Abstract
Background and AimGranulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry.Methods and ResultsSpecific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining.ConclusionsTPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.
Highlights
Hepatocellular carcinoma (HCC) is a malignant neoplasm of hepatocytes and it accounts for more than 80% of primary liver cancers [1,2]
The curative treatment for HCC is surgical resection or liver transplantation, only a minority of HCCs are amenable to surgery as symptoms attributable to HCC usually develop in the late stages of the disease
The two cell lines were grown in AMEM medium containing 10% fetal bovine serum (FBS) and L-glutamine supplement at 37uC in 5% CO2
Summary
Hepatocellular carcinoma (HCC) is a malignant neoplasm of hepatocytes and it accounts for more than 80% of primary liver cancers [1,2]. HCC is a major global health problem. It shows significant regional variations with a very high incidence rate in Asia and Sub-Saharan Africa compared with the Western countries, where there is increasing incidence. Most of the HCC patients have advanced cirrhosis which leads to insufficient hepatic remnant and normal liver function after liver resection and surgical resection is not applicable for many patients. Another concern is the high recurrence rate after surgical resection. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry
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