Of the ten GBC patients enrolled in this study three were males and seven females. The mean age of the patients was 56 years (SD 14.68 months). Four patients had stage III and six patients had stage II GBC. A total of 891 asRNAs were identified as differentially expressed (DE-asRNAs) in cancer compared to normal gallbladder, of these 17 were found to be statistically significant and were subjected to further analysis (Table 1). Among the DE-asRNAs, 12 were up-regulated, (AFAP1-AS1, HMGA2-AS1, MNX1-AS1, UNC5B-AS1, SLC2A1-AS1, BBOX1-AS1, SOX21-AS1, ELFN1-AS1, TRPM2-AS, DNAH17-AS1, DCST1-AS1, and VPS9D1-AS1), while five were down-regulated, (MIR1-1HG-AS1, HAND2-AS1, PGM5P4-AS1, PGM5P3-AS1, and MAGI2-AS3).

The gene ontology analysis identified them to be involved in developmental processes, apoptosis, methylation, and cell proliferation (Supplementary Table 1). The pathway analysis revealed specific associations with different pathways for each asRNA (Supplementary Table 2). The HAND2-AS1, e.g., was linked with small-cell lung cancer and RNA degradation pathways, while MNX1-AS1 was enriched with WNT signaling, deadenylation-dependent mRNA decay, and the Eif4 pathway. UNC5B-AS1 was associated with pathways in cancer, basal cell carcinoma, and hedgehog signaling pathways. SLC2A1-AS1 was linked to RAP1 signaling and E2f-mediated regulation of DNA replication, while BBOX1-AS1 and VPS9D1-AS1 were associated with the hemostasis pathway. SOX21-AS1 was significantly involved in regulatory RNA pathways, the HIV-1 early elongation complex formation, RNA Pol III Transcription, and the influenza life cycle. Fourteen of the asRNAs (UNC5B-AS1, SLC2A1-AS1, BBOX1-AS1, SOX21-AS1, ELFN1-AS1, TRPM2-AS, DNAH17-AS1, DCST1-AS1, VPS9D1-AS1, MIR1-1HG-AS1, HAND2-AS1, PGM5P4-AS1, PGM5P3-AS1, and MAGI2-AS) are found to be associated with gallbladder cancer for the first time, while three have been previously reported (AFAP1-AS1, HMGA2-AS1, and MNX1-AS1) (Supplementary Table 3).

Phylogenetic analysis showed the similarities among significant DEasRNAs, as shown in Fig. 1. Five clusters were identified having 7, 5, 2, 2 As RNA, while TRPM2-AS was stand alone and was not part of any cluster. BLAST analysis revealed that 15 asRNA (AFAP1-AS1, HMGA2-AS1, MNX1-AS1, SLC2A1-AS1, BBOX1-AS1, ELFN1-AS1, TRPM2-AS, DNAH17-AS1, DCST1-AS1, VPS9D1-AS1, MIR1-1HG-AS1, HAND2-AS1, PGM5P4-AS1, PGM5P3-AS1, and MAGI2-AS) showed, variable degree of similarity with bacterial and viral genome except (UNC5B-AS1 and SOX21-AS1) as they were found to be fully conserved (Fig. 2 A to O). Two of these (VPS9D1-AS1 and SLC2A1-AS1) were found to have a high degree of similarity with the viral and bacterial genome. VPS9D1-AS1 showed similarity with human papilloma virus (HPV) type 16, Human immunodeficiency virus (HIV) Klebsiella pneumonae, and Basillus paralichemiformis. These results suggest that they were phylogenetically related to viral and bacterial genomes, suggesting horizontal gene transfer from both virus and bacteria at some point in evolution.

Neighbour-joining phylogenetic tree of significant asRNAs showing 5 clusters

Phylogenetic tree analysis showing ancestry with bacterial, viruses, fungi, Homo neanderthalensis, and Homo heidelbergensis DNA (A-O). Highlighted asRNAs indicate our DEGs, different colour nodes represent different species

The blast analysis of these significant asRNAs failed to show any similarity with fungi. The overlaps were found with genome of Chikungunya virus, Human immunodeficiency virus 1, Stealth virus 1, Staphylococcus aureus, Zika virus, Pasteurella multocida subsp., Klebsiella pneumoniae, Staphylococcus haemolyticus, Ralstonia solanacearum, Human endogenous retrovirus, Bacillus paralicheniformis, and Bradyrhizobium species.

The query cover in BLAST with Chikungunya virus was 34%, with an E value of (9.00E-116), and a percentage identity of (91.96) with a sequence of VPS9D1. Additionally, the query cover in BLAST with Chikungunya virus was 19%, with an E value of (4.00E-115), and a percentage identity of (93.22) with a sequence of SLC2A1.

The 15 enriched DEasRNAs targeted mRNAs interaction showed that the targeted mRNAs were involved in the p53 signaling pathway, endometrial cancer, pathway in cancer, microRNA in cancer, and PI3K-AKT signaling pathway Supplementary Fig. 1. A gene regulatory network of asRNAs targeting mRNAs, revealing a unique mechanism of gene regulation that plays a critical role in cancer development that involved genes in PIK3CA-AKT and NOTCH signaling pathway among others (Fig. 3). A complex cross talk of the major pathways was prepared and is presented in Fig. 4. The interplay of downstream EGFR, and Wnt pathway with DNA repair genes appears to be the responsible for gallbladder carcinogenesis.

asRNA targeted mRNA interaction network. Nodes in the network are represented by dots with various colors and shapes. The link between the two nodes is represented by the edge. Seeds (circle yellow), complex (Circle light blue), miRNA (circle dark pink), chemical (circle dark green), protein family (circle peach), small molecule (circle purple), stimulus (circle brown), phenotype (circle orange), and protein (circle pink)

Crosstalk between the significant asRNAs and the targeted mRNAs. Yellow color circle – Significant asRNAs, blue rectangular box—targeted mRNAs