反义核酸对人大肠癌CCL229细胞侵袭力的抑制作用

THE INHIBITORY EFFECT OF asODNs ON THE INVASION OF COLORECTAL CANCER CELL LINE CCL229

为观察反义寡核苷酸(asODN)对培养的高侵袭性人大肠癌CCL229细胞侵袭性的抑制作用,针对尿激酶型纤溶酶原激活物受体(re-ceptor of uPAR)mRNA的翻译起始区,合成了一段反义寡核苷酸(asODN),用脂质体将其导入培养的CCL229细胞中,通过逆转录-聚合酶链反应(RT-PCR)、流式细胞术、羊膜侵袭试验测定uPAR mRNA水平、细胞表面uPAR抗原表达及细胞侵袭力的变化,并在扫描电镜下观察细胞的形态改变。结果为:(1)RT-PCR检测asODNs组uPAR/β-actin比值为0.44±0.02,与对照组(0.81±0.01)相比,显著降低(P<0.05);(2)流式细胞术检测asODNs组癌细胞表面与uPA结合的受体和总受体的平均荧光指数分别为0.20±0.07、0.59±0.09,与对照组(分别为0.72±0.12、2.21±0.36)比较,明显下降(P<0.01,P<0.05);(3)体外侵袭试验结果,asODN组在48h和72h后穿过羊膜的细胞数分别为12±2、20±3,与对照组(分别为25±4、44±5)相比,明显减少;(4)扫描电镜观察表明细胞经asODNs作用后,表面伪足和针状突起明显减少。以上各指标在随机序列寡核苷酸(rODN)组和对照组相比,无明显变化。CCL229细胞表面uPAR的表达与癌细胞的侵袭力密切相关,asODNs能有效抑制CCL229细胞uPAR基因的表达,从而抑制相关的蛋白质合成,降低其侵袭性,实验结果在癌症的基因治疗上将具有一定意义。

To study the inhibitory effect of antisense oligodeoxynucleotide ( asODNs ) on colrectal cancer cell line CCL229 invasion in vitro. A 15-mer asODNs targeted against the translation start site of UPAR (urokinase-type plasminogen activator receptor) mRNA were introduced into CCL229 cells by lipid-mediated DNA-transfection and the variation of the levels of uPAR mRNA, uPAR antigen expression of the levels of uPAR mRNA, uPAR antigen expression on the cell sr-uface and invasion properties were observed by reverse transcription-polymerase chain reaction (RT-PCR), flowcytometry(FCM) and aminion invasion assay, the morphological feature of the cell after asODNs treatment was observed by scanning electron microscope (SEM). The results indicate (1) the uPAR/p-actin ratio was 0. 44 ?0. 02 for the asODNs treated cells, which is significantly lower compared with the control and rONDs treated cells (0. 81±0. 01 and 0. 750±0. 13respectively,P<0. 01), (2) the mean fluorescence index of uPAR combined with uPA and the whole uPAR on surface were 0. 20±0. 07 and 0. 59±0.09 respectively for asODNs treated cells, which is significantly lower compared with control cells (0. 72±0. 12 and 2. 21±0. 36 respectively, P<0. 05,P<0. 01); (3) the number of cells migrated the aminion (25±4, 44±5 for the control cells) obviously decreased after a-sODNs treatment,(12±2,20±3,P<0. 05);(4) the filopodia and microspikes on the CCL229 cell surface were decreased after asODNs treatment. The conclusion is that the expression of uPAR on the surface of CCL229 cell surface is responsible for invasity; the inhibitory effect of uPAR as ODNs were highly significant and this method may be of potential clinical interest in gene therapy of cancer.