"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer"

Abstract

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

Keywords: Epigenetic deregulation; Epigenetic networks; Long non-coding RNAs; Micro-RNAs; Non-small cell lung cancer; Transcription factor LRF.

Fig. 1

Flow diagram of experimental processes performed with NSCLC experimental models involving human cell lines and mouse tissues. To investigate the epigenetic landscape of human and mouse NSCLC, we have initially assessed the differential expression patterns of ZBTB7A human and Zbtb7a mouse genes in a variety of human cells and mouse tissues and in KRAS-mutant NSCLC (human cell lines and mouse tissues). ZBTB7A encodes LRF which has been characterized as an epigenetically motivated TF acting towards transcriptional regulation of a variety of ncRNA molecules during cell differentiation [12,13]. To highlight valid ncRNA networks underlying the NSCLC epigenetic profile, which involve LRF interaction with ncRNAs, we subsequently performed analysis of expression levels of six lncRNAs confirmed by recent literature [[18], [19], [20], [21], [22], [23]] for their implication in human lung cancer. Also, methylation screening in CpG islands flanking those six selected lncRNAs was determined to further correlate DNA methylation at 5′ site of TSS with lncRNA expression levels. LncRNAs are not conserved between humans and mice contrary to miRNA families and thus, mouse samples were subjected only to miRNA qualitative and quantitative analysis. Both mouse and human RNA samples were subjected to NGS analysis to determine miRNAs differentiated in NSCLC and normal tissue to yield the complete epigenetic field guided by aberrant miR expression levels. Finally, a comprehensive screening to assess miR/lncRNA/LRF networks differentiating NSCLC subtypes studied was conducted and a network of potential interrelationships was built.

Fig. 2

(A) Representative stereoscopic images of whole lungs (left panel) and microscopic images of hematoxylin/eosin-stained sections (right panel) of lung adenocarcinomas (LUAD) induced in FVB mice by 1 intraperitoneal injection of 1 g/kg urethane (six months latency). Dashed circles outline tumors. (B) Human cell lines subjected to analysis (C) lrf/Zbtb7a was significantly overexpressed in normal mouse lung tissue compared to other physiological tissues (liver and spleen) and comparable between mouse NSCLC and adjacent normal lung tissue. ***p < 0.001 (D) LRF/ZBTB7A levels were significantly lower in human HEK293 compared to BEAS-2B cells. Human BEAS-2B and A549 LUAD cells exhibited equal LRF/ZBTB7A expression levels, in contrast to H460 large cell carcinoma human cell line which presented significantly lower expression of LRF/ZBTB7A. **p < 0.01 (E) Methylation profiles of CpG islands flanking TSS of lncRNAs. DNA samples were isolated from A549, H460 and BEAS-2B human cell lines and were bisulfite treated before subjected to methylation analysis. Each box corresponds to a Cytosine followed by Guanosine across the CpG island sequence. Data are presented as mean percentage methylation.

Fig. 3

(A) Heatmap of mature mmu-miR profile comparisons between mouse NSCLC and paired normal lung tissue. Each column represents an RNA sample, and each row represents a single miR. NLT: Normal Lung Tissue (Sample 1 to 4), NSCLC_2A-4B: RNA samples originating from different mice with NSCLC. (B) Heatmap presenting differentially expressed mature forms of hsa-miRs derived from human cell lines A540 and H460 compared to normal epithelial cells BEAS-2B. The A and B sample types refer to the technical replicates used in the NGS analysis. The color scale in both heatmaps shows the relative miR expression level in certain slide: green indicates high relative expression levels; red indicates low relative expression levels. (C) Mouse chromosomal location 12qf1 [UCSC genome browser on Mouse (GRCm38/mm10)] encompassing miR clusters overexpressed in NSCLC tissue (Table 2). miRs in dark green are detected in our study as significantly upregulated, miRs in light green are detected but not differentially expressed with statistical significance (compared to normal lung tissue) and black are miR species below limit of detection.

Fig. 4

Interplay of lncRNA/miRNAs and ZBTB7A-mRNA in human NSCLC KRAS mutant cell lines A549 and H460, derived from the present study.

Fig. 5

miR and lncRNA species produced by this study as biomarkers of lung adenocarcinoma. Kaplan-Meier survival plots with median overall survival (OS), hazard ratios [20] with 95% confidence interval, and univariate log-rank probability values (P) from 513 patients with lung adenocarcinoma, stratified into low (black) and high (red) miRs and lncRNAs expression by the optimal cut-offs indicated. Data from Kmplot (http://kmplot.com).