基质交联分子1对前列腺癌DU145细胞迁移侵袭及上皮-间充质转化的影响

Effects of stromal interaction molecular 1 gene on cell migration and invasion and epithelial-mesen-chymal transition in prostate cancer DU145 cells

目的观察基质交联分子1（STIM1）对前列腺癌DU145细胞迁移、侵袭的影响并探讨其在上皮-间充质转化（EMT）过程中的作用。方法将携带STIM1基因的小干扰RNA（siRNA）慢病毒载体STIM1-pGCSIL-GFP转染人激素非依赖性前列腺癌DU145细胞（KD组），同时设阴性对照组（NC组）及空白对照组（CON组）。逆转录-聚合酶链反应（RT-PCR）及Westernblot验证抑制STIM1表达的有效性。采用划痕实验及Transwell迁移、侵袭实验检测抑制STIM1对肿瘤细胞运动及迁移、侵袭能力的影响。采用半定量RT-PCR及Westernblot分别在mRNA及蛋白水平检测EMT标志物E-钙黏蛋白（E-cadherin）、波动蛋白（Vimentin）、α-平滑肌肌动蛋白（α-SMA）在各组细胞中的差异。结果与对照组比较，转染STIM1-pGCSIL—GFP后，DU145细胞内STIM1的mRNA及蛋白水平均明显降低。划痕实验显示KD组DU145细胞的运动能力明显降低。细胞迁移结果显示，CON、NC、KD组每个视野细胞数分别为（92．0±14．2）、（87．3±2．3）、（60．8±10．3）个，KD组数目明显减少（P〈0．05）；细胞侵袭实验提示，CON、NC、KD组每视野细胞数分别为（79．6±3．7）、（80．0±3．5）、（45．8±5．8）个，KD组侵袭数目明显减少（P〈0．05）。RT-PCR和Westernblot结果显示，KD组上皮标志物E—cadherin mRNA及蛋白表达水平增高（分别为NC组的1．38、1．72倍）；间质标志物Vimentin的mRNA及蛋白表达水平降低（分别为NC组的0．67、0．74倍），α-SMA mRNA及蛋白表达水平降低（分别为NC组的0．23、0．54倍，P〈0．05）。结论抑制前列腺癌DU145细胞中STIM1表达后，细胞运动及迁移、侵袭能力均明显降低，这可能与逆转EMT相关。

Objective To observe the effect of stormal interaction molecule 1 （STIM1） on cell migration and invasion in prostate cancer DUI45 cells, and explore whether STIM1 contribute to reverse the epithelial-mesenchymal transition （EMT） transformation. Methods DU145 cells were transfected with lentiviral vector STIMI-pGCSIL-GFP （KD group） and related negative vector （NC group）, normal DU145 cells without any interference were used as control （CON group）. After transfection, the expression of STIM1 in DU145 cells was detected by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. The cell mobility, migration and invasion ability of DU145 ceils were respectively measured by wound-healing assay and transwell migration and invasion assays. The expression of EMT markers [ E-eadherin, Vimentin, a-smooth muscle actin （α-SMA）] were detected by semi-quantitative RT-PCR and Western blotting analysis. Results After transfected with STIMI-pGCSIL-GFP, STIM1 expression in DU145 cells was decreased at both mRNA and protein levels （ P 〈 0. 05 ） , and the cell mobility was decreased. The number of migrated cells in group CON, NC and KD were 92. 0 ± 14. 2, 87. 3 ± 2. 3, 60. 8 ± 10. 3, respectively. The number of invaded cells in group CON, NC and KD were 79.6 ± 3.7, 80. 0 ± 3.5, 45.8 ± 5.8, respectively. These indicated that cell migration and invasion ability of transfected cells were decreased （P 〈 0. 05 ）. Epithelial marker E-cadherin increased and mesenchymal markers Vimentin, α-SMA decreased in transfected cells that detected by both RT-PCR and Western blotting analysis （P 〈 0. 05）. Conclusion STIM1 inhibition could decrease the cell mobility, cell migration and invasion. This phenomenon may due to STIM1 could reverse the EMT transformation in DU145 cells.