多聚嘧啶区结合蛋白1通过上皮间质转化途径促进结直肠癌的转移与侵袭

Polyrimidine tract binding protein 1 promotes metastasis and invasion of colorectal cancer through epithelial-mesenchymal transition pathway

目的分析多聚嘧啶区结合蛋白1(PTBP1)在结直肠癌中的表达水平,并探讨其对结直肠癌细胞侵袭及上皮-间质转化(EMT)的影响。方法(1)下载TCGA数据库中结直肠癌转录组数据集,用Limma包分析结直肠癌组织(n=479)和正常组织(n=42)中PTBP1的表达及其与患者临床预后的相关性。(2)第1转染组和第2转染组为SW480细胞中分别转染对照慢病毒(ShCtrl)5μL和PTBP1敲低慢病毒(ShPTBP1组)5μL;第3转染组和第4转染组为LoVo细胞中分别转染ShCtrl和ShPTBP1各5μL;第5转染组和第6转染组为HCT116细胞中分别转染对照质粒(Vector)5μg和PTBP1过表达质粒(plasmid+PTBP1组)5μg;第7转染组和8转染组为HT29细胞中分别转染Vector和plasmid+PTBP1各5μg。以Transwell小室检测上述不同转染组细胞的侵袭能力,用蛋白质印迹法检测各组细胞中的PTBP1、E-cadherin、Vimentin和Snail蛋白表达水平。结果(1)PTBP1在结直肠癌组织和正常组织中的表达量分别为62.05±30.21和39.38±10.06,2组间比较差异有统计学意义(P<0.05),并与临床不良预后有关。(2)第1至第8转染组的侵袭细胞百分比分别为(100.00±6.28)%,(45.77±1.68)%,(100.00±7.27)%,(41.05±5.68)%,(100.00±6.55)%,(157.65±4.24)%,(100.00±9.80)%和(189.51±4.66)%;这8组的PTBP1蛋白表达水平分别为1.22±0.10,0.80±0.09,0.93±0.04,0.69±0.05,0.96±0.04,1.11±0.01,0.92±0.04和1.17±0.04;这8组的E-cadherin蛋白表达水平分别为0.36±0.05,0.87±0.12,0.64±0.00,1.00±0.06,0.60±0.02,0.49±0.02,0.53±0.02和0.30±0.01;这8组的Vimentin蛋白表达水平分别为0.82±0.02,0.65±0.05,0.72±0.04,0.54±0.04,0.46±0.06,0.74±0.04,0.45±0.06和0.74±0.05;这8组的Snail蛋白表达水平分别为0.96±0.04,0.74±0.05,0.72±0.04,0.57±0.01,0.55±0.01,0.82±0.09,0.58±0.02和0.90±0.09。以上指标:第2转染组与第1转染组比较,或第4转染组与第3转染组比较,或第6转染组与第5转染组比较,或第8转染组与第7转染组比较,差异均有统计学意义(均P<0.05)。PTBP1表达与Vimentin和Snail呈正相关,而与E-cadherin呈负相关。结论PTBP1在结直肠癌组织中异常高表达,且与癌细胞的侵袭能力呈正相关,其机制可能与诱导EMT途径有关。

Objective To analyze the expression of polyrimidine tract binding protein 1(PTBP1)in colorectal cancer(CRC)and to explore the effect of PTBP1 on cell invasion and epithelial-mesenchymal transformation(EMT).Methods(1)The data set of CRC transcriptome were downloaded from the cancer genome atlas(TCGA)database.PTBP1 expression between CRC tissues(n=479)and adjacent normal tissues(n=42)were analyzed by limma method,the correlations between PTBP1 and TNM stage and prognosis were observed too.(2)Transfection-1 and-2 groups were transfected with control lentivirus(Shctrl)5μL or PTBP1-knockdown lentivirus(Sh PTBP1)5μL in SW480 cells.Transfection-3 and-4 groups were transfected with Shctrl 5μL or Sh PTBP15μL in Lo Vo cells.Transfection-5 and-6 groups were transfected with control plasmid(Vector)5μg or PTBP1-overexpression plasmid(plasmid+PTBP1)5μg in HCT116 cells.Transfection-7 and-8 groups were transfected with Vector 5μg or plasmid+PTBP15μg in HT29 cells.The ability of cell invasion was evaluated by Transwell assay.The expressions of PTBP1,E-cadherin,Vimentin and Snail were detected by Western blot in the above transfected cells,respectively.Results(1)The PTBP1 expression level in CRC tissues and adjacent normal tissues were 62.05±30.21 vs 9.38±10.06,respectively,with significant difference between the two groups(P<0.05).PTBP1 expression was positively correlated with poor prognosis of CRC patients.(2)The percentage of invasive cells in transfection 1-8 groups were(100.00±6.28)%,(45.77±1.68)%,(100.00±7.27)%,(41.05±5.68)%,(100.00±6.55)%,(157.65±4.24)%,(100.00±9.80)%,(189.51±4.66)%,respectively;the expression of PTBP1 protein levels in the 8 groups were 1.22±0.10,0.80±0.09,0.93±0.04,0.69±0.05,0.96±0.04,1.11±0.01,0.92±0.04,1.17±0.04,respectively;the expression of E-cadherin protein levels in the 8 groups were 0.36±0.05,0.87±0.12,0.64±0.00,1.00±0.06,0.60±0.02,0.49±0.02,0.53±0.02,0.30±0.01,respectively;the expression of Vimentin protein levels in the 8 groups were 0.82±0.02,0.65±0.05,0.72±0.04,0.54±0.04,0.46±0.06,0.74±0.04,0.45±0.06,0.74±0.05,respectively;the expression of Snail protein levels in the 8 groups were 0.96±0.04,0.74±0.05,0.72±0.04,0.57±0.01,0.55±0.01,0.82±0.09,0.58±0.02,0.90±0.09.Compared between transfection-2 and transfection-1 groups,or transfection-4 and transfection-3groups,or transfection-6 and transfection-5 groups,transfection-8 and transfection-7 groups,the difference of the factors were significant(all P<0.05).The expression of Vimentin and Snail were decreased following the downregulation of PTBP1;while E-cadherin expression was upregulated.Conclusion PTBP1 was abnormally upregulated in CRC tissues.PTBP1 promotes cell invasion through EMT process in CRC cells.