Gene expression changes induced by LKB1 expression in human lung adenocarcinoma cell lines

LKB1 is a tumor suppressor lost in approximately 30% of lung adenocarcinomas. It is a serine-threonine kinase involved in regulating metabolism, proliferation, and cell polarity. We have characterized its association with mRNA expression profiles in resected tumors and in cell lines, but little is known about the direct effects of LKB1 on the regulation of these genes. This study investigates the effects of LKB1 activity on mRNA expression in two LKB1-mutant lung adenocarcinoma cell lines, H2122 and A549. Wild-type LKB1 has been stably expressed in these cell lines using a pBABE retrovirus as well as an empty pBABE control and a kinase-dead mutant of LKB1 (K78I) control (Addgene). Samples submitted are two cell lines, three experimental conditions, and three replicates, for a total of 17 samples (one sample was excluded for poor RNA quality). Gene expression of these samples are analyzed to determine transcriptional regulatory effects of LKB1 expression. Results of this analysis are compared to our analysis of resected human tumors to determine gene patterns that are differentially expressed between LKB1-deficient and LKB1-wild-type tumors whose expression is also affected by restoration of LKB1 in vitro. RMA gene expression was taken from two cell lines stably expressing LKB1 or controls of K78I mutant LKB1 or empty pBABE vector. Log2 average expression differences are calculated and compared to results from analysis of gene expression associated with LKB1 loss in resected human tumors.LKB1（肝激酶B1）是一种抑癌基因，在约30%的肺腺癌中发生缺失。其属于丝氨酸-苏氨酸激酶，参与调控代谢过程、细胞增殖与细胞极性。我们已明确其在手术切除肿瘤及细胞系中与信使核糖核酸（mRNA）表达谱的关联，但目前对LKB1直接调控这些基因的分子机制仍知之甚少。本研究以两株LKB1突变型肺腺癌细胞系H2122与A549为模型，探究LKB1活性对mRNA表达的影响。我们通过pBABE逆转录病毒载体，在上述细胞系中稳定表达野生型LKB1，同时设置空pBABE载体对照与LKB1激酶失活突变体（K78I）对照，相关载体均购自Addgene。本次实验样本包含两株细胞系、三种实验条件，各设三次生物学重复，共计17例样本（1例因RNA质量不佳被排除）。通过对上述样本的基因表达进行分析，以明确LKB1表达的转录调控效应。将本次分析结果与我们对切除的人类肿瘤样本的分析结果进行比对，以筛选出在LKB1缺陷型与LKB1野生型肿瘤中差异表达、且其表达水平可通过体外恢复LKB1表达得到调控的基因表达特征。本研究采用稳健多阵列平均（RMA）算法，对两株分别稳定表达LKB1、K78I突变型LKB1或空pBABE载体的细胞系进行基因表达量分析；计算基因表达量的log₂平均差异，并将其与切除的人类肿瘤样本中与LKB1缺失相关的基因表达分析结果进行比对。