Investigation Of The Roles Of DRC3f And ERF3b In Progression Of Liver Disease

There are over 360 million individuals are chronically infected with HBV in the world and a part of them are at risk of developing and dying from HBV-related chronic liver disease, including cirrhosis and hepatocellular carcinoma. Though propagation of Global Baby Immune Programme induced the incidence of chronic hepatitis to a lower level, the incidence of hepatocellular carcinoma will still increase in the coming twenty years.In previously work, two low molecular peptides had been found by matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS). The peptide with 16 amino acids, which exists at a higher level in sera of mild and moderate degrees chronic hepatitis B (CHB) patients than that in severe degree CHB patients, was identified as C3f-desArg (DRC3f). Complement system is very important in human immune system. Complements normally circulate in peripheral blood as inactive forms and have high metabolic rate. Except for involving unspecific defenses and specific immune response, complements also can be activated in inflammation and induce tissue damage. In the process of complement activation, complement C3, which exists at a high level, can be activated into C3b. Then C3b was decomposed into inactive iC3b and C3f by factor I with the help of complement receptor I and factor H. C3f was further decomposed into DRC3f. It has been reported that complement C3 and cleavage fragments including iC3b, C3f-desArg and its smaller derivatives played an important role in some disease progression.The other peptide with 37 amino acids, which exists at higher level in sera of CHB patients than that in hepatocellular carcinoma (HCC) patients, was identified as eukaryotic peptide chain release factor 3b (eRF3b). In eukaryotes, two release factors, eRF1 and eRF3 are required to complete protein synthesis. eRF1 recognizes all three stop codons by direct interaction at the decoding A site and activates the peptidyl transferase center, which triggers the hydrolysis of the peptidyl tRNA, generating a free full-sized polypeptide. eRF3 is a GTPase that stimulates eRF1 activity in a GTP-dependent manner. In mammals, two distinct genes encoding eRF3, namely GSPT1 and GSPT2, have been identified. The products of these genes, eRF3a and eRF3b, share 87% sequence identity and differ only in their N-terminal domains. Except involved in translation termination, eRF3 also involve in cell cycle progression and tumorigenesis though the mechanism are far from being understood. The results of most studies showed that eRF3a is the main factor in translation termination. However in mammals, eRF3b can fulfill all the functions of eRF3a in translation termination.Part One: Peptide C3f-desArg (DRC3f) promotes the proliferation of QSG-7701 cell in vitroObjective: As a marker of complement activation, complement C3f and DRC3f also can serve as biomarkers of liver damage. But the involved pathological mechanism is still not clear. Therefore, in present study, the role of DRC3f in cell proliferation will be investigated.Methods: After QSG-7701 Cells were incubated with sera containing DRC3f and synthesis peptide DRC3f, Tetrazolium test was used to analyze the effect. RNA was extracted from the cells stimulated by DRC3f. Expression of transforming growth factorβ(TGFβ1) and collagenⅠ(COLⅠ) were analyzed by quantitive RT-PCR.Results: After incubated with sera from hepatitis B patients and health individuals, it was found that the QSG-7701 cells growed faster in mild hepatitis group and moderate hepatitis group than that of severe hepatitis group and health individuals. There was significantly difference (F= 55.715, P<0.01). When the QSG-7701 cells were again incubated with sera filtrated by 3Kd filter, the same results were obtained (F=26.325, P<0.01). For further confirming, we stimulated the QSG-7701 cells by synthesis peptide DRC3f and stellate cell LX-02 was used as control. It was found that the peptide could enhance the growth of QSG-7701 cells but not for LX-02 cells. The results of RT-PCR indicated that the expression of TGFβ1 and COLⅠwere deduced with dose response of DRC3f. Compared with unstimulated group, the ratios of TGFβ1 to 18S rRNA were 0.96 in 62.5ng. mL-1 group and 0.42 in 500ng. mL-1 group. The ratios of COLⅠto 18S rRNA were 0.93 in 62.5ng. mL-1 group and 0.57 in 500ng. mL-1 group.Conclusion:DRC3f can stimulate the growth of hepatic cells. Expression of TGFβ1 and COLⅠwere deduced with dose response of DRC3f. Part Two: Expression of eRF3b in liver tissues and cultured hepatocytes in vitroObjective: In process of tumorigenesis, variations in tumor cells such as increasing synthesis of proteins, over expressing of some proteins related to cell cycle often have relations with translating factor. A series of evidence have confirmed the role of translating initial factor and translating elonging factor in tumorigenesis, but few studies aimed at releasing factor. The aim of study was to find the expression of eRF3b in human liver tissues and different cell lines.Methods: The expression of eRF3b in human hepatocarcinoma and adjacent normal tissues was analyzed by immunohistochemical method. Fluorescence quantitative PCR was performed to study the expression of GSPT2, ERF1 and GSPT1 in normal hepatic cell line QSG-7701, hepatocellular carcinoma cell line HepG2 and HepG2.215 cell line that merged HBV. The results derived from qRT-PCR were analyzed based on threshold cycle (Ct) values of a method called 2-ΔΔCt method in accordance with 18s rRNA gene to standardization. After the cells were dissolved by RIPA, Western Blot was performed, in which theα-tublin was served as standard.Results: The results of immunohistochemistry on Paraffin-embedded tissue sections showed that eRF3b was positive express in parenchyma cells and interstitial cells. The results of immunohistochemistry on culture cell showed that eRF3b protein was distributed in cytoplasm. The expression level of GSPT2 mRNA detected by fluorescence quantitative PCR was higher in HepG2 cells than in other two cell lines, while ERF1 and GSPT1 mRNA expressed at a higher level in HepG2.215 cells than other two cell lines. In western blot, the relative expression of eRF3b toα-tublin was 0.73 in HepG2 cells, which was higher than 0.25 in QSG-7701 cells and 0.24 in HepG2.215 cells.Conclusion: eRF3b was positively expressed in liver tissues and cultured cells. The relative expression was higher in HepG2 cells than that in QSG-7701 and HepG2.215 cells.Part Three: The effects of exogenous eRF3b on cell proliferation and cell cycle progressionObjective: To investigate the role of eRF3b in tumorigenesis through up- regulate or down-regulate its expression in HepG2 cells. Methods: The recombinant green fluorescent protein expressing plasmid of pEGFP-C2-GSPT2 and pEGFP-C2-GSPT2-111 were constructed. After transient transfected into HepG2 cells, the location of eRF3b was observed by fluorescence microscope. At the same time, short hairpin interfering RNA expressing plasmid pSilencer-GSPT2 and pSilencer-GSPT1 were constructed. The cells transfected pSilencer-GSPT1 was called eRF3a (-) and cells transfected pSilencer-GSPT2 was called eRF3b (-). The expression level of protein was analyzed by Western Blot. The effect of eRF3b on cell viability was assayed by tetrazolium test. The plate clone formation test was used to evaluate the effect on cell proliferation and the cell cycle was analyzed by flow cytometry.Results: Green fluorescent protein expressing plasmid pEGFP-C2-GSPT2 and pEGFP-C2-GSPT2-111 were constructed successfully. After transient transfection pEGFP-C2-GSPT2 and pEGFP-C2-GSPT2-111, HepG2 cells were observed under fluorescence microscope and the eRF3b protein located in cytoplasm. The transfection efficiency was 65.3% detected by flow cytometry. The transient transfection of pEGFP-C2-GSPT2 and pEGFP-C2-GSPT2-111 had no influences on cell cycle. Short hairpin interfering RNA expressing plasmid pSilencer2.1-GSPT2 and pSilencer2.1-GSPT1 was constructed successfully. After transient tranfection, HepG2 cells were lysed, protein was extracted at 0 h, 24 h and 54 h and Western Blot was done. The result showed that 58% protein was silenced at 54 h. In MTT test, the mean absorbencies of eRF3b (-) group, eRF3a (-) group and pSilencer control group were 0.1893, 0.147 and 0.259 separately (n=18). The results show that cell viability of RNA interfering group was lower than that of pSilencer control group. The cell viability of eRF3a (-) group was lower than that eRF3b (-) group. Plate clone formation test showed that the proliferation ability of eRF3b depletion cells was descending. Cell cycle analysis by flow cytometry indicated that eRF3b depletion changed the cell cycle. The percentage of G1 phase cells in eRF3b (-) group increased to 65% compared with 28.9% in pSilencer control group. The percentage of S phase cells in eRF3b (-) group decreased to 20.1% compared with 41% in pSilencer control group.Conclusion: eRF3b depletion inhibited the cell viability and cell proliferation and changed cell cycle progression. Our results provide a molecular basis for the role of eRF3b in cell cycle progression of HepG2 cells and predict a novel therapeutic target for treatment of HCC...