Decoding the role of novel long noncoding RNAs lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5 in regulating the expression of GAD1 and SLC6A12 in cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is an aggressive bile duct malignancy with a poor prognosis and limited treatment options. Recent studies highlight the role of metabolic and signalling pathways in tumour progression and resistance, including neurotransmitter-related pathways like gamma-aminobutyric acid (GABA). Key GABA-associated genes, such as Solute Carrier Family 6 Member 12 (SLC6A12), a GABA transporter and Glutamate Decarboxylase 1 (GAD1) involved in GABA synthesis, are implicated in cancer but remain poorly understood in CCA. This study aims to identify novel long non-coding RNAs (lncRNAs) specifically associated with cholangiocarcinoma (CCA) and to explore their potential mechanisms of action. By integrating transcriptomic data and interaction prediction tools, we focus on lncRNAs that are linked to key differentially expressed metabolic genes, thereby uncovering their possible roles in the metabolic reprogramming of CCA. Using RNA-Seq data from the Sequence Read Archive (SRA), differential expression analysis identified 84 differentially expressed metabolic genes (DEMGs) associated with metabolic pathways. Gene ontology and pathway analyses using DAVID and Reactome database revealed pathway enrichment due to DEGs, while protein interaction using STRING, functionally connected SLC6A12/BGT1 and GAD1/GAD67. Two novel downregulated long non-coding RNAs (lncRNAs), lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5, were identified based on expression correlations and genomic proximity to SLC6A12 and GAD1 genes. Interaction predictions using IntaRNA and lncTAR tools suggested lncRNA-mRNA interactions between the lncRNAs and mRNAs (SLC6A12 and GAD1). Transcription factor (TF) enrichment analysis using the CiiiDER tool and RNA-protein interaction predictions with the catRAPID tool revealed lnc-SLC6A12-1:3 functions as a regulatory scaffold, influencing the transcription of SLC6A12 and GAD1 by recruiting TFs such as IRF1, THAP1, FOSL1, and NR4A1. Whereas lnc-SLC6A12-7:5 did not show strong binding to TFs. In Ideal conditions, lnc-SLC6A12-1:3 enhances SLC6A12 expression by promoting IRF1 and FOSL1 activity but antagonises THAP1 and NR4A1, leading to the checked expression of GAD1. These interactions highlight a complex regulatory network where lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5 differentially modulate transcription factor activity, balancing the expression of these key genes in CCA. For the first time, this in silico study reveals that two novel long non-coding RNAs, lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5, regulate the expression of SLC6A12 and GAD1 through cis and trans binding interactions, respectively. Based on these interactions, we hypothesise that these lncRNAs may contribute to the modulation of the GABAergic pathway, which plays a crucial role in fulfilling the high energy demands of cholangiocarcinoma cells. Further experimental validation and investigation into the regulation of SLC6A12 and GAD1 are required to gain deeper insights into CCA pathogenesis and to identify potential therapeutic targets.

Keywords: Cholangiocarcinoma; GAD1; Lnc-SLC6A12-1:3; Lnc-SLC6A12-7:5; LncRNA; SLC6A12.