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目的 确认卵巢癌细胞ERCC1基因VIII外显子剪接变异的存在 ,探讨该剪接变异对ERCC1基因表达的作用及其对卵巢癌细胞对铂类药物耐药性的影响。方法 采用Northern杂交及RNA酶保护分析确认人卵巢癌细胞内ERCC1基因调控区在RNA水平存在剪接变异。分别构建野生型及缺失VIII外显子的ERCC1表达载体 ,转染中国仓鼠卵巢 4 3 3B细胞 ,采用Westernblot检测ERCC1表达水平研究该剪接变异对ERCC1基因表达水平的影响 ,采用MTT和细胞克隆形成实验研究该剪接变异细胞对铂类药物耐药性的影响。结果 卵巢癌细胞ERCC1基因VIII外显子存在剪接变异。Westernblot结果显示该剪接变异不显著影响ERCC1表达水平 ,但CHO 4 3 3B细胞MTT及细胞克隆形成实验表明 ,该剪接变异显著影响细胞对顺铂的耐药性。结论 本研究首次证明人卵巢癌细胞、组织ERCC1基因VIII外显子存在剪接变异 ,该变异不显著影响ERCC1基因的绝对表达水平 ,但影响细胞对顺铂的耐药性
Objective To confirm the existence of splicing mutations in exon VIII of ERCC1 gene in ovarian cancer cells and to investigate the effect of the splicing variant on the expression of ERCC1 gene and its effect on the drug resistance of ovarian cancer cells to platinum drugs. Methods Northern blotting and RNAse protection analysis confirmed the existence of splicing variants at the RNA level in ERCC1 gene regulatory region of human ovarian cancer cells. The ERCC1 expression vector of wild type and deletion of exon VIII was constructed and transfected into Chinese hamster ovary 4 3 3B cells respectively. The expression of ERCC1 was detected by Western blotting. The effect of this splicing variant on the expression of ERCC1 gene was investigated. MTT and cell clone formation assay The effect of the splicing variant on the drug resistance of platinum drugs was investigated. Results There was a splicing mutation in exon VIII of ERCC1 gene in ovarian cancer cells. Western blot analysis showed that the splicing variant did not significantly affect the expression of ERCC1, but the MTT assay and cell clone formation assay of CHO 4 3 3B cells showed that this splicing variant significantly affected the cell resistance to cisplatin. Conclusions This study, for the first time, shows that there is a splicing mutation in the exon VIII of ERCC1 gene in human ovarian cancer cells, which does not affect the absolute expression level of ERCC1 gene but affects the cell resistance to cisplatin