RT‐PCR detection of fibronectin EDA+ and EDB+ mRNA isoforms: Molecular markers for hepatocellular carcinoma

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Abstract
Alternative splicing of fibronectin pre-mRNA has been shown to be independently regulated at the EDA and EOB regions in a tissue and developmental stage-specific manner. In this study, RT-PCR approaches were developed for the detection of EDA and EDB FN mRNA isoforms in hepatocarcinoma cells (SK-Hep-I) grown in vitro and in human liver biopsies. While EDA+ and EDB+ isoforms were not present in control adult liver, they were detectable in the hepatocarcinoma cells and in fetal liver. The RT-PCR analysis, extended to biopsies of malignant and non-malignant hepatic tissues, showed that FN mRNAs containing the EDA and EDB sequences were present in the 14 hepatocelfular carcinomas (HCCs) tested but absent in the non-tumorous liver tissues (i.e., normal parenchyma, nonspecific reactive and chronic hepatitis, steatosis). The EDB+ FN mRNA isoforms were also detected in 3 cases of benign neoplasm (hepatocellular adenoma, HCA, I; nodular focal hyperplasia, NFH, 2), while the EDA+ was only detectable in I of the 2 cases of NFH. In addition, both EDA+ and EDB+ isoforms were expressed in 5 out of 9 cirrhotic livers surrounding the tumors. This molecular analysis, which can also be performed on small liver biopsies (2 mg), may therefore be a useful additional tool in the diagnosis of HCC.