Expression profiling of ubiquitin-related genes in LKB1 mutant lung adenocarcinoma

Abstract

Liver kinase B1 (LKB1) is a tumor suppressor, and there is a very high proportion of LKB1 mutation in lung adenocarcinoma. The function of LKB1 is closely related to that of ubiquitin related genes. Our objective is to analyze the changes in ubiquitin-related genes in LKB1 mutant lung adenocarcinoma. We searched The Cancer Genome Atlas (TCGA) and obtained gene expression profiles from 230 lung adenocarcinoma patients, which were then analyzed using R software. Kaplan-Meier curves and Cox proportional hazards regression were applied to estimate survival. Real-time reverse transcription PCR was used to verify gene expression. Gene function was explored by gene set enrichment analysis. There were significantly expressed differences in the ubiquitin-related gene SH3RF1 between the LKB1 mutant and wild-type lung adenocarcinoma patients (p = 9.78013E-05). Patients with LKB1 mutation and high expression of SH3RF1 had a better prognosis than the low expression group (HR 0.356, 95% CI 0.136-0.929, p = 0.035). SH3RF1 can influence cell cycle, apoptosis, DNA replication and the p53 signaling pathway. SH3RF1 might have great clinical value act as a diagnostic biomarker and indicator to evaluate the prognosis of LKB1 mutant lung adenocarcinoma patients. This gene also can become a new treatment target for LKB1 mutant lung adenocarcinoma patients.

Figure 1

Flow sheet of the target genes screening process.

Figure 2

(a) STK11 gene alterations. Blue represents deep deletion, black represents truncating mutation (putative driver), black green represents missense mutation (putative driver) and green represents missense mutation (putative passenger). Genetic alterations were found in 43 of 230 lung adenocarcinoma patients (19%). The aberrant expression threshold was defined as z-score ± 2.0 from the TCGA RNA Seq V2 data. This OncoPrint was analysed by cBioPortal. (b) The heatmap of 116 ubiquitin related differential expression genes in 230 lung adenocarcinoma patients. The first row refers the LKB1 mutation information. Blue represents 187 LKB1 wild type patients and yellow represents 43 LKB1 mutation patients. c, the scatter plot of 9 ubiquitin related differential expression target genes. LKB1 WT represents the LKB1 wild type patients and LKB1 MU represents the LKB1 mutation patients.

Figure 3

The Kaplan–Meier survival curve of 9 ubiquitin related differential expression target genes. WT represents the LKB1 wild type patients and MU represents the LKB1 mutation patients. All p values were two sides and less than 0.05 were considered significant.

Figure 4

(a) western blot in A549 cell line. Cell lysates were collected, and anti-LKB1 antibody was used to detect LKB1 expression. GAPDH was used as the loading control. Negative sign refers to A549 stable transfection cell line transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-MCS-3Flag control plasmids. Positive sign represents A549 stable transfection cell line transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-stk11 plasmids. (b) negative sign refers to A549 transient transfection cell line transfected with PCDNA3.0 vector control plasmid. Positive sign represents A549 transient transfection cell line transfected with LKB1 (K78I) kinase-dead mutant plasmid. (c) Quantitative real-time PCR in A549 stable transfection cell line. TRIM2 and SH3RF1 primers were separately used to detect TRIM2 and SH3RF1 expression. CT refers to A549 stable cell line transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-MCS-3Flag. LKB1 refers to A549 stable cell line transfected with pLenti-EF1a-mcherry-P2A-Puro-CMV-stk11 plasmids. TRIM2, p = 0.0346. SH3RF1, p = 0.0089. All p values were two sides and less than 0.05 were considered significant. (d) Quantitative real-time PCR in A549 transient transfection cell line. TRIM2 and SH3RF1 primers were separately used to detect TRIM2 and SH3RF1 expression. CT refers to A549 transient transfection cell line transfected with PCDNA3.0 vector plasmid. LKB1 KD refers to A549 transient transfection cell line transfected with LKB1 (K78I) kinase-dead mutant plasmid. NS = not significant. (e) The GSEA results for SH3RF1 in LKB1 mutate NSCLC patients. Inclusion criteria: NOM p-val < 0.05 and FDR q-val < 0.25.