。Huh7-KO细胞株处于G1期的细胞比例明显高于Huh7-WT细胞系(P=0.0166),而处于S期的细胞比例明显低于Huh7-WT细胞系(P=0.0002)。浓度为1000 U/ml的IFN-γ对Huh7-KO细胞株组的细胞抑制作用明显高于Huh7-WT细胞系组(P=0.0091)。结论在Huh7肝癌细胞株中敲除PBRM1可以导致G 1/S阻滞从而抑制细胞增殖。高浓度IFN-γ可以更加有效地杀伤PBRM1缺失的肿瘤细胞。

Objective To explore the effect of polybromo1(PBRM1)gene knock-out by clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR associated protein 9(Cas9)system on the function of hepatocellularcarcinoma cell.Methods We used CRISPR/Cas9 technique to knock out PBRM1 gene in Huh7 cell line.Sanger sequencing and Western blotting were performed to verify the knockout of the target region.Based on the results of RNA-seq,the function of the differentially expressed gene between Huh7 knock-out(KO)cells and Huh7 wild-type(WT)cell line were enriched.The change of cell cycle was detected by flow cytometry.The cell proliferation was explored by CCK-8 assay and clone forming test.In addition,Huh7-KO cells and Huh7-WT cell line were treated with different concentrations of Interferon-γ(IFN-γ)to verify the change of IFN-γ response pathway.Results PBRM1 gene knock-out cell line was successfully constructed.The growth curve and colony forming efficiency of Huh7-KO cells significantly slower than those of Huh7-WT cell line(P<0.0001).The down-regulated genes in Huh7-KO cells were mainly related to cell cycle.And the up-regulated genes were involved in immune response.The proportion of Huh7-KO cells in G1 phase was higher than that in Huh7-WT cells(P=0.0166),while the proportion of Huh7-KO cells in S phase was significantly less(P=0.0002).The inhibition effect of 1000 U/ml IFN-γ on Huh7-KO cells group was significantly higher than that of Huh7-WT cell line group(P=0.0091).Conclusion PBRM1 knockout in Huh7 hepatocellular carcinoma cell line inhibits cell proliferation through G 1/S phase arrest.PBRM1 deficient tumor cells are killed by high concentration of IFN-γmore effectively.