GSE1 promotes the proliferation and migration of lung adenocarcinoma cells by downregulating KLF6 expression

Background: Lung cancer, particularly lung adenocarcinoma, remains one of the most lethal malignancies globally. Identifying aberrant molecular pathways and potential therapeutic targets is pivotal for improving patient outcomes. This study focused on the role of GSE1 (genetic suppressor element 1) in lung adenocarcinoma, elucidating its interaction mechanisms and downstream effects. Objectives: 1. Determine the expression levels of GSE1 in lung adenocarcinoma tissues and its implication on cancer cell behaviors. 2. Investigate the effects of GSE1 depletion on the proliferation and migration capabilities of A549 and H1299 lung adenocarcinoma cell lines. 3. Elucidate the potential cellular partners of GSE1, with a particular focus on its interaction with histone deacetylase 1 (HDAC1) and the broader BRAF-HDAC complex (BHC). 4. Analyze the transcriptomic changes following GSE1-knockdown in A549 cells to pinpoint its broad molecular influence. 5. Examine the possible cooperative role of GSE1 and HDAC1 in modulating the expression of significant genes, especially the tumor suppressor gene KLF6. Methods: GSE1 expression was assessed in lung adenocarcinoma tissues and correlated with cell behaviors. The effects of GSE1 depletion were studied in A549 and H1299 cells. Immunoprecipitation assays were utilized to confirm GSE1's interactions. Transcriptomic analysis identified differentially expressed genes post-GSE1-knockdown, with a focus on genes harboring HDAC1 binding sites. The relationship between GSE1 and KLF6 expression was verified using RT-qPCR and western blotting. Results: GSE1 was found to be upregulated in lung adenocarcinoma. Its depletion inhibited proliferation and migration in both A549 and H1299 cells. GSE1 was demonstrated to interact with HDAC1 and other BHC components. Following GSE1-knockdown, 207 genes were upregulated and 159 were downregulated, with 140 genes showcasing HDAC1 binding sites. Among these genes, GSE1 was revealed to inhibit the transcription of the tumor suppressor gene KLF6. Conclusion: GSE1 plays a central role in promoting non-small cell lung cancer progression, potentially through its cooperation with HDAC1. This interaction appears to downregulate KLF6 expression, highlighting a novel molecular pathway that can be targeted for therapeutic interventions in lung adenocarcinoma. Objective: The primary goal of this experiment is to understand the effects of GSE1 knockdown on the transcriptome of A549 cells and identify the genes and pathways influenced by GSE1. 1. Sample Preparation: - Cell Type: Human lung adenocarcinoma cell line, A549. Sample Groups: GSE1-knockdown: A549 cells transfected with GSE1 siRNA, designed to specifically reduce the expression of GSE1. Control: A549 cells transfected with negative control siRNA, which should not interfere with the gene expression and serves as a comparison baseline. Culturing Conditions: Cells were cultured in 6-well plates with a seeding density of 2 × 10^5 cells/well. After 24 hours of growth, cells underwent transfection. 2. Transfection: Following 24-hour growth, A549 cells were transfected with either GSE1 siRNA (for GSE1 knockdown) or negative control siRNA. 48 hours post-transfection, cells were washed with PBS to prepare for RNA extraction. 3. RNA Extraction and Validation: Total RNA was isolated from both sample groups using the TRIzol reagent. The extracted RNA's quantity and integrity were validated using a NanoDrop One Microvolume UV-Vis Spectrophotometer. The effectiveness of GSE1 knockdown at the protein level was confirmed via western blotting. 4. RNA Sequencing: From the validated RNA samples, cDNA libraries were prepared using the TruSeq RNA Sample Preparation Kit. These libraries were then sequenced on a HiSeq 4000 instrument, generating paired-end reads (50SE) at a depth of approximately 20 million sequences. 5. Data Processing and Analysis: Initial raw sequencing data underwent quality control to remove low-quality reads, adapters, and Poly-N sequences. Cleaned reads were then mapped to the human reference genome using STAR software, allowing a maximum of two mismatches per read. The expression levels of genes (quantified as RPKM) were determined, followed by the identification of differentially expressed genes (DEGs) between the GSE1-knockdown and control groups using DESeq2 software. Significant DEGs were filtered based on a false discovery rate (FDR) ≤ 0.001 and a difference in RPKM ratio ≥ 0.5.