IRE1α上调人骨肿瘤细胞XBP1启动子转录活性的研究  

Study on Upregulation of XBP1 Promoter Transcription Activity by IRE1α in Bone Neoplasm Cells

作　　者：李祥柱[1,2] 赵文君[1,2] 刘艳娜[1,2] 周菁华[1,2] 郭风劲[1,2] 

机构地区：[1]重庆医科大学细胞生物学及遗传学教研室,重庆市400016 [2]重庆医科大学发育生物学与模式动物平台,重庆市400016

出　　处：《医学分子生物学杂志》2012年第3期171-177,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81171697）,重庆市自然科学基金（No.2011jjA10047）.

摘　　要：目的研究内质网跨膜蛋白肌醇酶1α（inositol-requiring enzyme 1α，IRE1α）对其下游转录因子XBP1启动子转录活性的影响。方法在成功构建人IREla基因全长重组质粒的基础上，设计合成并构建靶向IRE1α基因的siRNA干扰载体（pS1；pS2）；采用巢式PCR扩增XBP1启动子，插入到pGL3．Basic载体，构建携带XBP1启动子的报告基因重组质粒pGL3-XBP1。分别将siIRE1α干扰质粒与pGL3-XBPl报告基因重组质粒共转染人SW1353细胞和Saos-2细胞中，48h后采用双荧光报告系统检测IRE1α对XBP1启动子转录活性的调控作用。结果酶切及测序结果证实siRNA1干扰质粒和XBPl启动子真核表达载体均构建成功，双荧光报告系统检测显示SW1353细胞和Saos-2细胞中，直接载体pcDNA3．15（-）IRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显高于其他实验组（P〈0．5），并随着IRE1α转染剂量的增加逐渐达到饱和；而silRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显降低。免疫印迹结果显示，过表达IRE1α明显上调剪接型XBPlS蛋白的表达。结论人恶性骨肿瘤细胞中，过表达IRE1α明显上调XBP1启动子的转录与表达，并能有效促进XBPIU剪切生成XBPIU；通过siRNA方法抑制IRE1α后，XBP1启动子的转录与表达受到抑制。本实验为进一步研究骨肿瘤细胞中IRE1α调控XBP1启动子转录与剪接的分子机制奠定基础，为临床骨肿瘤的诊断与治疗提供一定的实验依据。Objective To study the effect of transmembrane protein of the endoplasmic reticu- lum inositol enzyme lot （inositol-requiring enzyme lot, IRE1α） on transcriptional activity of its downstream transcription factor XBP1. Methods Based on a successfully built full-length human IRE1α recombinant, the siRNA interference sequences and plasmids targeting IRE1α （pS1 and pS2） were designed and constructed. The promoter recombinant of pGL3-XBP1 and its truncation were constructed by the nested PCR. After 48 h, these recombinants were co-transfected into SW1353 and Saos-2 cells, respectively. The transcriptional activity of XBP1 was detected by dual fluorescent reporter system. At the same time, the expression of XBP1U/XBP1S was identified by western blot. Results The siIRE1α interference plasmid and the XBP1 promoter recombinant were successfully constructed. In SW1353 and Saos-2 cells, luciferase activity of IRE1α eo-transfected with pGL3-XBP1 was significantly higher than that of control experimental groups （ P 〈 0. 5 ）, gradually saturating with the increase of transfeetion dose of IREl1α, whereas the luciferase activityof silRE1α co-transfected with pGL3-XBP1 were significantly reduced, And the western blot results showed that overexpression of IRE1α significantly increased the expression of XBP1S in SW1353 and Saos-2 cells. Conclusion In human malignant bone cells, overexpression of IRE1α significantly increases the transcription and expression of XBP1 promoter, and can contribute effectively to spli- cing XBP1 U. Suppression of IRE1α by siRNA can inhibit the transcription and expression of XBP1 promoter.

关 键 词：IRE1α XBP1启动子 转录活性 骨肿瘤 

分 类 号：Q75[生物学—分子生物学]