含三方基序54调控胰腺癌增殖、侵袭和转移的机制

Tripartite motif containing 54 regulates the proliferation,invasion and metastasis of pancreatic cancer

目的探讨含三方基序54(TRIM54)在胰腺癌中的调控机制。方法胰腺癌细胞株(ASPC-1、BXPC-3、MIA-PaCa-2、PANC-1、SW1990)和正常胰腺上皮导管细胞(HPDE)株购自美国模式培养物集存库(ATCC)。采用实时荧光定量聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blot)检测TRIM54在人胰腺导管上皮细胞(HPDE)和胰腺导管腺癌(PDAC)细胞中的表达。通过转染质粒(shRNA)敲低TRIM54的表达。细胞计数试剂盒(CCK-8)检测细胞的增殖能力。Transwell和细胞划痕实验检测细胞侵袭迁移能力的变化。两组之间的比较采用独立样本t检验,多组样本之间的比较采用方差分析。结果通过RT-PCR检测,HPDE中TRIM54信使RNA(mRNA)表达水平明显低于胰腺癌细胞(ASPC-1、BXPC-3、MIA-PaCa-2、PANC-1、SW1990)(1.00±0.02比2.05±0.04比1.37±0.03比1.62±0.02比3.18±0.10比3.59±0.07,F=1042.04,P<0.05)。Western blot结果显示,HPDE中TRIM54蛋白表达水平明显低于胰腺癌细胞(ASPC-1、BXPC-3、MIA-PaCa-2、PANC-1、SW1990)(1.00±0.02比2.04±0.05比1.18±0.01比2.06±0.02比2.42±0.02比2.90±0.06,F=1360.92,P<0.05)。质粒成功敲低TRIM54的表达,实验结果表明敲低TRIM54的PANC-1和SW1990增殖、侵袭、迁移能力减低。Western blot实验表明敲低TRIM54后PANC-1细胞NC组N-钙黏蛋白(N-cadherin)高于sh-TRIM54组(1.00±0.03比0.58±0.03比0.74±0.02,F=233.26,P<0.05),SW1990细胞NC组N-cadherin高于sh-TRIM54组(0.95±0.02比0.66±0.02比0.77±0.02,F=127.64,P<0.05),PANC-1细胞NC组Vimentin高于sh-TRIM54组(1.00±0.03比0.71±0.02比0.79±0.03,F=130.09,P<0.05),SW1990细胞NC组Vimentin高于sh-TRIM54组(0.94±0.02比0.69±0.03比0.66±0.02,F=167.04,P<0.05)。而PANC-1细胞NC组E-cadherin低于sh-TRIM54组(1.00±0.02比1.08±0.04比1.20±0.03,F=34.21,P<0.05),SW1990细胞NC组E-cadherin低于sh-TRIM54组(0.95±0.03比1.36±0.03比1.28±0.03,F=218.35,P<0.05)。回复实验表明WNT激动剂增强了敲低TRIM54的PANC-1和SW1990增殖、侵袭、迁移能力。此外,添加WNT激动剂后PANC-1细胞中sh-TRIM54组N-cadherin和Vimentin低于sh-TRIM54+SKL2001组(N-cadherin:0.67±0.02比0.93±0.01,t=18.02,P<0.05;Vimentin:0.59±0.02比0.91±0.03,t=15.99,P<0.05),SW1990细胞中sh-TRIM54组N-cadherin和Vimentin低于sh-TRIM54+SKL2001组(N-cadherin:0.76±0.01比0.88±0.02,t=10.77,P<0.05;Vimentin:0.68±0.02比1.07±0.03,t=19.78,P<0.05)。而PANC-1细胞中sh-TRIM54组E-cadherin高于sh-TRIM54+SKL2001组(1.31±0.03比1.15±0.02,t=7.32,P<0.05),SW1990细胞中sh-TRIM54组E-cadherin高于sh-TRIM54+SKL2001组(1.20±0.06比1.01±0.06,t=4.20,P<0.05)。结论TRIM54在胰腺癌中高表达且通过调控WNT通路来促进胰腺癌细胞的增殖、侵袭和转移。

Objective To investigate the regulatory mechanism of tripartite motif containing 54(TRIM54)in pancreatic cancer.Methods Pancreatic cancer cell lines(ASPC-1,BXPC-3,MIA-PaCa-2,PANC-1,SW1990)and human pancreatic ductal epithelial(HPDE)cell lines were purchased from the American type culture collection(ATCC).Real-time quantitative polymerase chain reaction(RT-PCR)and Western blotting were used to detect the expression of TRIM54 in HPDE cells and pancreatic ductal adenocarcinoma(PDAC)cells.The expression of TRIM54 was knocked down by plasmid(shRNA)transfection.Cell counting kit-8(CCK-8)assay was used to detect the proliferation of cancer cells.Transwell and wound healing assays were utilized to observe the changes of cell invasion and migration.Independent sample t test was performed for comparison between two groups,while analysis of variance was used for comparison among multiple groups.Results The result of RT-PCR showed that the mRNA expression of TRIM54 in HPDE cells was significantly lower than that in pancreatic cancer cells(AsPC-1,BxPC-3,MIA PaCa-2,PANC-1,SW1990)(1.00±0.02 vs.2.05±0.04 vs.1.37±0.03 vs.1.62±0.02 vs.3.18±0.10 vs.3.59±0.07,F=1042.04,P<0.05).Similarly,the result of immunoblotting showed that the protein expression of TRIM54 in HPDE cells was significantly lower than that in pancreatic cancer cells(1.00±0.02 vs.2.04±0.05 vs.1.18±0.01 vs.2.06±0.02 vs.2.42±0.02 vs.2.90±0.06,F=1360.92,P<0.05).The abilities of proliferation,invasion and migration of PANC-1 and SW1990 cells were impaired after TRIM54 depletion by plasmids transfection.For PANC-1 cells,the N-cadherin level in NC group was higher than that in sh-TRIM54 group(1.00±0.03 vs.0.58±0.03 vs.0.74±0.02,F=233.26,P<0.05).N-cadherin protein level decreased in sh-TRIM54 group as compared with NC group in SW 1990 cells(0.95±0.02 vs.0.66±0.02 vs.0.77±0.02,F=127.64,P<0.05).Vimentin in NC group was higher than that in sh-TRIM54 group(1.00±0.03 vs.0.71±0.02 vs.0.79±0.03,F=130.09,P<0.05),while Vimentin in NC group of SW1990 cells was higher than that in sh-TRIM54 group(0.94±0.02 vs.0.69±0.03 vs.0.66±0.02,F=167.04,P<0.05).In PANC-1 cells,E-cadherin expression in NC group was lower than that in sh-TRIM54 group(1.00±0.02 vs.1.08±0.04 vs.1.20±0.03,F=34.21,P<0.05).The E-cadherin expression in NC group of SW1990 cells was lower than that in sh-TRIM54 group(0.95±0.03 vs.1.36±0.03 vs.1.28±0.03,F=218.35,P<0.05).The recovery experiment implied that WNT agonists enhanced the proliferation,invasion and migration of PANC-1 and SW1990 cells with TRIM54 knockdown.In addition,the expression of N-cadherin and Vimentin in sh-TRIM54 group was lower than that in sh-TRIM54+SKL2001 group(N-cadherin:0.67±0.02 vs.0.93±0.01,t=18.02,P<0.05;Vimentin:0.59±0.02 vs.0.91±0.03,t=15.99,P<0.05).Besides,the expression levels of N-cadherin and Vimentin in sh-TRIM54 group were lower than those in sh-TRIM54+SKL2001 group in SW1990 cells(N-cadherin:0.76±0.01 vs.0.88±0.02,t=10.77,P<0.05;Vimentin:0.68±0.02 vs.1.07±0.03,t=19.78,P<0.05).In PANC-1 cells,E-cadherin expression in sh-TRIM54 group was higher than that in sh-TRIM54+SKL2001 group(1.31±0.03 vs.1.15±0.02,t=7.32,P<0.05).The E-cadherin expression in sh-TRIM54 group was higher than that in sh-TRIM54+SKL2001 group in SW1990 cells(1.20±0.06 vs.1.01±0.06,t=4.20,P<0.05).Conclusion TRIM54 is highly expressed in pancreatic cancer and promotes the proliferation,invasion and migration of pancreatic cancer cells by regulating the WNT signaling pathway.