体外RNA干扰抑制骨桥蛋白表达对肾癌细胞生长和侵袭的影响

Downregulation of osteopontin by RNA interference resulting in suppression of the proliferation and invasion of renal cancer cells in vitro

目的观察体外RNA干扰（RNAi）抑制骨桥蛋白（OPN）表达对人肾癌细胞生长和侵袭能力的影响，并探讨其机制。方法构建短发夹RNA（shRNA）重组质粒，转染肾癌ACHN细胞株，筛选获得稳定转染的细胞，逆转录-聚合酶链反应（RT—PCR）检测OPNmRNA表达水平；Westernblot检测OPN、基质金属蛋白酶（MMP）-2和MMP-9蛋白的表达；流式细胞术、噻唑蓝（MTF）比色法和Transwell实验分别检测细胞周期变化、细胞生长和和侵袭能力。结果与未转染的ACHN细胞（4．16±0．12）比较，转染重组质粒后的细胞（ACHN／OPN）中OPNmRNA的表达量（1．68±0．15）下降了59．6％（P〈0．05），而OPN、MMP-2和MMP-9蛋白的表达量（51．15±6．03、89．26±5．15、167．85±13．42）较转染前（214．01±12．08、187．53±9．56、233．12±15．78）分别下降了76．1％、52．4％和28．O％（P〈0．05），细胞分裂出现S期停滞，凋亡二倍体数量增加，细胞的生长、迁移和侵袭能力受到明显抑制。结论OPN在人肾癌细胞的生长和侵袭过程中发挥重要作用，这些过程与MMP-2和MMP-9的表达密切相关。

Objective To investigate the effects of osteopontin short hairpin RNA （OPN shRNA） on the proliferation and invasion of ACHN cellsin vitro and the mechanism. Methods OPN shRNA recombinant plasmids were constructed and transfected into ACHN cells. OPN mRNA expression level was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Protein expression levels of OPN, matrix metalloproteinases （MMP）-2 and MMP-9 were assayed by Western blotting. The cell cycle was assessed by flow cytometry. MTr method and Transwell chamber test were used to observe the abilities of proliferation and invasion of ACI-IN cells. Results Compared with the untreated ACHN cells （4. 16 ±0. 12）, OPN mRNA expression in the ceils transfected with recombinant plasmid （ 1.68 ± 0. 15） was decreased by 59.6% （P〈0.05）. Compared with the control group （214.01 ± 12.08, 187.53 ±9.56, 233. 12 ± 15.78）, the＇protein expression levels of OPN, MMP-2 and MMP-9 in the cells transfected with recombinant plasmid （51.15 ± 6. 03, 89. 26 ± 5.15, 167. 85 ± 13.42） were knocked down by 76. 1%, 52.4% and 28. 0% , respectively （P 〈 0. 05 ）. The cells were blocked in S phase and apoptotic hypodiploid was increased significantly. Recombinant plasmid significantly suppressed the proliferation and invasion of ALAN cells. Conclusion OPN plays an important role in the proliferation and invasion of human renal cancer ceils. These processes are correlated with the expression of MMP-2 and MMP-9.