MMP-2特异性RNA干扰对人胰腺癌细胞粘附和侵袭的作用

Role of RNA interference targeting MMP-2 on pancreatic carcinoma cell invasion and adhesion

目的探讨靶向基质金属蛋白酶-2(MMP-2)的RNA干扰对人胰腺癌细胞粘附和侵袭的作用。方法设计和构建靶向MMP-2的特异性小干扰RNA(siRNA)表达质粒,将质粒转染入人胰腺癌BxPC-3细胞系中,根据转染质粒靶序列的不同分为5组,即pGPU6-1、pGPU6-2、pGPU6-3、pGPU6-4和阴性对照组pGPU6(-),并设仅加入转染剂的空白对照组。应用RT-PCR和Westernblot法检测各组BxPC-3细胞转染后MMP-2蛋白及mRNA表达水平,应用MTT法及流式细胞术检测各组细胞增殖和凋亡水平,以平板粘附模型和Transwell小室模型检测各组转染后肿瘤细胞粘附和侵袭能力,比较各组间差异。结果与空白对照组和阴性对照组相比,pGPU6-1、pGPU6-2、pGPU6-3组MMP-2mRNA抑制率分别为75.3%、64.5%、51.6%和74.7%、63.7%、50.5%,差异均有统计学意义;MMP-2蛋白表达抑制率分别为79.1%、64%、52.3%和78%、62.2%、50.5%,差异均有统计学意义;粘附抑制率分别为63.1%、42.9%、25.6%和62.2%、41.6%、23.9%,且穿膜细胞数量明显降低,差异均有统计学意义。对照组与pGPU6-4组的MMP-2蛋白及mRNA表达水平无明显差异,在肿瘤细胞粘附和侵袭能力方面也未见明显差异。MTT法及流式细胞术的结果并没有提示各组间肿瘤细胞增殖和凋亡水平的差异。结论靶向MMP-2的RNA干扰能够明显抑制胰腺癌BxPC-3细胞蛋白水平及mRNA水平的表达,从而抑制肿瘤细胞粘附和侵袭的能力,但并没有导致干扰后肿瘤细胞增殖和凋亡能力的改变。

Objective To investigate effects of RNA interference（RNAi）on expression of matrix metalloproteinase-2（MMP-2）gene,invasion and adhesion of human pancreatic cancer cells line,BxPC-3.Methods RNA interference was performed by using the vector（pGPU6）-based small interference RNA（siRNA）plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cells line,BxPC-3.Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experiment siRNA plasmids as pGPU6-1,pGPU6-2,pGPU6-3,pGPU6-4 and one negative control siRNA plasmid as pGPU6（-）.MMP-2 expression was measured by reverse transcription polymerase chain reaction（RT-PCR） and Western blot.Cell proliferation and apoptosis were examined by MTT and flow cytometry,respectively.The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.Results The expression of MMP-2 was inhibited and the inhibitory effects of different sequence were varied.pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasion and adhesion were significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 group compared to pGPU6（-） and blank control group.However,no differences concerning cell proliferation and apoptosis were observed after transfection between experiment groups and control groups.Conclusion RNA interference against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cells line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis.