3.1POLR2K expression in bladder cancer
POLR2K, as an important gene in the RNA process machinery, is the only subunit common to all three polymerases that dose dependently improves the assembly of the Pol III pre-initiation complex [33, 34]. As Pol III products are required for protein synthesis and cancers have unusually high levels of Pol III activity , it is possible that the up-regulation of POLR2K could facilitate Pol III assembly and thus contribute to cell proliferation and cancer development .Inspired by this ,we used GEPIA to perform survival analysis for POLR2K in various types of cancers, and found that POLR2K overexpression was significantly related to poor survival(Supplementary Figure 1). Therefore, we want to further clarify the role of POLR2K in BLCA and understand the influences on cancer development, expression patterns and regulatory networks of POLR2K in BLCA.
Using TCGA and the Gene Expression Omnibus (GEO) database, we firstly assessed POLR2K transcription levels in BLCA studies. Analysis of the Oncomine database revealed an increase in DNA copy number variation (CNV) and mRNA expression of POLR2K in BLCA tissues compared with normal tissues (p ˂ 0.01). POLR2K ranked within the top 17% of transcriptome profile and within the top 9% of DNA CNVs (Figure 1), when the fold differences were adjusted to 2. In addition, correlation analysis of POLR2K with clinic pathological features of 408 bladder cancer patients from the Cancer Genome Atlas database was conducted by UALCAN website. And the results consistently suggested high transcription levels of POLR2K in BLCA. Compared with healthy people, the level of transcription for POLR2K was much higher in bladder cancer patient based on the sub-group such as disease stages, gender, smoking condition, histological subtypes, and nodal metastasis status (Figure 2). Furthermore, Kaplan-Meier survival analysis displayed that high levels of POLR2K was positively related to poor progression-free survival (PFS) and overall survival (OS) in bladder cancer patients (Supplementary Figure 1).Therefore, POLR2K expression could become a potential diagnostic indicator in BLCA.
3.2 Alterations of POLR2K in bladder cancer
3.2.1 Type and frequency of POLR2K alterations in bladder cancer
POLR2K is a gene encoding one of multiple subunits of RNA polymerase II (Pol II), whose biological function is to synthesize mRNA. This subunit could be also found in another two polymerases, RNA polymerase III (Pol III) and RNA polymerase I (Pol I). POLR2K could be utilized to synthesize diverse function non coding RNAs, small RNAs, mRNA progenitors, and ribosomal RNA progenitors. In order to investigate the type and frequency of POLR2K changes within bladder cancer, we then used the ciBioPortal to analyze sequence data from BLCA patient tissues in the TCGA database. Compared with normal healthy tissues, POLR2K level was altered in 141 of 408 (35%) BLCA patient tissues (Figure 3a). These alterations includes mRNA up-regulation in 139 cases (34.5%), amplification in 59 cases (14.7%), multiple alterations in 55 cases (13.7%) mRNA and down-regulation in 2 cases (0.49%) (Supplementary Fig 2). Therefore, amplification is identified in BLCA, as the most prevalent type of POLR2K CNV.
3.2.2 Interaction network of POLR2K alterations in bladder cancer
To investigate biological interaction network of POLR2K in BLCA, we used Common Pathways to obtain POLR2K–neighboring genes, and then used Cbioportal to inquire the alteration frequencies of these genes. (Figure 3b and Supplementary Fig 2). The neighboring genes of POLR2K make KDMSA (35%), KMT2D (33%) together with INTS8 (32%) as one of the most frequent alterations in BLCA. With the help of Cytoscape, analysis of GO category annotations implied these genes encoding proteins localized primarily to, or functioned at, RNA polymerase complex, the spliceosomal complex, and MLL3/4 complex. These proteins are fundamentally related to posttranscriptional gene expression processes (i.e. snRNA and dsRNA processing, transcription-coupled nucleotide-excision repair, etc), but they were also enriched such as “U2-type spliceosomal complex” and “basal transcription machinery binding” (Figure 4a–4c). As expected, KEGG pathway analysis indicated that POLR2K is connected to Spliceosome, RNA polymerase, mRNA surveillance and Basal transcription factors (Figure 4d). As a result, the interaction network of POLR2K alterations is related to several RNA metabolic processes and the gene expression regulation.
3.3 Enrichment analysis based on POLR2Kfunctional network in bladder cancer
3.3.1 GO annotation and KEGG enrichment analysis for co-expression genes related to POLR2Kin bladder cancer
Using LinkedOmics online tool, we took advantage of TCGA database to explore mRNA sequencing data comprising 408 patients of BLCA. From the volcano plot depicted in Figure 5a, significant negative correlation with POLR2K was suggested among 2395 genes (dark green dots), while significant positive correlation was indicated among 2635genes (dark red dots) (P-value<0.01, false discovery rate FDR< 0.05). The heat map displayed 50 significant genes whose mRNA expression positively or negatively correlated with POLR2K in BLCA (Figure 5b, 5c). This result demonstrated that POLR2K had an extensive influence on the transcriptome. Supplementary Figure 3a–3c show scatter plots for each gene. Significantly, POLR2K expression is positively related to the expression of ZNF706 (positive rank #1, Pearson correlation = 0.7887, p = 8.055e-88), YWHAZ (Pearson correlation = 0.6695, p = 2.224e-54), and COX6C(Pearson correlation = 0.6638, p = 3.552e-53), which mirrors alterations in mitochondrial composition as well as apoptosis, DNA repair, and transcriptional regulation. Further exploration by GSEA analysis demonstrated differentially expressed genes were primarily related to mitochondrial protein complex, spliceosome complex, replication fork and condensed chromosome, whereas those genes are involved mainly in mitochondrial gene expression, mRNA/ncRNA processing, DNA replication and cell cycle. These genes also serve as constituent of mitochondria and ribosomes (Figure6a–6c and Supplementary Tables 1–3).
Interestingly, GSEA analysis implied that these genes were also located in MHC protein complex, secretory granule membrane, cell leading edge and extracellular matrix, where they participated in the positive regulation of cell motility, antigen binding and immune response-regulating signaling pathway. They were also involved in response to type I interferon, I-kappaB kinase/NF-kappaB signaling, Toll-like / NOD-like receptor signaling pathway and JAK-STAT signaling pathway, all of which were associated with tumor immune escape. Also, GSEA analysis of KEGG pathway showed highly significant enrichment in cell cycle, DNA replication, ribosome and spliceosome, as well as in Natural killer cell mediated cytotoxicity and Cytokine-cytokine receptor interaction (Figure 6d, 6e and Supplementary Table 4). Taken together, POLR2K function networks were mainly responsible for gene expression, mRNA surveillance, cell cycle, DNA replication while also involved in tumor immune response and survival, which demonstrated significant deregulation of cancer related pathways.
3.3.2 POLR2K networks of transcription factor, miRNA and kinase targets and in bladder cancer
Transcription factor, miRNA and Kinase target network of associated gene dataset created by GSEA were used to examine targets of POLR2K in BLCA. The significant kinase-target networks were associated mainly with mitogen-activated protein kinase 6 (Kinase_MAPK6), homeodomain interacting protein kinase 2（Kinase_HIPK2）and mitogen-activated protein kinase 7 (Kinase_MAPK7) (Table 1 and Supplementary Tables 5–7); the miRNA-target network was generally related to AACTGGA_MIR145, TTGCCAA_MIR202, ACTTTAT_MIR507, GTGCAAT_MIR25_MIR32_MIR92_MIR363_MIR367 and TATTATA_MIR374; the transcription factor-target network was involved fundamentally in the cell cycle regulation factors including V$E2F1_Q6, V$FOXO1_02 and V$CDC5_01, whereas also involved in the IRF Transcription Factor (IRF) family including V$IRF7_01, V$IRF1_01, and V$ISRE_01. Correlation among genes for the Kinase MAPK7, miR-145 and E2F1_Q6 respectively was uncovered by subsequent protein-protein interaction networks created by GeneMANIA. These genes enriched for transcription factor E2F1 have been linked primarily to modulating cell cycle checkpoint, DNA repair and DNA replication, while associated with MCM complex, G1/S transition of mitotic cell cycle and telomere maintenance (Figure 7). Furthermore, genes enriched for MAPK7 are related mainly to the regulation of innate immune response, toll-like receptor family signaling and stress-activated protein kinase signaling cascade (Supplementary Figure 4). And genes enriched for miRNA-374 can be related primarily to modulation of Notch signaling pathway, activating transcription factor binding and modulation of transcription in other organism involved in symbiotic interaction (Supplementary Figure 5)
4. Validation by Experiments
4.1 In vitro knockdown of POLR2Kinhibits bladder cancer cell proliferation and progression
Since increased POLR2K level is related to malignant proliferation, POLR2K might have critical effects on one or multiple steps of bladder cancer progression. To validate the effects of POLR2K on cell proliferation in bladder cancer cell, siRNA was used to knockdown POLR2K in bladder cancer 5637 and T-24 cells. To avoid the off target effects of POLR2K-siRNA, we used siRNA1 and siRNA2 to knockdown POLR2K, in the following experiments. The knockdown efficiency was assessed by RT-qPCR, and Western blotting (Figure. 8a-8c).The MTT experiment indicated that the viability of 5637 and T24 cells were significantly reduced after POLR2K knockdown (Figure. 8d-8e).In addition, Colony formation was performed to evaluate the growth capability of siRNA-transfected 5637 and T24 cancer cells. The results showed that there were significantly fewer colonies in the POLR2K knockdown group than in the Non-control group (Figure. 8f-8g), demonstrating that decreased expression of POLR2K could slow down bladder cancer cell growth. Taken together, these results indicated that in vitro knockdown of POLR2K suppressed bladder cancer cell proliferation and progression.
4.2 In vitro knockdown of POLR2K decreased bladder cancer cell invasion and migration
Since POLR2K overexpression is positively related with metastasis stage in BLCA patients based on our bioinformatics analysis (Figure 2f), we want to validate whether POLR2Kcould have an influence on invasion and migration in BLCA cells in vitro. Therefore, Wound Healing and Transwell experiments were performed in siRNA-transfected 5637 and T24 cell lines, results showing that migration and invasion ability of the POLR2K knockdown group was remarkably suppressed compared with that of Non-control group (8h-8j).Taken together, these results indicated that in vitro knockdown of POLR2K suppressed bladder cancer cell invasion and migration.
4.3 In vitro knockdown ofPOLR2K lead to decreased E2F1 level and G1 arrest.
Based on analysis of POLR2K functional network in bladder cancer, we have found that POLR2K is related with cell cycle (Figure 6e) and may be involved fundamentally in the V$E2F1_Q6 dataset. To validate this, q-PCR and Western Blot were used to examine E2F1 level after knocking down POLR2K.The results showed that upon POLR2K knockdown, E2F1 is diminished both in mRNA and protein level at 48 hours (Figure 8k-8m). In consistence with this, Flowjo analysis showed that G1 arrest occurs after knocking down POLR2K according to the PI staining (Figure 8n-8o). These findings show that POLR2K could modulate the expression of E2F1 in BLCA. Interestingly, we found that E2F1 could in return regulate POLR2K expression according to HA-E2F1_MCF-7_hg19_1 Gene Set in ENCODE Transcription Factor Binding Site Profiles(https://maayanlab.cloud/Harmonizome/gene_set/HA-E2F1_MCF-7_hg19_1/ENCODE+Transcription+Factor+Binding+Site+Profiles). The corresponding binding sequence was shown in Figure 8p. Furthermore, TCGA database showed a significantly positive correlation between POLR2K and E2F1 (Figure 8q), whose result is similar with ours based on tissue micro-assays (Supplementary Figure 6). Taken together, these results suggests that the POLR2K may interact with E2F1 in a positive loop where malignant phenotypes of bladder cancer cells could be aggregated.
4.4 PORL2K is a positive indicator in BLCA
To validate the clinical relevance of our bioinformatics findings, we detected POLR2K and E2F1 expression patterns in bladder cancer patients by immune-staining of a tumor tissue microarray. Consistent with our bioinfomatics findings in bladder cancer, the level of E2F1 and POLR2K was significantly increased compared with the matched non-cancerous tissues (Figure 8s-8u), and the expression of E2F1 was related to POLR2K expression in bladder cancer (Supplementary Figure 6.). These results indicate a hint that POLR2K could be a tumor promoter within the bladder cancer.