MRP3基因参与卵巢癌细胞拓扑替康耐药机制形成的研究

The study on the expression of MRP3 gene involved in Topotecan resistance in the human ovarian carcinoma cell line SKOV3/TPT.

目的:分析人卵巢癌SKOV3/TPT多药耐药的分子机制,寻找可能参与拓扑替康(TPT)耐药机制的新基因。方法:采用大剂量间歇诱导法,对SKOV3细胞株用TPT反复冲击诱导培养,获得稳定的SKOV3/TPT;利用Affymetrix U133A基因表达谱芯片,比较SKOV3及SKOV3/TPT细胞的基因表达;从中选择高表达的MRP3基因进行RT-PCR验证。再将MRP3反义及正义寡核苷酸片段分别用脂质体转染进耐药细胞,用MTT法、流式细胞仪及半定量RT-PCR检测体外转染后细胞内TPT的荧光强度、耐药指数及MRP3 mRNA表达的改变。结果:基因芯片检测发现,有7个差异表达基因可能参与了SKOV3/TPT细胞的耐药,其中4个基因被上调,3个基因被下调,MRP3为上调最明显的基因。MRP3反义寡核苷酸转染SKOV3/TPT细胞后,可明显降低细胞内TPT的浓度及耐药指数(P<0.05),细胞内MRP3 mRNA的表达较未转染细胞下降了60.16%(P<0.05)。结论:SKOV3/TPT细胞耐药表型的形成涉及多个基因表达的改变。MRP3的高表达导致细胞内药物浓度降低,可能是卵巢癌细胞对TPT耐药的主要原因。

Objective：To study the molecular mechanism underlying multidrug resistanee and identify unknown genes that might be involved in drug resistance development in SKOV3/TPT cells. Methods：The TPT -resistant human ovarian carcinoma cell subline SKOV3/TPT was established with SKOV3 exposed to high-level concentration of TPT intervally and repeatedly. The differential expression of associated genes in SKOV3/TPT compared with SKOV3 were determined by Affymetrix U133A Gene Chips. PCR were performed to tested the transcription level of MRF3 gene which was over expressed in SKOV3/TPT . The antisense-phosphorothioate oligonucleotide （ASODN） including the translation initiation site of MRP3 mRNA and the sense oligonucleotide （SODN）were transferred into SKOV3/TPT cells by Lipofect-2000 （LF）. The resistance index of topotecan, intracellular fluorescence intensity of TPT and MRP3 mRNA expression in SKOV3/TPT cells after transfected were detected respectively by methyl thiazolyl tetrazolium （MTT） assay, flow cytometry （ FCM ）, and semiquantitative RT-PCR. Results：Gene chip revealed that there were seven differential expressed genes in SKOV3/TPT cells. MRP3, MRP7, SCN11A and EBP were up-regulated, MRP4, FIJ12443 and SEMA3C were down-regulated. RT-PCR were used to confirm the results. After transfer of MRP3-ASODN/LF into SKOV3/TPT cells,the resistance index to topotecan was decreased from 10.67 to 3. 165.The intracellular TPT fluorescence intensity was obviously increased from 4.55 ± 0. 75 to 9.05 ± 1.13 （ P 〈 0.05 ）. The mRNA level of MRP3 was reduced to 60.16 % （ P 〈 0.05 ）. Conclusions ：There are some differentially expressed genes in SKOV3/TPT cells. The overexpression of MRP3 which mediated drug efflux may play an important role in the induction of TPT-resistance in ovarian cancer cell line.