Comprehensive quantitative analysis of alternative splicing variants reveals the HNF1B mRNA splicing pattern in various tumour and non-tumour tissues

Jan Hojny,Marta Kalousova,Romana Michalkova,Quang Hiep Bui,Eva Krkavcova,Petra Kleiblova,Kristyna Nemejcova,Ivana Struzinska,Michaela Bartu,Pavel Dundr

doi:10.1038/s41598-021-03989-z

Jan Hojny, Marta Kalousova + Show 8 more

Open Access

https://doi.org/10.1038/s41598-021-03989-z

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Abstract

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.

Highlights

Abbreviations CRC Colorectal carcinoma clear cell renal carcinoma (ccRCC) Clear cell renal carcinoma prostate carcinoma (PC) Prostate carcinoma high-grade serous carcinoma (HGSC) High-grade serous carcinoma T Tumour NT Non-tumour p Deletion/intronization of 5′ exon part Δ Deletion/intronization of whole exon alternative splicing variants (ASVs) Alternative splicing variant NGS Next-generation sequencing RNA-Seq RNA next-generation sequencing droplet digital PCR (ddPCR) Droplet digital PCR RNA Quality Number (RQN) RNA quality number
Our previous work showed a loss of the Hepatocyte nuclear factor-1-beta (HNF1B) protein expression in several solid tumours, and suggests that HNF1B may act in a tumour suppressive fashion in colorectal carcinoma (CRC)[15], clear cell renal carcinoma and chromophobe renal cell carcinoma[16], prostate carcinoma (PC)[17], and high-grade serous carcinoma (HGSC)[18]
Considering the highest overall HNF1B mRNA expression in the NT kidney tissue compared to other tissues[7], these variants were not analysed in other sample sets, and we considered them as minor HNF1B ASVs

Summary

Introduction

Abbreviations CRC Colorectal carcinoma ccRCC Clear cell renal carcinoma PC Prostate carcinoma HGSC High-grade serous carcinoma T Tumour NT Non-tumour p Deletion/intronization of 5′ exon part Δ Deletion/intronization of whole exon ASV Alternative splicing variant NGS Next-generation sequencing RNA-Seq RNA next-generation sequencing ddPCR Droplet digital PCR RQN RNA quality number. Our previous work showed a loss of the HNF1B protein expression in several solid tumours, and suggests that HNF1B may act in a tumour suppressive fashion in colorectal carcinoma (CRC)[15], clear cell renal carcinoma (ccRCC) and chromophobe renal cell carcinoma[16], prostate carcinoma (PC)[17], and high-grade serous carcinoma (HGSC)[18]. 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. 1), which included alternatively spliced exon 3p (exon 3 which lacks 78 bp at 5′; known as variant NM_01165923.4), Δ7 (deletion/intronization of exon 7; predicted in XM_011525161.1), Δ7–8 (known in transcript NM_001304286.2 where was described in combination with 3p variant), Δ8 (predicted in XM_011525164.1 and XM_011525160.1), Δ5–8 and Δ6–8 (both not previously described in databases). HNF1B ASVs 3p, Δ7, Δ7–8, Δ5–8, and Δ6–8 ASVs were chosen for subsequent precise quantitative characterization in a wide spectrum of tumour (T) and non-tumour (NT) samples in a number of tissues presented in this work

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