尿路上皮癌抗原1基因沉默可抑制人胰腺癌细胞的体外迁移和侵袭

Inhibitory effects of urothelial cancer associated 1 gene silencing on migration and invasion of human pancreatic cancer cells in vitro

目的 :检测尿路上皮癌抗原1(urothelial cancer associated 1,UCA1)在胰腺癌组织和细胞系中的表达情况,探讨靶向沉默UCA1基因表达对胰腺癌PANC-1细胞体外迁移和侵袭的影响及其可能的分子机制。方法 :应用实时荧光定量PCR法检测UCA1在胰腺癌组织及其癌旁组织和胰腺癌细胞系(PANC-1、SW1990、Bx PC-3和Capan-2)中的表达水平。分别构建插入针对UCA1基因的特异性sh RNA(UCA1-sh RNA)和阴性对照sh RNA(negative control-sh RNA,NC-sh RNA)的重组表达质粒,并将它们转染至胰腺癌PANC-1细胞中,随后采用实时荧光定量PCR法检测UCA1表达的变化。应用细胞划痕愈合实验和Transwell侵袭实验分别检测沉默UCA1基因表达对PANC-1细胞体外迁移和侵袭能力的影响。蛋白质印迹法检测UCA1基因沉默对上皮-间质转化相关分子钙黏蛋白(E-cadherin)和波形蛋白(vimentin)表达的影响。结果 :UCA1在胰腺癌组织中的表达水平明显高于癌旁组织(P<0.000 1),且UCA1在伴有转移的癌组织中表达水平高于无转移者(P=0.002)。4株胰腺癌细胞系中均能检测到UCA1 m RNA表达,其中PANC-1细胞中相对高表达(P<0.01)。UCA1-sh RNA转染PANC-1细胞后,UCA1的表达水平明显降低(P<0.01)。相较于NC-sh RNA转染组,UCA1-sh RNA转染后的PANC-1细胞体外迁移(P<0.05)和侵袭能力(P<0.01)均明显降低。UCA1-sh RNA转染组细胞中vimentin表达水平明显下调(P<0.01),而E-cadherin表达水平则明显上调(P<0.01)。结论 :UCA1在胰腺癌中高表达,通过sh RNA转染靶向沉默该基因表达可明显抑制胰腺癌细胞的体外迁移和侵袭,其机制可能与UCA1调控上皮-间质转化有关。

Objective： To determine the expression of urothelial cancer associated 1 （UCA1） in human pancreatic cancer tissues and cell lines, and investigate the effects of UCA1 gene silencing on migration and invasion of pancreatic cancer cell line PANC-1 in vitro and its possible molecular mechanism. Methods： The expression level of UCA1 in pancreatic cancer tissues and the corresponding para-carcinoma tissues as well as 4 types of human pancreatic cancer cell lines （PANC-1, SWl 990, BxPC-3 and Capan-2） was detected by real-time fluorescent quantitative-PCR. The shRNA expression vector targeting UCA1 gene （UCA1-shRNA） or negative control shRNA （NC- shRNA） was transfected into PANC-1 cells. Then the change of UCA1 expression level was detected by real-time fluorescent quantitative-PCR. The migration and invasion capacities of PANC-1 cells were detected by wound healing assay and Transwell invasion assay, respectively. The protein expressions of vimentin and E-cadherin, which were the markers of epithelial to mesenchymal transition （EMT）, were detected by Western blotting. Results： The expression level of UCA1 in pancreatic cancer tissues was significantly higher than that in their corresponding para-carcinoma tissues （P 〈 0.000 1）, and the expression level of UCA1 in pancreatic cancer tissues with metastasis was also higher than that in pancreatic cancer tissues without metastasis （P = 0.002）. The expression of UCA1 mRNA was detectable in all of four pancreatic cancer cell lines, and UCA1 expression level in PANC-1 cells was the highest among the four cell lines （P 〈 0.01）. The expression of UCA1 was decreased in PANC-1 cells after transfection with UCAI-shRNA （P 〈 0.01）. The abilities of migration （P 〈 0.05） and invasion （P 〈 0.01） of PANC-1 cells in UCAI-shRNA transfection group were obviously reduced as compared with those of NC-shRNA group. The expression level of vimentin protein was significantly down-regulated in UCAI-shRNA transfection group as compared with that in the NC-shRNA group （P 〈 0.01）, whereas the expression level of E-cadherin protein was up-regulated （P 〈 0.01）. Conclusion： UCA1 is highly expressed in pancreatic cancer tissues. The silencing of UCA1 gene expression by shRNA transfection can inhibit migration and invasion abilities of human pancreatic cancer PANC-1 cells in vitro. This effect may be associated with EMT regulated by UCA1.