反义SKP2对胆管癌细胞系QBC中p27^(kip1)表达的影响

The effects of SKP2 antisense oligodeoxynucleotides on the expression of p27^(kip1)in cholangiocarcinoma QBC cell fine

目的通过 S 期激酶相关蛋白(SKP2)反义寡核苷酸(ASODN)阻断泛素-蛋白酶体降解途径,观察对胆管癌细胞 QBC 中 SKP2和 p27^(kip1)表达的影响,探讨泛素-蛋白酶体途径在胆管癌发生发展中的作用。方法 SKP2反义寡核苷酸和胆管癌细胞 QBC 共培养,脂质体转染,逆转录-聚合酶链反应(RT-PCR),Western blot 法检测 SKP2和 p27^(kip1) mRNA 和蛋白在 QBC 中的表达。结果 SKP2 ASODN 作用于 QBC 后,SKP2在 mRNA 和蛋白的表达较对照组和 NSODN 组均显著降低(P<0.05),p27^(kip1)mRNA 表达差异无统计学意义(P>0.05),蛋白表达则较对照组和 NSODN 组明显增高(P<0.05)。结论 SKP2介导的泛素-蛋白酶体途径在调节胆管癌细胞 QBC 中 p27^(kip1)的降解中发挥重要作用,SKP2反义寡核苷酸在显著抑制 QBC 中 SKP2 mRNA 和蛋白的表达的同时,促进 p27^(kip1)蛋白表达,但对 p27^(kip1) mRNA 无明显影响,提示 SKP2对 p27^(kip1)的调节主要在转录后水平。SKP2作为胆管癌基因治疗中有效的潜在靶基因,在胆管癌的基因治疗方面具有重要意义。

Objective To investigate the effect of SKP2 antisense oligodeoxynueleotide （ASODN） on the expression of p27^kip1 and SKP2 gene in QBC cells and its probable mechanism. Methods QBC ceils were co-cultured With SKP2 ASODN. The expression levels of SKP2 mRNA and protein were determined by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. Results （ 1 ） After treatment with SKP2 ASODN, the expression levels of SKP2 mRNA and protein were down-regulated. The expression level of SKP2 mRNA and protein in ASODN treatment group was lower than in control groups （P 〈 0.05 ）. （2） Although p27^kip1 mRNA remained unchanged, its protein level was up-regulated as compared with control groups （ P 〈 0.05 ）. Conclusion SKP2 mediated the ubiquit- in-proteosome pathway plays a critical role in degrading eyelin-dependent kinase inhibitor p27^kpi1 in QBC cells. SKP2 ASODN can inhibit the expression of SKP2 mRNA and protein,but increase the level of p27^kiv1 protein, suggesting SKP2 as target gene seems to offer a potential basis for the development of new cancer gene therapy modality and a useful tool