That DNA-directed RNA polymerases (Pol I, Pol II and Pol III) is differentially expressed and deregulated has been detailed in multiple cancers . POLR2K, a subunit of this polymerases, participates in multiple steps of transcription .In this paper, we initially discovered that POLR2K was overexpressed in 408 BLCA samples from the TCGA and that high POLR2K expression may serve as an indicator of poor survival. Subsequently, further studies of the expression of POLR2K and its regulatory network will be necessary to obtain additional insight into the possible function of POLR2K in BLCA. Therefore, we conducted bioinformatic analyses of sequence data and hoped to motivate the future research of bladder cancer.
It has been a wearing problem for years since early detection approach of BLCA eluded many clinicians. Cystoscopy and urine cytology are currently employed to diagnose BLCA . However, cystoscopy is an invasive method and also low sensitivity for bladder carcinoma in situ. Urine cytology is a non-invasive and has a higher specificity but lower sensitivity for low-grade urothelial tumors. In spite of the search for urinary biomarkers for the early and non-invasive detection of BLCA, no available biomarkers are currently employed in clinical practice. Thus, potential BLCA biomarkers are urgently required to increase the early diagnosis of BLCA. We also demonstrated that CNVs and mRNA expression of POLR2K are much higher in bladder cancer tissues compared with normal tissues by analyzing transcriptional sequence data from clinical patient tissues. We discovered that POLR2K overexpression exists in many cases of bladder carcinogenesis. We think that POLR2K research could move the field of liquid biopsy biomarkers forward, which deserves additional clinical and research attention. And it needs to be verified whether POLR2K could be detectable in liquid biopsy.
It has been reported that CNVs could directly influence gene expression and have drastic phenotypic consequences due to altering gene dosage or disrupting coding sequences. [23, 24]. This work discovered that amplification was the main type of POLR2K change and POLR2K copy number was augmented, which was related to poor survival including overall survival (OS) and disease-free survival (DFS) (Supplementary Figure 1). In light of these findings, we inferred that alterations in chromosomal structure might be involved in the altered POLR2K expression as well as POLR2K dysfunction in BLCA. POLRAK genetic change could lead to alterations in numerous downstream signals that may ultimately result in carcinogenesis for POLR2K plays a critical role in multiple biological functions. In fact, neighboring gene networks in close proximity to POLR2K display amplification with varying strengths in BLCA. Meanwhile, associated functional network are found to be related to spliceosome signaling, mRNA surveillance and ribosome signaling. Therefore, the network of POLR2K alterations is related to posttranscriptional modulation, which is involved in protein translation as well as RNA splicing, in consistence with several other published results about the biological roles of POLR2K [25, 26]. Furthermore, related functional networks are also involved in positive regulation of cell motility, antigen binding and immune response-regulating signaling pathway. Therefore, the networks based on POLR2K genetic change is also associated with the tumor immune response, which is closely related to the mechanism by which tumor escapes from host immune system.
To reveal critical network of transcription factors, miRNAs and target kinases, GSEA analysis is performed. Our results imply that the functional network of POLR2K is involved generally in the ribosome, spliceosome, mRNA/ncRNA processing, cell cycle and DNA replication. Just the same as the mutation webwork, the functional association network that integrates the effects of POLR2K transcription alteration participates in RNA metabolic processes and gene expression regulation. As represented above, we conclude that POLR2K has a profound influence on the maintenance of short introns splicing [27, 28].
Oncogenic kinases play a critical role in coupling intracellular signaling pathways with extracellular signals, which promote cancer progression in all stages . We discovered that POLR2K in BLCA is related to a network of kinases including Kinase_MAPK7, Kinase_MAPK6 and Kinase_HIPK2. All of these kinases regulate mitosis, gene expression and apoptosis. Indeed, HIPK2 has been recognized as a signaling molecule which acts in various signal transduction pathways and cellular processes such as cell proliferation and apoptosis, transcriptional regulation and antiviral responses[30, 31] while MAPK7 could facilitate tumor escape from immune surveillance [32, 33] . Deregulated activity of HIPK2 may affect the genome integrity, leading to cancer development . In BLCA, POLR2K might modulate DNA replication, repair, and gene expression via HIP2 kinase.
“Continuous proliferation” has been proposed as top one of 10 hallmark features of tumors . One crucial explanation is that cell cycle-associated proteins, if aberrantly expressed, could contribute to cell cycle disorder in tumor cells, which results in decreased differentiation, abnormal proliferation and rapid progression in cancer cells. E2F1 is among key links in cell cycle modulation web-work. Abnormal E2F1 expression proactively is related to the tumor formation and progression of BLCA . There is one study has published that elevated levels of E2F1 is involved in shorter survival of bladder cancer patients , and another study showed that the POLR2K network of transcription factor targets is related to E2F1. Thus, these analyses suggest that E2F1 may be a critical target of POLR2K which modulates cell cycle and cancer progression in BLCA patients by acting through E2F1 transcription factor. Interestingly, according to known transcription factor binding site motifs from the TRANSFAC, we found that E2F1 could directly bind the promoter region of POLR2K likely to modulate the expression of POLR2K (http://amp.pharm.mssm.edu/Harmonizome/gene_set/E2F1/TRANSFAC+Predicted+Transcription+Factor+Targets). More convincingly, POLR2K gene is verified, by ChIP-seq in MCF-7 cell line , with transcription factor binding evidence in the HA-E2F1_MCF-7_hg19_1 transcription factor binding site profile from the ENCODE Transcription Factor Binding Site Profiles dataset (http://amp.pharm.mssm.edu/Harmonizome/gene_set/HA-E2F1_MCF7_hg19_1/ENCODE+Transcription+Factor+Binding+Site+Profiles). Thus, this analyses indicate that E2F1 could interact with POLR2K to modulate the survival of cancer cells.
Cancer and chronic inflammation could be linked together by activating innate immune responses through NLR and TLR signals . Chronic inflammation, mainly due to aberrant inflammasome or NF-κB activation, is closed coupled with cancer through TLRs-involved cytokine production. The IFN-regulatory factor family proteins are transcription factors with varying biological functions, which might render a microenvironment for immune evasion and tumor progression . Therefore, our analysis could represent that POLR2K might play a vital role in tumor microenvironment where POLR2K act as a chronic inflammation keeper and act as an immune surveillance regulator.
Our data mining also recognized some miRNAs that were related to POLR2K. These miRNAs are short non-coding RNA molecules that post-transcriptional regulate protein expression. Distinct miRNAs alterations could be used to characterize BLCA [43, 44]. The particular miRNAs in this paper are associated with cancer occurrence, metastasis and invasion. Indeed, miR-145, miR-202 and miR-374 has been proposed as diagnostic, prognostic or therapeutic marker of BLCA [45, 46] .miR-202 and miR-347 participate in invasion, metastasis and cancer progression[46, 47], while miR-145 modulates suppressor of cytokine signaling 7 (socs7) to enhance IFN-β expression, thus contributing to BLCA apoptosis. We speculate that deregulation of these miRNAs would be in consistence with the phenotype of POLR2K overexpression in BLCA, which should be further verified by experiments.
Our study presents striking evidence for the significance of POLR2K in urinary bladder carcinogenesis and demonstrate its potential as an early indicator in BLCA. This study imply that POLR2K overexpression in BLCA has profound impacts on tumor immune surveillance and on multiple steps of gene expression and of the cell cycle, thus contributing to immune surveillance evasion ultimately. POLR2K is particularly associated with some tumor-related transcription factors such as E2F1 and IRF family, miRNAs such as miRNA-145 and kinases such as HIPK2. Our study deploys current online websites to conduct bioinformatics analysis of bladder cancer. This strategy has superiorities in terms of simplicity and large sample size, which enables us to perform more large-scale POLR2K genomics research and functional studies free of charge, compared with classic chip screening.
Meanwhile, by using the TCGA database, we also face some limitations. The first one is that the BLCA samples in TCGA database include three ethnic group (Caucasian, African-american and Asian). Different genetic background of BLCA patients can influence gene expression profiles. The second limitation is that the BLCA samples is relatively small in stage 1(e.g., the relatively low number of severe cases), which may have limited the statistical significance of some correlations. Thus, adequate inclusion of each ethnic group and BLCA stages should be included in further prospective studies to know more about the generalisability of this study. The third one is that transcriptional sequencing could directly present information about protein level or protein activity, which should be tackled in subsequent studies with experiments. The fourth limitation is that the infiltration of tumor-associated normal cells has an profound influence on the analysis of clinical tumor samples by genomic methods, such as copy number data or gene expression profiles, and therefore, biological interpretation of the analysis results deserves considerable attention considering sample heterogeneity, which could be the reason why the 50 most frequently altered neighbor genes enriched in the CD56+NKCells and 721_B_lymphoblasts (Supplementary Table 8).