Knock-down of sorcin induces up-regulation of MDR1 in HeLa cells
Knock-down of sorcin induces up-regulation of MDR1 in HeLa cells
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DOI：
10.1248/bpb.30.1065
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发表时间：
2007-06-01
影响因子：
2
通讯作者：
Sakaeda, Toshiyuki
中科院分区：
文献类型：
--
作者：
Kawakami, Megumi;Nakamura, Tsutomu;Sakaeda, Toshiyuki
在我们之前的研究中，通过将mdr1的典型底物硫酸长春花碱逐步作用于人宫颈癌细胞系HeLa-Ohio(HeLa)，建立了mdr1/P糖蛋白高表达的多药耐药亚系Hvr100-6。对它们的基因和蛋白质表达谱进行了分析，通过基因芯片分析鉴定出148个差异表达基因。蛋白质组学分析证实，在Hvr100-6细胞中，22 kDa的可溶性抗性相关钙结合蛋白--山梨素表达上调。为了阐明NDR1和Sorcin之间的关系，用针对其mRNAs的小干扰RNA(SiRNAs)处理HeLa细胞。治疗后第1天和第2天，mdr1基因的siRNA表达水平分别下降86%和61%，而sorcin基因的表达水平无明显变化。反之，在治疗后1-3天，核糖核糖核酸抑制其表达达80-90%。有趣的是，抑制山梨素诱导NDR1mRNA的表达水平增加了3倍以上。以罗丹明123为探针评价NDR1外排功能的结果显示，与干扰siRNA处理的HeLa细胞相比，山梨素的siRNA处理的HeLa细胞有增加NDR1外排功能的趋势。山梨素基因敲除的HeLa细胞中caspase-3的活性和表达水平均高于干扰siRNA处理的细胞。因此，我们证明山梨素可能是mdr1表达的部分抑制因子。此外，本研究表明，索尔辛通过caspase-3功能障碍抑制细胞凋亡。
In our previous study, the MDR1/Pglycoprotein-overexpressing multidrug resistant subline, Hvr100-6, was established from the human cervical carcinoma cell line HeLa-Ohio (HeLa) by stepwise exposure to an anti-microtubule agent, vinblastine sulfate, a typical substrate of MDR1. Their gene and protein expression profiles were analyzed herein, and 148 genes were identified to be differentially expressed by cDNA microarray analysis. The up-regulation of sorcin, a soluble resistance-related calcium-binding protein of 22 kDa, was confirmed in Hvr100-6 cells by the proteome analysis. To clarify the relationship between NDR1 and sorcin, HeLa cells were treated with small interfering RNAs (siRNAs) targeted for theirs mRNAs. The siRNA for MDR1 mRNA resulted in its decrease by 86% and 61% on the days 1 and 2 after the treatment, whereas the expression level of sorcin mRNA was not changed. On the other hand, the siRNA for sorcin mRNA suppressed its expression by 80-90% on days 1-3 after the treatment. Interestingly; suppression of sorcin induced a more than 3-fold increase in the expression level for NDR1 mRNA. An efflux function of NDR1 evaluated with using rhodamine 123 as a probe showed a tendency to be increased in HeLa cells treated with siRNA for sorcin, compared with that in the cells treated with scramble siRNA. The activity and the expression of caspase-3 in the sorcin knock-down HeLa cells were relatively higher than those in the cells treated with scramble siRNA. Thus, we demonstrated that sorcin might be a partial suppressor of MDR1 expression. Furthermore, the present study suggested that sorcin repressed apoptosis via dysfunction of caspase-3.