*2.1. MAP1B Is the Most Significantly Upregulated Gene Associated with Microtubule Bundle Formation in UBUC Transcriptomes*

The UBUC transcriptome dataset includes 93 tissue samples, with 78 categorized as deeply invasive tissues (pT2–pT4) and 15 categorized as noninvasive or superficial (pTa and pT1) tissues. Metastasis was detected in 28 patients and absent in 49 patients. Through transcriptome profiling, we identified 11 probes spanning six transcripts associated with microtubule bundle formation (GO:0001578). Among these expressed genes, we found that tumors with increased *MAP1B* expression and decreased *MARK4* had a more advanced pT status and a higher incidence of metastatic events (Figure 1A). Our main goal was to find the most significant upregulated genes associated with advanced disease. Therefore, we choose *MAP1B* for further validation. Table 1 shows the *MAP1B* gene (Probe: 226084\_at, 214577\_at) upregulation with up to 1.2832-, 0.3773- and 0.9436-, 0.3943- fold log ratios in advanced and metastatic UC, respectively. Furthermore, we found through survival analysis that increased *MAP1B* expression

was significantly related to poor prognosis in patients with UBUC (Figure 1B). As shown in Figure 1c,d, the *MAP1B* transcripts level was significantly higher among tumors with high pT status (pT2–pT4) than in noninvasive tumors (pTa–pT1) in both the UTUC and UBUC groups (both *p* < 0.01). Our findings indicate that *MAP1B* is associated with tumor aggressiveness.

**Figure 1.** Analysis of gene expression in urinary bladder urothelial carcinoma (UBUC) using a published transcriptome dataset (GSE31684). (**A**) Cluster analysis of genes focusing on the GO microtubule bundle formation class (GO:0001578) revealed that *MAP1B* was one of the most significantly upregulated genes associated with more advanced pT status and metastatic disease. Tissue specimens from cancers with a distinct pT status are illustrated at the top of the heat map, and the expression levels of upregulated and downregulated genes are represented as a continuum of brightness of red or green, respectively. Specimens with no change in messenger RNA (mRNA) expression are shown in black. (**B**) Kaplan–Meier plots showing the prognostic significance of *MAP1B* expression for the survival of UBUC. Using a QuantiGene assay, *MAP1B* mRNA expression was significantly increased in both (**C**) upper tract urothelial carcinoma (UTUC) and (**D**) UBUC at advanced primary pT stages.

#, Meta., distal metastasis developed during follow-up; Non-Meta.: no metastatic event developed.

#### *Cancers* **2020**, *12*, 630

 to cancer

**Table 1.**

Summary of di

fferentially

 expressed genes associated with microtubule

 bundle formation (GO: 0001578) and showing positive associations
