Preliminary Verification Of Thymosin β4as A New Candidate Marker Of Hepatocellular Carcinoma And Studies On Its Influence On The Biological Behavior Of Hepatocellular Carcinoma Cells

Background&ObjectiveThe magnetic bead separation coupled with matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) isincreasingly being applied to the areas of biomarker discovery. Using this novel system,we have found a distinguished differentially expressed protein peak in sera of patientswith hepatocellular carcinoma (HCC), which was identified as thymosin β4(Tβ4). Inrecent years, many studies have indicated that Tβ4is overexpressing in breast cancer,lung cancer, pancreatic cancer, colorectal cancer and other tumor types. It has closelyrelated with tumor occurrence, development and metastasis. The mechanism may involvestimulating tumor angiogenesis, promoting tumor cell epithelial mesenchymaltransformation, enhancing tumor cell migration, invasion and metastasis ability, as wellas inducing tumor cell anti-apoptosis and drug resistance. However, until now, there isn’tany data regarding the relationship between Tβ4and HCC. In this project, we willconduct a large-scale analysis using more HCC samples and exprerimental evidence tofurther confirm the role of Tβ4in promoting HCC development. Finally, the results fromour studies will provide theoretical and experimental basis for the development of a newTβ4-targeting model for personalized diagnosis and therapy of HCC.Methods1. Using the magnetic bead separation coupled with MALDI-TOF MS to discoverdistinguished differentially expressed proteins in sera of patients with HCC.2. Using a variety of experimental methods to detect the expression of Tβ4in severalhepatoma cell lines.(1) Using indirect immunofluorescence laser confocal assays to detect the localization ofTβ4in hepatoma cells. (2) Using quantitative real-time-PCR to determine Tβ4mRNAlevel in immortalizedliver cell line and several HCC cell lines.(3) Using flow cytometry to evaluate the Tβ4protein level in immortalized liver cell lineand several HCC cell lines.3. Tβ4was measured with a newly developed commercial enzymelinked immunosorbentassay in serum with healthy people, chronic hepatitis B, HBV-infected cirrhosis andHBV-infected hepatocellular carcinoma.4. Detection of the expression of Tβ4in HCC specimens, and futher analyzed therelationship between Tβ4expression in HCC specimens and HCC sera, as well as,clinical pathologic features of HCC.(1) Immunohistochemistry (IHC) was used to detect the expressions of Tβ4in clinicalHCC tissues.(2) Analysis of the correlation between Tβ4expression in HCC tissues and Tβ4levels inHCC sera as well as between clinical pathologic features of HCC.5. Effects of Tβ4on proliferation, migration, and invasion of HCC cells.(1) MTT assays was performed to determine cells proliferation activity.(2) Wound healing assays was performed to detect cells migration activity.(3) Transwell assays was performed to evaluate cells invasion activity.6. Using adherent model and anoikis model, hepatoma cells were cultured and thefollowing assays were performed.(1) Using Annexin V/FITC to assess the level of apotosis after different treatments.(2) Using Western Blot analysis to determine the expression level of apoptotic proteins,anti-apoptotic proteins, cell survival signaling pathway key molecule ERK and nucleartranscription factor NF-κB after different treatments.Results1. Using the magnetic bead separation coupled with MALDI-TOF MS, we have firstly discovered a distinguished differentially expressed protein Tβ4in sera of patients withHCC.2. The localization of Tβ4in hepatoma cell was in the cytoplasm.3. The level of Tβ4expression in several hepatoma cell lines was different, which wasnoticeably upregulated in most of the tested HCC cells, compared to that of chang livercells, especially highly metastatic hepatoma cell MHCC-97H was about double than thechang liver cells.4. The levels of Tβ4in CHB, HBV-Cirrhosis and HBV-HCC patients presented risingtrend with the progression of the liver disease. The Tβ4in HBV-HCC patients wasmarkedly increased as compared with that in healthy group.5. Tβ4more efficiently enhances proliferation, migration,and invasion of HCC cells invitro.6. The results of clinical research of HCC specimens:(1) Tβ4expression in tumor tissues was higher than paratumor tissues from HCCpatients.(2)A positive correlation between serum Tβ4levels and Tβ4expressions in tumor tissuesfrom HCC.(3) Tβ4expressions in tumor tissues correlated well with serum AFP, TNM stage andvascular invasion (P <0.05).7. Tβ4endowed hepatoma cells resistance to anoikis through activation of ERK.ConclusionTβ4was suggested to be closely related to hepatocellular carcinoma developmentand metastasis. The level of Tβ4expression presented rising trend with the progression ofthe liver disease and step by step influenced the level of activation of a series of signalways in intracellular. As well as, increased the ability of hepatoma cells proliferation,migration and invasion. Finally, promoted tumor unlimited growth and tumor angiogenesis. The results of these experiments gave preliminary validation of Tβ4as acandidate marker of HCC. Thus, understanding the role in cancer development, whichcan provide theoretical and experimental basis for the development of a newTβ4-targeting model for personalized diagnosis and therapy of HCC.