Abstract
Background: Natural antisense long non-coding RNAs (lncRNAs) are regulatory RNAs transcribed from the opposite strand of either protein coding or non-coding genes, able to modulate their own sense gene expression. Hence, their dysregulation can lead to pathologic processes. Cancer is a complex class of diseases determined by the aberrant expression of a variety of factors, among them, the oncofetal chromatin architectural proteins High Mobility Group A (HMGA) modulate several cancer hallmarks. Thus, we decided to investigate the presence of natural antisense lncRNAs in HMGA1 and HMGA2 loci, and their possible involvement in gene expression regulation.Methods: We used FANTOM5 data resources, FANTOM-CAT genome browser and Zenbu visualization tool, which employ 1,829 human CAGE and RNA-sequencing libraries, to determine expression, ontology enrichment, and dynamic regulation of natural antisense lncRNAs in HMGA1 and HMGA2 loci. We then performed qRT-PCR in different cancer cell lines to validate the existence of HMGA2-AS1 transcripts. We depleted HMGA2-AS1 transcripts with siRNAs and investigated HMGA2 expression by qRT-PCR and western blot analyses. Moreover, we evaluated cell viability and migration by MTS and transwell assays, and EMT markers by qRT-PCR and immunofluorescence. Furthermore, we used bioinformatics approaches to evaluate HMGA2 and HMGA2-AS1 correlation and overall survival in tumor patients.Results: We found the presence of a promoter-associated lncRNA (CATG00000088127.1) in the HMGA1 gene and three antisense genes (RPSAP52, HMGA2-AS1, and RP11-366L20.3) in the HMGA2 gene. We studied the uncharacterized HMGA2-AS1 transcripts, validating their existence in cancer cell lines and observing a positive correlation between HMGA2 and HMGA2-AS1 expression in a cancer-derived patient dataset. We showed that HMGA2-AS1 transcripts positively modulate HMGA2 expression and migration properties of PANC1 cells through HMGA2. In addition, Kaplan-Meier analysis showed that high level of HMGA2-AS1 is a negative prognostic factor in pancreatic cancer patients.Conclusions: Our results describe novel antisense lncRNAs associated with HMGA1 and HMGA2 genes. In particular, we demonstrate that HMGA2-AS1 is involved in the regulation of its own sense gene expression, mediating tumorigenesis. Thus, we highlight a new layer of complexity in the regulation of HMGA2 expression, providing new potential targets for cancer therapy.
Highlights
The advent of next-generation high throughput sequencing highlighted a new regulation layer in which RNA is a fundamental player
HMGA1 antisense transcription is concentrated in the promoter region of HMGA1 where CATG00000088127.1 gene is located and annotated in FANTOM-CAT as “Promoter-associated long non-coding RNAs (lncRNAs)” (p_lncRNA_divergent) (Supplemental Figure 1), characterized to be bidirectional transcribed
We observed three independent Transcription Start Sites (TSS) in antisense orientation relative to HMGA2 transcription, which promoted the transcription of three genes, annotated in FANTOM-CAT as “Other RNAs” (RPSAP52 and RP11-366L20.2, named HMGA2-AS1), and “Enhancer lncRNA” (RP11-366L20.3) (Figure 1)
Summary
The advent of next-generation high throughput sequencing highlighted a new regulation layer in which RNA is a fundamental player. Natural antisense lncRNAs are characterized by being transcribed from the opposite strand of a protein-coding gene [5,6,7,8]. Natural antisense lncRNAs can stimulate or reduce the gene expression of the sense transcripts at multiple levels, assuming a functional role in physiological and pathological processes [8,9,10,11]. Natural antisense long non-coding RNAs (lncRNAs) are regulatory RNAs transcribed from the opposite strand of either protein coding or non-coding genes, able to modulate their own sense gene expression. Their dysregulation can lead to pathologic processes. We decided to investigate the presence of natural antisense lncRNAs in HMGA1 and HMGA2 loci, and their possible involvement in gene expression regulation
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