Transcriptomic profiling was done in 10 patients with adenocarcinoma of the gallbladder, of these 7 were females with a mean age of 56 (SD14) months. Six had stage II and three had stage III cancer. Transcriptomic profiling identified 775 lncRNAs of which 16 were significant with more than 2 fold log change. Of these 7 were upregulated and 9 were down regulated. VarElect recognized 12 of these lncRNA (LINC02441, LINC00668, LINC02331, LINC01224, LINC02595, LINC01354, LINC02086, LINC00705, LINC02884, LINC01914, LINC02565 and LINC00924) while 4 were novel (LINC01819, LINC02562, LINC02254, and LINC02038) and were never reported in cancer earlier.
Functional analysis of these lncRNAs showed them to be primarily involved in RNA/mRNA metabolic process, macromolecule biosynthetic process, nucleic acid metabolism, nitrogen compound synthesis, cellular biosynthesis and regulation, and aromatic compound metabolism etc. KEGG analysis showed them to be associated with splicosome, mRNA/RNA surveillance, transport, and degradation, ribosome biogenesis, WNT and TGF beta signaling pathways. Two of these had been earlier identified in prostate and thyroid cancer, however, all 16 lncRNA were first time identified in gallbladder cancer.
On cross species blast, all of these were found to be present in vertebrates with variable degree of similarities, however, 7 of these (LINC01819, LINC00705, LINC00668, LINC02595, LINC01354, LINC02884, and LINC01224) were found to be having very high similarity with bacterial genomes and were phylogenetically very closely related to the bacterial genome, suggesting horizontal gene transfer from bacteria at some point of evolution (Supplementary table 1, Fig. 1, 2). These overlaps were found with the genome of Bacillus paralicheniformis, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus epidermidis, Shigella flexneri, Salmonella enterica, Ralstonia solanacearum, Mycobacterium tuberculosis, and Pasteurella multocida among others. The coverage was as high as 20% seen between Staphylococcus aureus with LINC01224. While for rest of 9 lncRNA no phylogenetic similarity was observed and these genes were found to be conserved over time. Phylogenetic analysis with genome of Homo heidelbergensis and Homo neanderthalensis revealed significant overlap suggesting that these genes were present in them as well suggesting the gene insertion during evolution of man (Fig. 3).
Integrated analysis of the functions of ncRNA with KEGG analysis showed that these 7 lncRNA were involved in prolactin signaling pathway, estrogen signaling pathway, ferroptosis, PI3K-AKT pathway, chemokine pathway and focal adhesion pathway additional Fig. 1. The regulation of function occurred mainly through a set of genes that are detailed in Table 1. The miRNA and other lncRNA interactions with coding genes in select pathways is given in supplementary tables 2–11. The gene-gene interactions are detailed in Fig. 4.
Table 1
Pathways identified by integrated non coding RNA and KEGG analysis for 7 non conserved genes with bacterial gene overlap.
gene-set | genes | hsaid | p-adjust | p-value | pathway |
AKT3,RELA,PRL,SOCS6,SRC,TH,TNFRSF11A | 7 | hsa04917 | 0.004256126045487145 | 0.00042561260454871455 | Prolactin signaling pathway |
AFDN,AKT3,IGF2,RASGRP2,GNAQ,GRIN2B,MAGI2,RAPGEF4,SRC,TIAM1 | 10 | hsa04015 | 0.0381614449746874 | 0.011448433492406221 | Rap1 signaling pathway |
PCBP2,ACSL5,FTH1,LPCAT3 | 4 | hsa04216 | 0.0381614449746874 | 0.008367138203526666 | Ferroptosis |
AKT3,KRT19,CREB3L1,GNAQ,HSPA1A,KCNJ3,SRC | 7 | hsa04915 | 0.04243573572500861 | 0.021217867862504305 | Estrogen signaling pathway |
RELA,CREB3L1,GRIN2B,TH | 4 | hsa05030 | 0.04243573572500861 | 0.018950969816884503 | Cocaine addiction |
LAMA1,AKT3,ITGB4,RELA,COL6A3,IFNAR1,COL2A1,CREB3L1,PPP2R3B,RB1,WNT9A,WNT9B | 12 | hsa05165 | 0.04720125057204981 | 0.04397158095887674 | Human papillomavirus infection |
LAMA1,AKT3,IGF2,ITGB4,RELA,COL6A3,IFNAR1,COL2A1,CREB3L1,IL2RB,MAGI2,PPP2R3B,PRL | 13 | hsa04151 | 0.04720125057204981 | 0.031553416183672266 | PI3K-Akt signaling pathway |
AKT3,CXCL16,CXCR5,RASGRP2,RELA,CCL18,GNAQ,SRC,TIAM1 | 9 | hsa04062 | 0.04720125057204981 | 0.03605916433239382 | Chemokine signaling pathway |
LAMA1,AKT3,RELA,COL2A1,RB1 | 5 | hsa05222 | 0.04720125057204981 | 0.039336121630164055 | Small cell lung cancer |
LAMA1,AKT3,ITGB4,COL6A3,MYL12A,PPP1R12C,COL2A1,SRC | 8 | hsa04510 | 0.04720125057204981 | 0.04720125057204981 | Focal adhesion |