Cigarette smoke modulates methylation levels of LEF1-AS1 and impedes its expression: An experimental study

Abstract

Introduction: Cigarette smoke (CS) contains carcinogenic substances and influences genetic regulation and epigenetic modifications, such as DNA methylation. It plays a role in the development of various cancers, including colon, bladder, lung cancer, and leukemia. Long non-coding RNAs play a significant role in controlling several pathways in the cell, including lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1), which is found overexpressed in lung, oral, glioblastoma, and colon cancers and downregulated in leukemias. We investigated the impact of CS on DNA methylation of the promoter region of LEF1-AS1 as well as its expression in endothelial cells.

Methods: This experimental study was designed to investigate the effects of cigarette smoke on the methylation status of the promoter region of LEF1-AS1 in smoker and non-smoker samples and its expression in relevant cell models. To measure the alternations of DNA methylation, extracted DNA samples from 64 male subjects (32 smokers and 32 non-smokers) were bisulfite-treated and amplified using polymerase chain reaction (PCR) with methylation-specific PCR primers. Furthermore, to define the impact of CS on LEF1-AS1 expression, human umbilical vein endothelial cells (HUVECs) were fed with media containing CS for 3 and 6 hours. The expression analysis of LEF1-AS1 was performed using the GTEx (Genotype-Tissue Expression) database, including an assessment of its expression in various cancers such as lung and brain cancers. The functional analysis of the LEF1-AS1 gene was conducted across multiple tissues using data from the GENT2 databases, along with meta-survival and functional enrichment analysis.

Results: The results indicated an average increase of 19.8% in DNA methylation of the promoter region of LEF1-AS1 in the samples from the smokers compared with those from the non-smokers, as well as a significant reduction of LEF1-AS1 expression level in the HUVECs (45% and 83%) after treatment with CS (3 and 6 Hours), respectively. LEF1-AS1 expression varied significantly across tumor types when compared to their normal counterparts. Some cancers, such as lung and brain, showed increased expression, suggesting cancer-specific overexpression of LEF1-AS1. Variability in expression across cancers and normal tissues implies potential heterogeneity in gene regulation. A meta-survival analysis of the LEF1-AS1 gene (e.g. GSE31546, GSE31548, GSE19188), revealed hazard ratios (HR) ranging widely, with some studies (e.g. GSE31546, HR=12.02) suggesting increased risk, though confidence intervals often included 1, indicating uncertainty. Low heterogeneity (I²=16%, p=0.26) suggests consistency among studies, but the overall findings lack strong statistical significance.

Conclusions: Our findings indicate that CS alters LEF1-AS1 DNA methylation and causes an inhibition of LEF1-AS1 expression.

Keywords: DNA methylation; HUVECs; LEF1-AS1; cigarette smoke; mRNA expression.

Figure 1

Relative RNA expression in NHBE cells with and without cigarette smoke exposure. Box plots depict median values (central line), interquartile range (IQR; box boundaries), and whiskers (1.5×IQR) of four replicate petri dish of NBEA cells. The NHBE group (control) shows higher LEF1-AS1 RNA expression compared to NHBE+CS (cigarette smoke-treated)

Figure 2

Differences in the global DNA methylation pattern of LEF1-AS1 between non-smokers and smokers. The results of the MSP analysis of LEF1-AS1 in 64 samples from non-smokers (N.S) and smokers (S) are displayed as a box plot. Each box plot indicates the mean methylation level of the 32 samples from the N.S and 32 samples from the S

Figure 3

Effects of CS on LEF1-AS1 expression in HUVECs. RNA expression of LEF1-AS1 in the HUVECS after incubation with CS-free DMEM (control) and CS for 3 (CS+3) and 6 (CS+6) hours. Mean ± SD of the results from three independent experiments

Figure 4

Comparative tissue-wide expression profile of LEF1-AS1 across different cancer types (represented by ‘Cancer’) and their corresponding normal tissues (represented by ‘Normal’). The data are shown in box plots, where the y-axis represents the log₂-transformed expression levels of LEF1-AS1 in various tissue types