Abstract:Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor crucial for the development of several tissues, and a promising biomarker of certain solid tumours. Thus far, two HNF1B alternative splicing variants (ASVs) have been described, however, the complete spectrum, prevalence and role of HNF1B ASVs in tumorigenesis are unclear. Considering the equivocal data about HNF1B ASVs and expression presented in literature, our aim was to characterize the spectrum of HNF1B mRNA splicing variants across differ… Show more
“…One of the possible explanations for this difference may lie in to the fact that the expression on a protein level and mRNA level may be difficult to correlate for several reasons including translation rates, translation rates modulations, modulations of protein half-life, protein synthesis delay, and protein transport 55 . Moreover, in our recent study, we have shown that there are several transcriptional variants of HNF1B which have not been recognized yet (contrary to the previous knowledge about three possible HNF1B isoforms) 56 . The positive correlation between HNF1B and EZH2 expression in our study was also reflected in the observed positive correlation between AR expression and expression of both EZH2 and HNF1B.…”
Section: Discussioncontrasting
confidence: 67%
“…After RNA quality characterization, 3.75 µg of the total RNA of each sample (where available) was treated by DNase I (Thermo Fisher) prior to one-step cDNA synthesis in 40 µl reaction using SuperScript III Reverse Transcriptase (Thermo Fisher) with random hexamers (Roche) as described previously 56 . All RNA samples were processed according to the Digital MIQE Guidelines 65 .…”
Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target. Hepatocyte nuclear factor 1 beta (HNF1B) is an important tissue specific transcription factor, which is essential for the embryonic development of several organs including the kidney, pancreas, liver, biliary tract, genital tract, and gastrointestinal system 1,2. Heterozygous germline mutations are associated with several congenital diseases of the kidney, pancreas, and urogenital tract, and the role of HNF1B in these diseases is well known 3-5. However, despite the increased awareness that HNF1B plays an important role in the carcinogenesis of several solid tumors, the exact significance of HNF1B in carcinogenesis is not yet fully understood 6-9. The regulatory mechanisms and pathways in which HNF1B is involved in the process of carcinogenesis are not clear, but it appears that HNF1B may act as either oncogene or oncosuppressor gene based on the type of tumor and its histogenesis 6,7,10. In the prostate, an increased expression of HNF1B seems to be protective against prostate cancer, and HNF1B therefore has been attributed an oncosuppressive role 10. However, the levels of protein expression of HNF1B in prostate cancer are not well known, and the mechanisms playing role in its regulation are of interest. According to current knowledge, HNF1B is commonly inactivated in prostate cancer, especially due to HNF1B promoter methylation, which occurs in about 50% of cases 11. Another recently suggested possible mechanisms of HNF1B inactivation is the effect of the enhancer of zeste homolog 2 (EZH2), the overexpression of which has been suggested to downregulate the expression of HNF1B 12,13. EZH2 is a gene belonging to the category of Polycomb
“…One of the possible explanations for this difference may lie in to the fact that the expression on a protein level and mRNA level may be difficult to correlate for several reasons including translation rates, translation rates modulations, modulations of protein half-life, protein synthesis delay, and protein transport 55 . Moreover, in our recent study, we have shown that there are several transcriptional variants of HNF1B which have not been recognized yet (contrary to the previous knowledge about three possible HNF1B isoforms) 56 . The positive correlation between HNF1B and EZH2 expression in our study was also reflected in the observed positive correlation between AR expression and expression of both EZH2 and HNF1B.…”
Section: Discussioncontrasting
confidence: 67%
“…After RNA quality characterization, 3.75 µg of the total RNA of each sample (where available) was treated by DNase I (Thermo Fisher) prior to one-step cDNA synthesis in 40 µl reaction using SuperScript III Reverse Transcriptase (Thermo Fisher) with random hexamers (Roche) as described previously 56 . All RNA samples were processed according to the Digital MIQE Guidelines 65 .…”
Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target. Hepatocyte nuclear factor 1 beta (HNF1B) is an important tissue specific transcription factor, which is essential for the embryonic development of several organs including the kidney, pancreas, liver, biliary tract, genital tract, and gastrointestinal system 1,2. Heterozygous germline mutations are associated with several congenital diseases of the kidney, pancreas, and urogenital tract, and the role of HNF1B in these diseases is well known 3-5. However, despite the increased awareness that HNF1B plays an important role in the carcinogenesis of several solid tumors, the exact significance of HNF1B in carcinogenesis is not yet fully understood 6-9. The regulatory mechanisms and pathways in which HNF1B is involved in the process of carcinogenesis are not clear, but it appears that HNF1B may act as either oncogene or oncosuppressor gene based on the type of tumor and its histogenesis 6,7,10. In the prostate, an increased expression of HNF1B seems to be protective against prostate cancer, and HNF1B therefore has been attributed an oncosuppressive role 10. However, the levels of protein expression of HNF1B in prostate cancer are not well known, and the mechanisms playing role in its regulation are of interest. According to current knowledge, HNF1B is commonly inactivated in prostate cancer, especially due to HNF1B promoter methylation, which occurs in about 50% of cases 11. Another recently suggested possible mechanisms of HNF1B inactivation is the effect of the enhancer of zeste homolog 2 (EZH2), the overexpression of which has been suggested to downregulate the expression of HNF1B 12,13. EZH2 is a gene belonging to the category of Polycomb
“…Normalized NGS data (ratio of individual splicing reads) showed a similar relative expression of the studied variants when compared to ddPCR quantitative analysis (Table 3 ). Moreover, we did not identify any novel HNF1B splicing variants by capture RNA-Seq other than those reported in our previously published work, where deep sequencing of individual HNF1B exon-exon junctions was used, which could potentially miss the ASVs 19 . Figure 5 Representative example of the HNF1B splicing pattern based on the capture RNA-Seq of a kidney T sample visualised as a sashimi plot in IGV (Broad Institute).…”
Section: Resultsmentioning
confidence: 76%
“…The HNF1B reference transcript comprises nine coding exons and produces full length 557 amino acid protein (RefSeq NM_000458). In our previous work, we performed mainly qualitative analyses and described 45 splicing events in a limited number of samples (eight representative samples per analysed tissue pool) 19 . By this approach we have identified predominant and predominant-candidate HNF1B ASVs (Fig.…”
Section: Introductionmentioning
confidence: 99%
“…POU H POU homeodomain. The scheme of HNF1B transcripts is based on previous findings 19 and actual RefSeq database information. …”
Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor and putative biomarker of solid tumours. Recently, we have revealed a variety of HNF1B mRNA alternative splicing variants (ASVs) with unknown, but potentially regulatory, functions. The aim of our work was to quantify the most common variants and compare their expression in tumour and non-tumour tissues of the large intestine, prostate, and kidney. The HNF1B mRNA variants 3p, Δ7, Δ7–8, and Δ8 were expressed across all the analysed tissues in 28.2–33.5%, 1.5–2%, 0.8–1.7%, and 2.3–6.9% of overall HNF1B mRNA expression, respectively, and occurred individually or in combination. The quantitative changes of ASVs between tumour and non-tumour tissue were observed for the large intestine (3p, Δ7–8), prostate (3p), and kidney samples (Δ7). Decreased expression of the overall HNF1B mRNA in the large intestine and prostate cancer samples compared with the corresponding non-tumour samples was observed (p = 0.019 and p = 0.047, respectively). The decreased mRNA expression correlated with decreased protein expression in large intestine carcinomas (p < 0.001). The qualitative and quantitative pattern of the ASVs studied by droplet digital PCR was confirmed by next-generation sequencing, which suggests the significance of the NGS approach for further massive evaluation of the splicing patterns in a variety of genes.
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