多嘧啶束结合蛋白1在肾透明细胞癌增殖、侵袭中的多组学机制研究

Multi-omics of polypyrimidine tract binding protein 1 in clear cell renal cell carcinoma

目的采用多组学方法探究多嘧啶束结合蛋白1(PTBP1)在肾透明细胞癌中的作用以及机制。方法通过慢病毒载体导入CRISPR-Cas9方法构建PTBP1条件性敲除的人肾透明细胞癌细胞(786-O-PTBP1 KO),该细胞系购自中国典型培养物保藏中心。细胞计数试剂盒(CCK-8),迁移和侵袭实验(Transwell)评估PTBP1敲除对于肾透明细胞癌增殖、迁移和侵袭能力的影响。对照组786-O细胞(786-O-WT)和PTBP1条件性敲除786-O细胞(786-O-PTBP1 KO)分别进行全长转录组测序(RNA-Seq)和液相色谱-质谱联用分析(LC-MS/MS),检测肾透明细胞癌细胞在敲除PTBP1后出现的差异表达基因、转录本与蛋白;交联免疫共沉淀结合高通量测序(CLIP-Seq)检测剪切因子PTBP1在肾透明细胞癌中发挥作用的结合靶点RNA。通过Human Protein Atlas和Gene card数据库分析6个靶点的临床相关性。统计分析组间比较采用独立样本t检验。结果KO组迁移细胞计数明显低于WT组(116比182,t=14.73,P<0.01);KO组侵袭细胞计数低于WT组(199比179,t=4.34,P<0.01);KO组细胞增殖倍数在24、48、72、96时均低于WT组细胞(0.438比0.493,t=2.78,P<0.05;0.831比1.070,t=10.86,P<0.01;1.354比1.804,t=20.15,P<0.01;2.127比2.280,t=8.91,P<0.01),差异有统计学意义。1876个RNA-Seq差异基因与132个质谱分析差异基因的交集基因为78个,从中筛选出在352个CLIP-Seq结合峰中有显著结合的6个靶点基因RNA;临床数据库提示6个靶标中,N-乙基马来酰亚胺敏感因子(NSF)是PTBP1发挥促癌作用的目标靶点。结论PTBP1明显促进肾透明细胞癌的增殖、迁移和侵袭,其机制可能与下游靶点之一的NSF及可溶性NSF附着蛋白受体(SNARE)介导的囊泡运输与自噬有关。

ObjectiveTo investigate the role and mechanism of polypyrimidine tract binding protein 1(PTBP1)in clear cell renal cell carcinoma(ccRCC)by multi-omics methods.MethodsPTBP1 conditionally knockout in human ccRCC cells(786-O-PTBP1 KO)were constructed by introducing CRISPR-Cas9 with lentiviral vector.The cell line was purchased from China Center for Type Culture Collection.Cell counting kit-8(CCK-8)and Migration and Invasion assays(Transwell)were used to evaluate the effects of PTBP1 knockout on the proliferation,migration and invasion of ccRCC cells.The wild type of 786-O cells(786-O-WT)and the PTBP1 conditional knockout 786-O cells(786-O-PTBP1 KO)were analyzed by full-length transcription-sequencing(RNA-Seq)and LC-MS/MS,respectively.The differentially expressed genes,transcripts and proteins of ccRCC after PTBP1 knockout were detected by Cross-linked immunocoprecipitation-binding high-throughput sequencing(CLIP-Seq),which was applied to detect target RNAs which bind the shear factor PTBP1 to plays a role in ccRCC.The interaction of PTBP1 with the predicted binding targets was detected by gel electrophoresis mobility shift assay(RNA-EMSA).Independent-samples t-test was used for comparison between groups.Independent-samples T test was used for comparison between two groups.ResultsThe number of migrating cells in KO group was significantly less than that in WT group(116 vs.182,t=14.73,P<0.01).The number of invasive cells in KO group was less than that in WT group(199 vs.179,t=4.34,P<0.01),and cell proliferation in KO group at 24,48,72 and 96 h was significantly lower than that in WT group(0.831 vs.1.070,t=2.78,P<0.05;1.354 vs.1.804,t=10.86,P<0.01;2.127 vs.2.280,t=8.91,P<0.01).There were 78 different genes expressed by both RNA-Seq and MS analysis,and 6 target gene RNAs with significant binding in CLIP-Seq were screened out.Clinical data suggests that among the 6 targets,N-ethylmaleimide-sensitive factor(NSF)is the target where PTBP1 plays a role in promoting cancer.ConclusionPTBP1 significantly promotes the proliferation,migration and invasion of ccRCC,and its mechanism may be related to NSF and soluble NSF attachment protein receptor(SNARE)mediated vesicular transport and autophagy.