3.1 Overview of sequencing results and verification by quantitative real-time PCR
Compared with the human genome, the ratio of reads aligned to gene regions, coding regions, splice sites, introns, and non-coding regions was normal, genome coverage was good, and sequencing quantity was sufficient, as shown in Fig. S1.
A total of 84043 lncRNAs were detected, of which 1030 lncRNAs were newly predicted. By observing the differences in transcript length, number of exons, and expression levels between lncRNAs and mRNAs, it was shown that the lncRNAs conformed to the general characteristics, as shown in Fig. S2.
The quantitative real-time PCR results of the selected 12 genes were highly consistent with the sequencing results, suggesting that the sequencing experiment had high reliability. As shown in Fig. 1.
3.2 Transcriptome changes from normal gallbladder to gallbladder with chronic inflammation
A total of 851 different mRNAs were identified, of which 385 were upregulated and 466 were downregulated. There were 322 different lncRNAs, of which 103 were upregulated and 219 were downregulated. The expression of mRNAs and lncRNAs showed obvious differences between the two groups, and the expression of samples in the same group showed good homogeneity, as shown in Fig. 2.
GO enrichment of differentially expressed mRNAs
There were 759 GO items with q-value ≤ 0.05. By further summarizing the top 100 mRNAs with a larger rich factor, it was found that different mRNAs were distinctly related to inflammation (35 items), metabolism (24 items) including lipid metabolism (10 items) and sex hormone metabolism (four items). The top three enriched factors were estrogen 16-α-hydroxylase activity, lipid hydroxylation, and the omega-hydroxylase P450 pathway, as shown in Fig. 3.
KEGG enrichment of differentially expressed mRNAs
There were 28 KEGG items with q-value ≤ 0.05, which were distinctly related to inflammation (six items), lipid metabolism (three items), steroid hormones metabolism (three items), amino acid and foreign substance metabolism (seven items). The top three enriched factors were phenylalanine, tyrosine and tryptophan biosynthesis, synthesis and degradation of ketone bodies, and steroid hormone biosynthesis, as shown in Fig. 3.
GO and KEGG enrichment of differentially expressed lncRNAs
Target genes were predicted by trans- and cis-regulation. There were 877 predicted target genes for the differentially expressed lncRNAs, of which 59 showed significant differences in expression.
There were 0 GO items with q-value ≤ 0.05 for target genes, and there were two KEGG items with q-value ≤ 0.05, which were homologous recombination, valine, leucine, and isoleucine degradation.
GO and KEGG enrichment analyses was also performed for differentially expressed target genes. There were 28 GO items with q-value ≤ 0.05, which were distinctly related to inflammation (17 items) and foreign substance metabolism (four items). There were seven KEGG items with q-value ≤ 0.05, and 16 items with p-value ≤ 0.05, which were mostly related to inflammation (nine items), lipid metabolism (two items), and tumor-related pathways (three items), as shown in Fig. S3.
3.3 Transcriptome changes from gallbladder with chronic inflammation to early GBC
A total of 176 different mRNAs were identified, of which 58 were upregulated and 118 were downregulated. There were 84 different lncRNAs that were identified, of which 20 were upregulated and 60 were downregulated. The expression of mRNAs and lncRNAs showed obvious differences between the two groups, and the expression of samples in the same group showed good homogeneity, as shown in Fig. 4.
GO enrichment of differentially expressed mRNAs
There were 116 GO items with q-value ≤ 0.05. Further cluster analysis revealed that the different mRNAs were distinctly related to immune activity (63 items) and connection between cells (30 items). The top three enriched factors were regulation of B cell receptor signaling pathway, regulation of humoral immune response, and regulation of complement activation, as shown in Fig. 5.
KEGG enrichment of differentially expressed mRNAs
There were seven KEGG items with q-value ≤ 0.05, and 24 items with p-value ≤ 0.05, which were mostly related to metabolism (13 items) and immune activity (six items), as shown in Fig. 5.
GO and KEGG enrichment of differentially expressed lncRNAs
Target genes were predicted by trans- and cis-regulation. There were 54 predicted target genes for differentially expressed lncRNAs, of which six showed significant differences in expression.
There were 0 GO items with q-value ≤ 0.05 for target genes and 76 items with p-value ≤ 0.05, which were mostly related to the modification and polymerization of proteins (36 items), connection and signal transduction between cells (23 items), and immune activity (seven items). There were 0 KEGG items with q-value ≤ 0.05, and 17 items with p-value ≤ 0.05, which were mostly related to immune activity (eight items) and signal transduction (six items), as shown in Fig. S4.
GO and KEGG enrichment analyses were also performed for the differentially expressed target genes. There were 0 GO items with q-value ≤ 0.05, and five items with p-value ≤ 0.05, which were related to development (four items) and connection between cells (one items). There was 0 KEGG items with q-value ≤ 0.05, and 0 items with p-value ≤ 0.05.
3.4 Transcriptome changes from early GBC to advanced GBC
A total of 26 different mRNAs were identified, of which 20 were upregulated and six were downregulated. There were 18 different lncRNAs, of which seven were upregulated and 11 were downregulated. The expression of mRNAs and lncRNAs showed obvious differences between the two groups, and the expression of samples in the same group showed good homogeneity, as shown in Fig. 6.
GO enrichment of differentially expressed mRNAs
There were 11 GO items with q-value ≤ 0.05. Further cluster analysis revealed that different mRNAs were all related to the transmembrane transport of substances (11 items), including the transmembrane transport of carboxylic acids (three items), ions (six items), and phospholipids (two items). There were 25 items with p-value ≤ 0.05, which were distinctly related to transmembrane transport of substances (17 items), cell membrane components (three items), and cell migration (three items). The top three enriched factors were carboxylic acid transmembrane transporter activity, carboxylic acid transmembrane transport, and organic anion transmembrane transporter activity, as shown in Fig. 7.
KEGG enrichment of differentially expressed mRNAs
There was only 1 KEGG item with q-value ≤ 0.05 or p-value ≤ 0.05, that was bile secretion, as shown in Fig. 7.
GO and KEGG enrichment of differentially expressed lncRNAs
Target genes were predicted by trans- and cis-regulation. There were 14 predicted target genes for the differentially expressed lncRNAs, none of which showed significant differences in expression.
There were three GO items with q-value ≤ 0.05 for target genes, and 50 items with a p-value ≤ 0.05, which were mostly related to RNA expression regulation (19 items), cell proliferation (five items), and cell migration (three items). There was only one KEGG item with q-value ≤ 0.05 or p-value ≤ 0.05, that was miRNAs in cancer, as shown in Fig. S5.