Multi-omics of polypyrimidine tract binding protein 1 in clear cell renal cell carcinoma
Objective To investigate the role and mechanism of polypyrimidine tract binding pro-tein 1(PTBP1)in clear cell renal cell carcinoma(ccRCC)by multi-omics methods.Methods PTBP1 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 Col-lection.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 ana-lyzed 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 pre-dicted 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 compari-son between two groups.Results The 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.Conclusion PTBP1 significantly pro-motes 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.
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