Identification and Characterization of Tropomyosin 3 Associated with Granulin-Epithelin Precursor in Human Hepatocellular Carcinoma

Background and AimGranulin-epithelin precursor (GEP) has previously been reported to controlcancer growth, invasion, chemo-resistance, and served as novel therapeutictarget for cancer treatment. However, the nature and characteristics of GEPinteracting partner remain unclear. The present study aims to identify andcharacterize the novel predominant interacting partner of GEP using co-immunoprecipitationand mass spectrometry. Methods and ResultsSpecific anti-GEP monoclonal antibody was used to capture GEP and its interactingpartner from the protein extract of the liver cancer cells Hep3B. The precipitatedproteins were analyzed by SDS-PAGE, followed by mass spectrometry and theprotein identity was demonstrated to be tropomyosin 3 (TPM3). The interactionhas been validated in additional cell models using anti-TPM3 antibody andimmunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressionswere then examined by real-time quantitative RT-PCR in clinical samples, andtheir transcript levels were significantly correlated. Elevated TPM3 levelswere observed in liver cancer compared with the adjacent non-tumorous liver,and patients with elevated TPM3 levels were shown to have poor recurrence-freesurvival. Protein expression of GEP and TPM3 was observed only in the cytoplasmof liver cancer cells by immunohistochemical staining. ConclusionsTPM3 is an interacting partner of GEP and may play an important role inhepatocarcinogenesis.