Upregulation of ABLIM1 Differentiates Intrahepatic Cholangiocarcinoma from Hepatocellular Carcinoma and Both Colorectal and Pancreatic Adenocarcinoma Liver Metastases

Abstract

Background: Altered gene expression in cancers holds great potential to improve the diagnostics and differentiation of primary and metastatic liver cancers. In this study, the expression of the protein-coding genes ring finger protein 135 (RNF135), ephrin-B2 (EFNB2), ring finger protein 125 (RNF125), homeobox-C 4 (HOXC4), actin-binding LIM protein 1 (ABLIM1) and oncostatin M receptor (OSMR) and the long non-coding RNAs (lncRNA) prospero homeobox 1 antisense RNA 1 (PROX1-AS1) and leukemia inhibitory factor receptor antisense RNA 1 (LIFR-AS1) was investigated in hepatocellular carcinoma, cholangiocarcinoma, colorectal liver metastases and pancreatic ductal adenocarcinoma liver metastases.

Methods: This study included 149 formalin-fixed, paraffin-embedded samples from 80 patients. After RNA isolation, quantification, reverse transcription and preamplification, real-time qPCR was performed. The gene expression between different groups was calculated relative to the expression of the reference genes using the ∆∆Cq method and statistically analyzed. The expression of the genes was additionally analyzed using the AmiCA and UCSC Xena platforms.

Results: In primary cancers, our results showed differential expression between primary tumors and healthy tissues for all the genes and lncRNA examined. Moreover, we found downregulation of RNF135 in hepatocellular carcinoma, downregulation of OSMR in colorectal liver metastases and upregulation of HOXC4 in cholangiocarcinoma compared to primary liver cancers and metastatic cancers. The major finding is the upregulation of ABLIM1 in cholangiocarcinoma compared to hepatocellular carcinoma, colorectal liver metastases, pancreatic ductal adenocarcinoma liver metastases and healthy liver tissue. We propose ABLIM1 as a potential biomarker that differentiates cholangiocarcinoma from other cancers and healthy liver tissue.

Conclusions: This study emphasizes the importance of understanding the differences in gene expression between healthy tissues and primary and metastatic cancers and highlights the potential use of altered gene expression as a diagnostic biomarker in these malignancies.

Keywords: ABLIM1; cholangiocarcinoma; colorectal carcinoma; colorectal liver metastases; gene expression; hepatocellular carcinoma; lncRNA; mRNA; pancreatic adenocarcinoma liver metastases; pancreatic ductal adenocarcinoma.

Figure 1

Expression of (A) RNF135, (B) EFNB2, (C) RNF125, (D) HOXC4 and (E) ABLIM1 in HCC, HCC NATs, CCA, CCA NATs, CRLM and PDLM. The expression is presented as the ∆Cq values and the significance between the different groups is shown. The whiskers of the boxplots represent the values of the upper and lower quartiles and the dots represent outliers. The line dividing the boxplot represents the median and the cross represents the mean. Legend: * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.001, **** p ≤ 0.0001; Cq, quantification cycle; HCC NAT, normal liver tissue adjacent to hepatocellular carcinoma; HCC hepatocellular carcinoma, CCA NAT, normal liver tissue adjacent to cholangiocarcinoma; CCA, cholangiocarcinoma; CRLM, colorectal liver metastases; PCLM, pancreatic ductal adenocarcinoma liver metastases.

Figure 2

Expression of OSMR and PROX1-AS1. The expression is plotted as the ∆Cq values and the significance between different groups is presented. The whiskers of the boxplots represent the values of the upper and lower quartiles and the dots represent the outliers. The line dividing the boxplot represents the median and the cross represents the mean. (A) OSMR expression in CRC, CRC NAT, CRLM, PCLM, HCC, HCC NAT, CCA and CCA NAT. (B) PROX1-AS1 expression in PDAC, PDAC NAT, PCLM, CRLM, HCC, HCC NAT, CCA and CCA NAT. Legend: * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.001, **** p ≤ 0.0001; Cq, quantification cycle; HCC NAT, normal liver tissue adjacent to hepatocellular carcinoma; HCC hepatocellular carcinoma, CCA NAT, normal liver tissue adjacent to cholangiocarcinoma; CCA, cholangiocarcinoma; CRC NAT, normal colon tissue adjacent to colorectal cancer; CRC, colorectal carcinoma; CRLM, colorectal liver metastases; PDAC NAT, normal pancreatic tissue adjacent to pancreatic ductal adenocarcinoma; PDAC, pancreatic ductal adenocarcinoma; PCLM, pancreatic ductal adenocarcinoma liver metastases.

Figure 3

Expression of ABLIM1 in the AmiCA and Xena platforms. (A) ABLIM1 expression in TCGA projects on the AmiCA platform. The log2 fold change between cancer and NAT for each cancer type is shown. (B) ABLIM1 expression in the Xena platform. The expression is plotted as the log2(norm_count+1) values and the significance between different groups is presented. The whiskers of the boxplots represent the values of the upper and lower quartiles and the dots represent the outliers. The line dividing the boxplot represents the median and the cross represents the mean. Legend: *** p ≤ 0.001, **** p ≤ 0.0001; HCC NAT, normal liver tissue adjacent to hepatocellular carcinoma; HCC hepatocellular carcinoma, CCA NAT, normal liver tissue adjacent to cholangiocarcinoma; CCA, cholangiocarcinoma.