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. 2016 Aug 26;17(1):683.
doi: 10.1186/s12864-016-3029-z.

Global Profiling of Alternative RNA Splicing Events Provides Insights Into Molecular Differences Between Various Types of Hepatocellular Carcinoma

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Free PMC article

Global Profiling of Alternative RNA Splicing Events Provides Insights Into Molecular Differences Between Various Types of Hepatocellular Carcinoma

Marie-Pier Tremblay et al. BMC Genomics. .
Free PMC article

Abstract

Background: Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data.

Results: Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control.

Conclusions: This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

Keywords: Gene dysregulation; Hepatitis B virus; Hepatitis C virus; Liver cancer; RNA splicing.

Figures

Fig. 1
Fig. 1
Alternative splicing of HBV- and HCV-associated hepatocellular carcinoma. a Classification of the TCGA RNA sequencing data for HCC and healthy tissues. b Overview of the strategy used to identify the changes in splicing for each type of HCC. c The splicing events list for HBV-associated HCC (THBV/NNoV) and HCV-associated HCC (THCV/NNoV) was filtered to keep only significant ASEs
Fig. 2
Fig. 2
Global profiling of alternative splicing event modifications in HBV- and HCV-associated HCC. a Alternative splicing event modifications in HBV-associated HCC (THBV/NNoV) and HCV-associated HCC (THCV/NNoV) are presented with their associated changes in gene expression. The graphs represent the relation between the difference of splicing for each hepatitis-associated HCC compared with healthy non-viral tissues, and the difference in gene expression for each of these alternative splicing event modifications. b Heatmap representations of isoform ratios (PSI values) for all tissues analyzed in the current study. The first heat map shows PSI values for HBV-associated HCC. HBV-associated HCC tissues (THBV) are shown in red, and the comparative healthy tissues are shown in green (NNoV). The second heat map shows PSI values for HCV-associated HCC. HCV-associated HCC tissues (THCV) are shown in blue, and the comparative healthy tissues are shown in green (NNoV)
Fig. 3
Fig. 3
AS modifications in transcripts encoded by tumor suppressors and oncogenes in HBV- and HCV-associated HCC. a Alterations in the AS patterns of transcripts encoded by tumor suppressors in HBV- and HCV-associated HCC. Some transcripts have multiple ASEs that are modified. Red bars indicate negative Delta PSI values, and blue bars represent positive Delta PSI values. b Alterations in the AS patterns of transcripts encoded by oncogenes in HBV- and HCV-associated HCC. The tumor suppressors and oncogenes were selected based on the TSGene [64] and allOnco databases (http://www.bushmanlab.org/links/genelists)
Fig. 4
Fig. 4
Validation of ASEs dysregulated in HCC. a Overview of the two isoforms encoded by OSBPL6, VPS13A and ZNF692 genes. Exons are depicted in red and the intervening introns are shown as thin black lines (not to scale). The primers used to detect the isoforms by RT-PCR assays are shown in gray and the sizes of the expected amplicons are also indicated. b cDNA obtained from tissues were analyzed by PCR using specific primers to detect both isoforms of the transcripts encoded by the OSBPL6, VPS13A and ZNF692 genes. Capillary electrophoregrams of the PCR reactions are shown. The positions and the amplitude of the detected amplicons are highlighted by red boxes. The positions of the internal markers are also indicated. The Delta PSI values for both the RNA-Seq and experimental validation assays are shown on the right
Fig. 5
Fig. 5
Comparison between HBV-, HCV-, HBV&HCV-, and non-viral-associated HCC. a The list displays the common differentially spliced transcripts for the various types of HCC with the corresponding Delta PSI values, the associated gene expression (in Log2), and the associated biological processes. b Comparison of the genes with dysregulated ASEs between HBV-, HCV-, HBV&HCV-, and virus-free HCC
Fig. 6
Fig. 6
RNA splicing factors in HCC. a Iris Graph representing the expression profile of splicing factors for HBV-associated HCC. Differences in gene expression levels are shown on a logarithmic color scale (Log2), from red (negative changes in expression) to blue (increase in gene expression). b Expression of splicing factors in both normal liver tissues and hepatocellular carcinomas. These images were extracted from the Human Protein Atlas database, according to its academic usage permission (see Ref. [65] and www.proteinatlas.org). The images show the results of immunohistochemical staining using specific antibodies (complete list and experimental protocol found at www.proteinatlas.org), followed by detection with horseradish peroxidase. Immunohistochemical staining was performed on both normal and HCC tissues for three different splicing factors (ESRP1, CWC27, and DDX41). c Misregulation of splicing factors alternative splicing in HBV- (THBV/NNoV, yellow), HCV- (THCV/NNoV, blue), HBV&HCV- (THBV&HCV/NNoV, green) and virus-free HCC (TNoV/NNoV, red). The delta PSI values are represented in red (negative delta PSI values) and in blue (positive delta PSI values). d Expression levels of hnRNPC in patients with or without tumors (left panel). Kaplan–Meier overall survival curve (right panel) for HCC patients expressing high (red) or low (blue) levels of hnRNPC. HNRNPC transcript level was negatively correlated with overall survival
Fig. 7
Fig. 7
Modifications to AS of 96 transcripts in response to knockdown of splicing factors with specific siRNAs. Using specific siRNAs, twelve splicing factors (U2AF2, SF3A2, RBM8A, RBM4, PRPF4B, NOVA1, KHSRP, HNRPU, HNRPL, HNRPH1, HNRPC, and HNRPA1) were individually knocked-down in different cell lines to assess their implication in splicing of 96 differents transcripts. Individual knockdowns and ASEs are shown to indicate which knockdowns caused a shift in alternative splicing in various cell lines (PC-3, SKOV3, NIH:OVCAR-3, MDA-MB-231, MCF7). Each column represents a distinct knockdown performed with specific siRNAs. The changes in PSI values are displayed. The map depicts the changes in PSI values in a color-coded scale. White areas indicate no shifts. Asterisks indicate transcripts for which AS was altered in HBV-associated HCC
Fig. 8
Fig. 8
Involvement of the HBx protein from HBV in AS. a Immunoblotting analysis using anti-HA antibody for the detection of HBx-HA protein from the cell lysates isolated after 5 days of transfection with pLenti6V5A-HBx. Control untransfected cells (T(−)) were also used in this assay. b List of ASEs common to HBV-associated HCC and HBx-expressing cells. c Example of an ASE modified following the expression of HBx. Overview of the two isoforms encoded by ENO3 gene. Exons are depicted in red and the intervening introns are shown as thin black lines (not to scale). The primers used to detect the isoforms by RT-PCR assays are shown in gray and the sizes of the expected amplicons are also indicated (top panel). RT-PCR reactions were performed on control cells (T(−)) and cells expressing HBx (HBx) using specific primers to detect both isoforms of the transcripts encoded by the ENO3 gene. Capillary electrophoresis was performed and an image of the detected reaction products is shown (lower panel). The positions of the expected amplicons are indicated by arrows. d Mass spectrometry analysis of cellular proteins interacting with HBx. The average H/L ratios of the HBx affinity purification-mass spectrometry experiments were plotted versus the total intensities. The red dots indicate cellular proteins involved in RNA splicing

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