shRNA抑制CD147基因表达对HT29细胞增殖、侵袭和致瘤能力的影响

Effect of the expression inhibition of CD147 by shRNA on the proliferation,invasion and tumorigenicity of HT29 cells

目的用RNA干扰(RNAi)技术抑制CD147基因的表达,检测其对人结直肠癌细胞系HT29细胞增殖、侵袭和致瘤能力的影响。方法设计合成CD147特异性的RNA干扰表达质粒pYr-mir30-shRNA,稳定转染HT29细胞,分别获得HT29/shRNA-control和HT29/shRNA细胞;RT-PCR和western blot分别检测CD147、MCT1、MCT4 mRNA和蛋白质的表达;明胶酶谱法检测MMP-2和MMP-9的活性;CCK8法分析细胞的增殖能力;Transwell小室检测细胞的侵袭能力;观察结直肠癌裸鼠皮下移植瘤的致瘤能力。结果与空白组比较,RNA干扰后的细胞中CD147、MCT1 mRNA(F分别为99.645和84.985)及蛋白质(F分别为73.675和19.842)的表达水平均降低(P均<0.01);MMP-2和MMP-9的活性均降低(P均<0.01);细胞增殖(t分别为7.491、15.023、14.584、6.637和11.211,P均<0.01)和侵袭(F=330.443,P<0.01)能力均降低,使裸鼠荷瘤能力下降(F=365.679,P<0.01)。结论 RNA干扰能有效抑制CD147基因的表达,抑制HT29细胞的增殖和侵袭能力。

Objective：To investigate the effect of the expression inhibition of CD147 gene by RNA interference on the proliferation, invasion and tumorigenicity of HT29 cells. Methods：A specific short-hairpin RNA （shRNA） expression vector pYr-mir30-shRNA was constructed and transferred into the HT29 cells. Then, the mRNA and protein expression levels of CD147, MCT1 and MCT4 were detected by real-time PCR and western blot, respectively. The activity of MMP-2 and MMP-9 was determined by gelatinase assay. The proliferation and invasion of HT29 cells were analyzed by CCK8 assay and Transwell test, respectively. In addition, the tumorigenicity of HT29 cells was observed by injecting them into nude mice. Results：Compared with the blank group, the CD147 and MCT1 mRNA （F=99.645 and 84.985, respectively, P〈0.01） and protein levels （F=73.675 and 19.842, respectively, P〈0.01） in shRNA interfering cells were significantly reduced. Meanwhile, the MMP-2 and MMP-9 activities and the proliferation, invasion and tumorigenicity of HT29 cells transferred by shRNA were also significantly decreased （P〈0.01）. Conclusion：RNA interference can effectively inhibit the expression of CD147, and the proliferation and invasion of HT29 cells.