Microarray analysis of gene expression profile in HCT116 colon cancer cells expressed the isoform A or isoform B of the Tazarotene-induced gene 1.

Tazarotene-induced gene 1 (TIG1), also named as retinoic acid receptor responder 1 (RARRES1), is a retinoid inducible type II tumor suppressor gene; the TIG1B isoform inhibits growth and invasion of cancer cells. Expression of TIG1B is frequently downregulated in various cancer tissues; however, the expression and activities of the TIG1A isoform has yet to be analyzed. This study investigated the effects of TIG1A and TIG1B isoforms on gene expression profiles of colon cancer cells. TIG1A, TIG1B and control stable clones derived from HCT116 colon cells were established using the GeneSwitch system. TIG1 isoform expression was induced upon 5 micro Molar of mifepristone (MFP) treatment for 24 hr. Biological triplicate samples were prepared and gene expression profiles were determined by microarray using human genome HGU133 plus 2 array (Affymatrix). Upon induction of TIG1A and TIG1B expression for 24 hr, a total of 129 and 55 genes were significantly altered, respectively. Of the genes analyzed, 23 and 6 genes were up- and downregulated, respectively in both TIG1A and TIG1B expressing cells. TIG1A, TIG1B and control stable clones derived from HCT116 colon cells were established using the GeneSwitch system. Inducible stable clones were plated in 15-cm dishes overnight in maintaining medium free of hygromycin and zeocin. Cells were refreshed with medium containing 5 micro molar of mifepristone and then incubated overnight. Cells were washed with ice-cold PBS and then harvested. Total RNA was prepared. Biological triplicate samples were prepared for control, TIGA and TIG1B stable cells. Samples of cRNA were prepared, labeled with biotin, and then hybridized to Human Genome U133 Plus 2.0 Array.

他扎罗汀诱导基因1（Tazarotene-induced gene 1, TIG1）又称视黄酸受体应答蛋白1（retinoic acid receptor responder 1, RARRES1），属于视黄酸诱导型II类肿瘤抑制基因；其中TIG1B亚型可抑制癌细胞的生长与侵袭。TIG1B的表达在多种癌组织中常出现下调，但TIG1A亚型的表达与活性尚未得到分析。

本研究探究了TIG1A与TIG1B亚型对结肠癌细胞基因表达谱的影响。采用GeneSwitch系统构建了源自HCT116结肠癌细胞的TIG1A、TIG1B及对照稳定克隆株。以5μM米非司酮（mifepristone, MFP）处理24小时，即可诱导TIG1亚型的表达。设置3份生物学重复样本，采用人类基因组HGU133 Plus 2.0基因芯片（Human Genome U133 Plus 2.0 Array, Affymetrix）检测基因表达谱。在诱导TIG1A与TIG1B表达24小时后，分别有总计129个和55个基因发生显著表达变化。在所有分析的基因中，TIG1A与TIG1B表达细胞中各有23个基因上调、6个基因下调。

研究人员再次构建了源自HCT116结肠癌细胞的TIG1A、TIG1B及对照稳定克隆株（采用GeneSwitch系统）：将诱导型稳定克隆接种于15cm培养皿中，于不含潮霉素（hygromycin）与Zeocin的维持培养基中过夜培养；更换含5μM米非司酮的培养基后继续孵育过夜。用冰预冷的磷酸盐缓冲液（PBS）洗涤细胞后收集样本，提取总RNA。为对照组、TIG1A组及TIG1B组稳定细胞株各制备3份生物学重复样本，制备互补RNA（cRNA）并以生物素标记，随后与人类基因组HGU133 Plus 2.0基因芯片进行杂交。