有丝分裂阻滞缺陷蛋白2选择性剪接体MAD2β在胃癌细胞多药耐药机制中的作用

目的观察有丝分裂阻滞缺陷蛋白2(mitotic arrest deficient protein 2, MAD2)基因的选择性剪接体MAD2β在人胃癌多药耐药性形成过程中的作用机制.方法从耐阿霉素人胃癌细胞系SGC7901/ADR中提取总RNA,通过RT-PCR获得MAD2β全长编码cDNA,并克隆至pUCm-T载体,正向亚克隆插入真核表达载体pcDNA3.1中.以脂质体介导pcDNA3.1/MAD2β真核表达载体转染胃腺癌细胞SGC7901.以体外药敏试验检测转染MAD2β的胃癌细胞及其对照组细胞对化疗药物的敏感性.通过流式细胞仪检测MAD2β基因转染组和对照组胃癌细胞在细胞周期中的分布和细胞内阿霉素的荧光强度.结果 RT-PCR扩增获得约0.53 kb片段,DNA序列测定证实为MAD2的一种选择性剪接形式,命名为MAD2β.重组正义真核表达载体pcDNA3.1/MAD2β瞬时转染SGC7901细胞,经MTT法证实,转染细胞SGC7901/MAD2β对化疗药物阿霉素、长春新碱和丝裂霉素的耐药性增强.与SGC7901/pcDNA3.1比较,阿霉素大剂量短期作用后,SGC7901/MAD2β细胞内的平均荧光强度比对照组低(P<0.05).结论 MAD2β基因在一定程度上增强了人亲本胃癌细胞SGC7901对化疗药物的耐受性,其可能的机制为肿瘤细胞在化疗药物作用下,有丝分裂出现异常,耐药细胞通过调控MAD2基因产生不同的选择性剪接形式, 降低MAD2的活性或改变其功能,以通过纺锤体检测点并继续存活.

Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell

OBJECTIVE: To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901. METHODS: RNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS. RESULTS: A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells. CONCLUSION: MAD2beta could increase the multidrug resistance of SGC7901 cell line.