Screening And Biological Functional Studies Of Biomarkers In Hepatocellular Carcinoma

Part1Screening and Identification of biomarkers in hepatocellular carcinoma by a proteomic approachOBJECTIVE:In order to search for a useful biomarker in hepatocellular carcinoma, this study will use a two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis to profile and identify the differentially expressed proteins between HCC tumor tissues with histological evidence and the adjacent non-tumour tissues. Clinical correlation of differentially expressed protein with different clinicopathological features in HCC was also evaluated.METHODS:For proteomics analysis,27pairs of human frozen HCC histologically-confirmed tissues and the adjacent non-tumor tissues were collected. Proteins from tumor and matched non-tumor tissues were extracted and seprated by2-DE proteomics profiling. The gels were stained using Coomassie Brilliant Blue, and then the protein spots were detected, quantified, and matched using PDQuest2D analysis software. After in-gel tryptic digestion, proteins identification was performed using the MALDI-TOF MS. The expression of peroxiredoxin3(PRDX3) at the mRNA and protein levels was confirmed by RT-PCR and western blot in HCC cell lines, and HCC samples, and further analysed by immunohistochemistry in119HCC tissue samples and36adjacent non-tumor/normal liver specimens of different clinical pathological stages.RESULTS:(1)22out of43dysregulated proteins were identified by using the2-DE-based proteomic profiling approach, including15upregulated proteins, including Formiminotransferase cyclodeaminase, Aldehyde dehydrogenase, mitochondrial, Retinal dehydrogenase1, Superoxide dismutase [Cu-Zn], Glutathione S-transferase A1, Calcineurin subunit B type1, Aminoacylase-1, T-complex protein11-like protein1, Prohibitin, Sulfotransferase1A2,3-mercaptopyruvate sulfurtransferase, Thioredoxin-dependent peroxide reductase,mitochondrial, Calumenin, Ketohexokinase; and7downregulated proteins,including CAP-Gly domain-containing linker protein4, Apolipoprotein A-I, Disks large homolog3, Peptidyl-prolyl cis-trans isomerase A, Nucleoside diphosphate kinase A, Cystatin-B. These proteins were functionally involved in diverse biological processes, including metabolism (9/22,40.9%), anti-apoptosis (4/22,18.2%), signal transduction (7/22,31.8%), redox regulation (5/22,22.7%), structural protein (3/22,13,6%), detoxification (2/22,9.1%), chaperone (3/22,13.6%), proteolysis (1/22,4.5%), DNA synthesis and cell differentiation (2/22,9.1%).Among these, thioredoxin-dependent peroxide reductase, mitochondrial peroxiredoxin3(PRDX3) is of great interest due to its high expression in HCC and relationship with oxidative stress in mitochondrial. (2) RT-PCR analysis showed that relative expression abundance of PRDX3at the mRNA level in HCC was0.79±0.14, which was significantly higher than that in non-tumor tissues (0.15±0.05)(p<0.05); The overall expression levels in the tumor tissues (0.82±0.13) were higher than those in the matched non-tumor tissues (0.52±0.09)(p<0.05), it was considered statistically significant.(3) Imunohistochemistry analysis showed that overexpression of PRDX3was associated with94.9%HCC, and correlated with poor differentiation (p<0.05),CONCLUSION:We successfully screened and identified22differential expressed proteins between tumor and non-tumor tissues. Among them, PRDX3was found overexpressed in HCC tissues, and its overexpression was correlated with tumor differentiation. Overexpression of PRDX3is closely associated with the progression of hepatocarcinoma, and may be a potential therapeutic target for HCC.Part2Biological functions of PRDX3gene expression in HepG2cell line by the method of RNA interferenceOBJECTIVE:Detection of PRDX3protein in cell lines and investigate the influnces of PRDX3expression on cell proliferation, migration and cell apoptosis.METHODS:The expression of PRDX3protein was detected in human hepatoma HepG2, Hep3B, QGY-7703, HuH7cell lines and the normal liver cell line QSG-7701by RT-PCR and western blot. The shRNA expressing vectors pRNAT-U6.1/Neo(SD1201) were transfected into HepG2cells, whereas the unrelated shRNA transfected HepG2cell lines and normal HepG2cell lines were negative control group and positive control group respectively. After PRDX3expression was inhibited by the method of RNA interference, MTT, flow cytometry, migration assay and colony conforming assay were used to investigate the biologic change of HepG2cells.RESULTS:(1) The expreesion of PRDX3in human hepatoma HepG2, Hep3B, QGY-7703, HuH7cell lines was significantly higher than that in the normal liver cell line QSG-7701.(2) The expressions of mRNA and protein of PRDX3in HepG2cells after transfection of PRDX3siRNA were markedly decreased compared with the other groups(p<0.05). MTT assay showed that the growth speed of HepG2/siPRDX3cells was slow compared to negative control HepG2/siNC. The colony conforming assay showed the colony numbers of HepG2/siPRDX3cells were decreased. Flow cytometry demonstrated the apoptosis cells of HepG2/siPRDX3cells increased, an cell migration assay showed that migration capability was decreased.CONCLUSION:The expression of PRDX3in human hepatoma cells can enhance the proliferation and protecting hepatoma cells from the apoptosis of cells. PRDX3has substantial clinical impact on the progression of hepatocarcinoma, and may be a potential therapeutic target for HCC.