The transcriptome sequencing of all tissue samples was performed based on the strand-specific library preparation to identify the expression levels of all genes. Differential expression analysis and a volcano plot demonstrated that 544 genes were found to be upregulated and downregulated in MIBC (Figure 1A and Table S1). According to their ontology, the 30 most significantly changed genes included phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling molecules (fibronectin 1 (FN1), collagen type VI alpha 2 chain (COL6A2), and collagen type I alpha 2 chain (COL1A2)), mitogen-activated protein kinase (MAPK) pathway-related molecules (transforming growth factor-beta 1 (TGFB1) and MDS1 and EVI1 complex locus (MECOM)), mitochondrial biogenesis regulators (metallothionein 1A (MT1A) and MT2A), exosomal proteins (tubulin beta 6 class V (TUBB6), TUBB3, galectin 1 (LGALS1), and interferon-induced transmembrane protein 3 (IFITM3)), biomolecule metabolism (cytidine deaminase (CDA), sphingosine kinase 1 (SPHK1), monoamine oxidase A (MAOA), and microsomal glutathione S-transferase 1 (MGST1)), and others. To identify the optimal number of clusters based on transcriptomic classification, Elbow plot analysis was applied for Thai MIBC transcriptome data. The results show that the three clusters were found to be optimal, as demonstrated in Figure 1B. The transcriptomic data of all MIBC tissue samples were then subjected to the classification of the MIBC groups by using principal component analysis by K-means clustering.In addition to the enrichment study, the data from differential gene expression (DEG) analysis also revealed the number of genes that were expressed differently between two clusters, as displayed in the Venn diagram (Figure 2A). These included the genes related to the calcium signaling pathway (bradykinin receptor B1 (BDKRB1), endothelin receptor type A (EDNRA), arginine vasopressin receptor 1A (AVPR1A), prostaglandin E receptor 3 (PTGER3), prostaglandin F receptor (PTGFR), neurotrophic receptor tyrosine kinase 3 (NTRK3), purinergic receptor P2X 1 (P2RX1, etc.), PI3K-Akt signaling pathway (COL6A2, COL1A2, integrin subunit alpha 8 (ITGA8), CAMP responsive element binding protein 5 (CREB5), COL6A3), MAPK signaling pathway (fibroblast growth factor 7 (FGF7), nerve growth factor (NGF), hepatocyte growth factor (HGF), angiopoietin 1 (ANGPT1)), or cyclic GMP-dependent protein kinase (cGMP-PKG) signaling pathway (potassium calcium-activated channel subfamily M regulatory beta subunit 1 (KCNMB1), KCNMA1, adrenoceptor alpha 2A (ADRA2A), ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), adenosine A1 receptor (ADORA1), protein kinase cGMP-dependent 1 (PRKG1)). The expression levels of each gene were demonstrated as volcano plots for each cluster (Figure 2B–D). Interestingly, all 37 genes were significantly upregulated in clusters A and C, but downregulated in cluster B. The most significantly expressed genes in cluster A included BDKRB1, EDNRA, AVPR1A, platelet-derived growth factor receptor beta (PDGFRB), and tenascin C (TNC), while COL6A3, COL1A1, COL6A2, PDGFRB, and PRKG1 were found to be the top five genes highly expressed in cluster C. For cluster B, the collagen-related genes (COL6A3, COL1A2, COL6A2), TNC, and fibroblast growth factor 2 (FGF2) were the statistically suppressed transcripts.