Gene expression profiling of Calu-6 lung cancer cells transfected with scrambled siRNA and LAPTM4B-specific siRNA. Homo sapiens

We recently characterized the adjacent airway field of cancerization in NSCLC by whole transcriptome expression analysis and demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) was an elevated field cancerization marker in NSCLCs and in adjacent but not in distant normal-appearing airways. We also found that LAPTM4B was up-regulated in NSCLCs compared to normal lung and promoted anchorage-dependent growth of lung cancer cells. Previous reports suggested that LAPTM4B is activated following metabolic and genotixc stress. The precise role of LAPTM4B in lung cancer cell survival and NSCLC pathogenesis is still elusive. Overall design: In the present study we sought to examine how LAPTM4B expression levels impact downstream expression profiles and cell signaling in order to gain better insights into the function of this putative oncogene in lung cancer. Calu-6 lung cancer cells were transfected with SMARTpool (Dharmacon)control/scrambled siRNA or siRNA targeting LAPTM4B. Following transfection, cells were cultured in the presence or absence of 10% fetal bovine serum (FBS). Three independent transfections for each condition were performed representing three biological replicates per condition (total number of samples = 12). RNA interference-mediated knock-down of LAPTM4B in samples was confirmed by quantitative real-time PCR. Total RNA was profiled using the Human Gene 1.0 ST platform.

本研究团队近期通过全转录组表达分析，对非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）的癌化邻域进行了特征分析，证实溶酶体跨膜蛋白4β（lysosomal protein transmembrane 4 beta, LAPTM4B）是NSCLC及其癌旁外观正常气道的上调癌化标志物，而在远端外观正常气道中无此表达上调现象。本研究同时发现，相较于正常肺组织，LAPTM4B在NSCLC中表达上调，并可促进肺癌细胞的锚定依赖性生长。既往研究表明，LAPTM4B可在代谢应激与基因毒性应激后被激活。目前，LAPTM4B在肺癌细胞存活及NSCLC发病机制中的具体作用仍有待阐明。

实验设计概述：
本研究旨在探究LAPTM4B表达水平如何影响下游表达谱与细胞信号通路，以深入解析这一推定癌基因在肺癌中的功能。将Calu-6肺癌细胞分别转染靶向LAPTM4B的小干扰RNA（small interfering RNA, siRNA），以及Dharmacon公司的SMARTpool阴性对照/乱序siRNA。转染完成后，将细胞分别置于含10%胎牛血清（Fetal Bovine Serum, FBS）与不含胎牛血清的培养基中培养。每种处理条件均设置3次独立转染，即3个生物学重复（总样本数共计12例）。通过实时定量PCR（quantitative real-time PCR, qRT-PCR）验证了样本中RNA干扰介导的LAPTM4B敲低效果。采用人类基因1.0 ST芯片（Human Gene 1.0 ST platform）对总RNA进行表达谱分析。