| [Background and aims] Primary liver cancer is a kind of high malignant and high mortality digestive system tumor. Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer; and metastasis and recurrence are the main factors affecting pateints'survival. Discovering the protein markers associated with tumor metastasis and recurrence of HCC and exploring the molecular mechanism have been the focus of the translational study for HCC. Besides alpha-fetoprotein (AFP), there is lack of effective tumor marker in clinical routine examination for liver cancers. Peripheral blood plasma contains large abundant proteins, and the pathological changes of tumor in liver can be reflected by the changes about type and/or quantity of proteins in the blood. Such characteristic changes may serve as indicators of real-time monitoring for development of the disease, so they are important for diagnosis and therapy. In this study, a novel approach was applied to discover the proteins released into the blood by solid tumors, and the resulting candidate protein markers were then validated with the tumor tissues and the blood samples derived from the patients with HCC.[Methods] We selected HCC tiussue for Primary organ cultures were estalished with the tumor and the adjacent tissues from 12 HCC patients with the epithelial cells specific serum-free medium, and then the conditional media (CM) were collected. The total proteins in 6 pairs of CM samples were separated by one-dimensional polyacrylamide gel electrophoresis, and analyzed with liquid chromatography-tandem mass spectrometry. DAVID bioinformatics resources (http://david.abcc.ncifcrf.gov/ home.jsp) were used for bioinformatics analysis to establish the HCC-related protein database. The interesting proteins from the database were selected for validation. Expression status of MMP1 and MMP7 was analyzed by immunohistochemical staining, with 71 paraffin-embedded tumor tissues from HCC patients. The circulating plasma protein levels of NID1, MMP1 and MMP7 were analyzed by enzyme-linked immunosorbent assay (ELISA), with a series of 230 blood samples, including 126 HCC patients, and 104 normal controls.[Results] Combining data of the CM samples from the tumor tissues and the adjacent tissues,1365 proteins were identified and a HCC-derived proteome database was generated. By subcellular location analysis, among these CM proteins,16% were annotated as extracellular or secreted proteins,3% were membrane associated proteins, 21% were cytoplasmic proteins. Immunohistochemistry resluts showed that MMP1 expresion in the surrounding nontumoral liver tissue were highly correlated with infiltration of liver capsule and survival of the patients. The protein expression of MMP7 in adjacent liver tissues was significantly higher than that in the tumor tissues. ELISA results indicated that the plasma protein levels of MMP1, MMP7 and NID1 were elevated in HCC patients compared to that of healthy subjects. The plasma levels of NID1 in HCC patients were significant negative correlated with infiltration of the liver capsule and background of hepatitis. The plasma levels of MMP7 in HCC patients were positive correlated with satellite tumor, number of tumors, and hepatitis. The preliminary results showed that combination of MMP1, NID1, and AFP had a higher sensitivity and specificity than any single marker.[Conclusions] The HCC-derived'secretory/released proteome'generated in this study provides a credible repertoire of potential biomarkers in blood for guiding both detection and prognosis of HCC. The plasma protein levels of MMP1, MMP7, and NID1 are correlated with the tumor metastasis and recurrence of hepatocellular carcinoma. A tumor marker panel (MMP1, NID1 and AFP) combind with clinical factors can improve the accuracy of diagnosis and prognosis.
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