CRL4B复合物抑制分泌型卷曲相关蛋白1促进胰腺癌的发生发展

CRL4B complex promotes the development of pancreatic cancer by inhibiting secreted frizzled related protein 1

目的探讨CRL4B复合物在胰腺癌发生发展中的作用及其分子机制。方法将胰腺癌细胞分为对照组(转染阴性对照慢病毒)、shCUL4B组(转染CUL4B慢病毒)、shDDB1组[转染DNA损伤结合蛋白1(DDB1)慢病毒]和shCUL4B+siSFRP1组[转染CUL4B慢病毒+分泌型卷曲相关蛋白1(SFRP1)-siRNA]。对敲低CUL4B和DDB1的胰腺癌细胞系进行RNA测序(RNA-seq),寻找CRL4B复合物调控的靶基因,采用实时荧光定量聚合酶链反应(qRT-PCR)检测靶基因mRNA的表达,染色质免疫共沉淀(ChIP)-PCR实验鉴定CRL4B复合物直接调控的靶基因,Western blot法检测上皮-间充质转化(EMT)标志物蛋白的表达水平,EdU细胞增殖实验检测细胞增殖能力,划痕实验和Transwell实验检测细胞迁移和侵袭能力。采用GEO、TCGA和GTEx数据库中胰腺癌相关肿瘤样本和正常组织样本的测序数据,分析CUL4B、DDB1和其下游靶基因的表达相关性。结果RNA-seq结果显示,CRL4B复合物调控的靶基因涉及多种恶性肿瘤相关信号通路。qRT-PCR检测结果显示,shCUL4B和shDDB1组待验证靶基因的mRNA表达水平均高于对照组,差异有统计学意义(均P<0.05)。ChIP-PCR实验结果显示,CRL4B复合物直接结合在NME1和SFRP1靶基因启动子区,敲低CUL4B的表达后,靶基因启动子区域组蛋白H2A第119位赖氨酸单泛素化富集减少。对照组PANC-1细胞增殖率为(32.10±3.58)%,高于shCUL4B组和shCUL4B+siSFRP1组[分别为(13.95±1.66)%和(22.38±0.77)%,均P<0.05];对照组AsPC-1细胞增殖率为(35.47±7.80)%,高于shCUL4B组和shCUL4B+siSFRP1组[分别为(19.60±3.58)%和(30.09±0.81)%,均P<0.05]。细胞划痕实验显示,对照组PANC-1细胞迁移率为(53.18±3.70)%,高于shCUL4B组和shCUL4B+siSFRP1组[分别为(17.46±2.62)%和(44.99±9.18)%,均P<0.05]。Western blot结果显示,对照组细胞上皮标志物α-catenin和γ-catenin的表达分别为(1.00±0.03和1.01±0.11),低于shCUL4B组(分别为1.44±0.01和1.21±0.06,均P<0.05),对照组细胞间充质标志物Fibronectin和Vimentin表达水平分别为1.01±0.14和1.02±0.18,高于shCUL4B+siSFRP1组(分别为1.53±0.13和1.22±0.07,均P<0.05)。对照组细胞的迁移率为(100.00±3.96)%,与shCUL4B组和shCUL4B+siSFRP1组[分别为(35.49±0.34)%和(107.06±2.77)%]比较,差异均有统计学意义(均P<0.05)。CUL4B和DDB1在胰腺癌中表达升高,且与SFRP1的表达呈负相关(r分别为-0.342和-0.264)。结论胰腺癌中CRL4B复合物转录抑制靶基因SFRP1进而促进胰腺癌的发生发展,且CRL4B复合物在胰腺癌中的高表达支持了其作为胰腺癌治疗的潜在靶点。

Objective To investigate the role of CUL4B-RING E3 ubiquitin ligase(CRL4B)complex in pancreatic tumorigenesis and the molecular mechanism.Methods Pancreatic cells were divided into control group(transfected with negative control lentivirus),shCUL4B group(transfected with CUL4B lentivirus),shDDBl group[transfected with DNA damage binding protein 1(DDB1)lentivirus],and shCUL4B+siSFRPl group(transfected with CUL4B lentivirus and SFRPl-siRNA).RNA-seq was performed in pancreatic cancer cell lines with CUL4B and DDB1 knocked down respectively,to identify the target genes regulated by CRL4B complex.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of target genes.Chromatin immunoprecipitation(ChIP)assay was used to identify the target genes directly regulated by CUL4B and DDB1.Western blot was used to detect the protein expression levels of the epithelial-mesenchymal transition(EMT)markers.The EdU cell proliferation test was used to detect cell proliferation ability.The scratch repair test and Transwell cell invasion test were used to detect cell migration and invasion ability.Finally,the sequencing data of pancreatic cancer-related tumor samples and normal samples in GEO,TCGA and GTEx databases were used to analyze the expression correlations of CUL4B,DDB1 and their downstream target genes.Results RNA-seq results showed that target genes regulated by CRL4B complex involved in a number of malignant tumor-related signaling pathways.qRT-PCR results verified that the mRNA expression levels of the target genes of CUL4B or DDB1 knockdown groups were higher than those of the control group,and the difference was statistically significant(P<0.05).ChIP-PCR results showed that CRL4B complex directly bound to the promoter regions of the target genes,NME1 and SFRP1,and the enrichment of monoubiquitination of lysine at 119 of histone H2A(H2AK119ubl)in the promoter region of target gene was reduced after CUL4B knockdown.The proliferation rate in PANC-1 cell line of the control group was(32.10±3.58)%,higher than(13.95±1.66)%in the shCUL4B group and(22.38±0.77)%in the shCUL4B+siSFRPl group(P<0.05).The proliferation rate in AsPC-1 cell line of the control group was(35.47±7.80)%,higher than(19.60±3.58)%in the shCUL4B group and(30.09±0.81)%in the shCUL4B+siSFRP 1 group(P<0.05).The scratch repair experiment showed that the migration rate of PANC-1 cell line control group was(53.18±3.70)%,higher than that(17.46±2.62)%in the shCUL4B group and(44.99±9.18)%in the shCUL4B+siSFRP 1 group(P<0.05).Western blot showed the expression levels of epithelial markers including a-catenin and 7-catenin in the control group were 1.00±0.03 and 1.01±0.11,respectively,lower than 1.44±0.01 and 1.21±0.06 in the shCUL4B group(P<0.05).The expression levels of mesenchymal markers including fibronectin and vimentin in the control group were 1.01±0.14 and 1.02±0.18,respectively,higher than 1.53±0.13 and 1.22±0.07 in the shCUL4B+siSFRP 1 group(P<0.05).The cell metastasis rate of the control group was(100.00±3.96)%,higher than the(35.49±0.34)%in the shCUL4B group and(107.06±2.77)%in the shCUL4B+siSFRP 1 group,the difference was statistically significant(P<0.05).The expressions of CUL4B and DDB1 were significantly upregulated in the pancreatic cancer tissues,and were negatively correlated with the expression of SFRP1(r=-0.342 and r=-0.264,respectively).Conclusions CRL4B complex inhibits the transcription of target gene SFRP1 and promotes the development of pancreatic cancer.Moreover,CRL4B complex is upregulated in pancreatic cancer,which provide a potential of therapeutic target for pancreatic cancer.