Alterations in total and in polysomal mRNA fraction in response to expression of RNA binding motif protein 35A

The gene coding for RNA binding motif protein 35A (RBM35A) is inactivated by frameshift mutations in an LS180 colon carcinoma cell line and in approximately in 50% of colon tumors with microsatellite instability. To get insight into the mechanism of action of these putative tumor suppressor gene we expressed functional copy of the RBM35A cDNA in the LS180 cells. We analyzed alterations in mRNA profiles in total and in polysomal fraction of mRNA in LS180 cells in response to expressing RBM35A gene under Tet off tetracycline inducible promoter. Tet-off LS180-RBM35A cells with restored expression of RBM35A protein (incubated without Dox for at least six days) and their parental cells maintained in the presence of Dox were grown to no more than 80% confluency to ensure good loading of polysomes. Total RNA was extracted using TRIZOL reagent (Invitrogen) according to manufacturer instructions. Polysomal fractions of mRNA of cells expressing or not expressing RBM35A protein were isolated using sucrose density gradient sedimentation. Ten micrograms of polysomal RNA from RBM35+ and RBM35- cells were used to synthesize double-stranded cDNA using a SuperScript II reverse transcriptase kit (Invitrogen). Biotinylated cRNA was synthesized from double-stranded cDNA using the Enzo Bioarray High Yield transcript labeling kit (EnzoLife Sciences), then purified using the GeneChip sample cleanup module (Affymetrix) and fragmented. Fragmented cRNA was hybridized to Affymetrix HG-U95Av2 GeneChips.

编码RNA结合基序蛋白35A（RBM35A）的基因，在LS180结肠癌细胞系以及约50%的微卫星不稳定型结肠肿瘤中，因移码突变而失活。为阐明该推定肿瘤抑制基因的作用机制，我们在LS180细胞中表达了RBM35A的功能性互补DNA（cDNA）拷贝。我们分析了在Tet-off四环素可诱导启动子下表达RBM35A基因后，LS180细胞总mRNA及mRNA多聚核糖体组分的转录组谱变化。将恢复表达RBM35A蛋白的Tet-off LS180-RBM35A细胞（在不含多西环素（Dox）的培养基中培养至少6天）及其在含Dox培养基中培养的亲本细胞，均培养至汇合度不超过80%，以保证多聚核糖体的完整性。按照厂商操作说明，采用TRIZOL试剂（Invitrogen）提取总RNA。通过蔗糖密度梯度离心法，分离表达与不表达RBM35A蛋白的细胞的mRNA多聚核糖体组分。取10 μg来自RBM35+与RBM35-细胞的多聚核糖体RNA，采用SuperScript II逆转录酶试剂盒（Invitrogen）合成双链cDNA。以双链cDNA为模板，使用Enzo Bioarray高产量转录标记试剂盒（EnzoLife Sciences）合成生物素标记的cRNA，随后通过GeneChip样品纯化模块（Affymetrix）完成纯化并片段化。将片段化的cRNA与Affymetrix HG-U95Av2基因芯片进行杂交。