Study On Construction Of Recombinant Decorin Eukaryotic Expressing Vector And Mechanism Of Decorin On HepG2 Hepatoma Cell

Many researches have indicated the relation between tumor cell and their microenvironment are very tight .Recently,the relationship between them become the focus and difficulty of many cancer researches. Decorin is one of the important moities of extracellular matrix (ECM) .It is a member of the small leucine-rich proteoglycan (SLRP) gene family and belongs to proteoglycans.A lot of researches have found decorin can inhibit the proliferation of various tumor cells.The inhibitory effects of decorin on tumor cells growth have been demonstrated both in vitro and in vivo. The transfected tumor cells with decorin gene failed to generate tumors in scid mices.The mature decorin molecule consists of a globular core protein and a cov- alently linked glycosaminoglycan chain including chondroitin sulfatase (cs) /de- rmatan sulfatase(ds). it's molecular weight is 92.5kda. The primary translation product of human decorin is 359 amino acids in length and can be divided into 4 functional domains.Decorin gene locates in human chromosome 12q21-q22 and the sequence length of decorin is 1080bp.The anti-tumor mechanisms of decorin have been investigated. The important mechanisms include the following aspects: (1) Decorin is a natural inhibitor of TGF-β. It reduces the biological activity of TGF-βand inhibits tumor progression and metastasis. (2) Decorin can inhibit tumor cell by which decorin links to P21 WAF1/ CIP1 and increase the P21 WAF1/ CIP1 translation product. (3)Decorin can induce apoptosis via activation of caspase-3. (4)Decorin induce Growth suppression by increasing the G0/G1 phase of the cell cycle and deceasing the S phase of the cell cycle.Now, many studies have discovered that the protein of decorin downregulate in patient of hepatoma carcinoma, howerver study on mechanism of decorin influencing HepG2 hepatoma cell is lacking. my researches successfully constructed the recombinant eukaryotic expression vector pcDNA3.1-decorin and stable transfects into HepG2 cell line in order to investigate the mechanism of anti-tumor of decorin. The results are as follows.1. The construction of eukaryotic expression vector pcDNA3.1-decorinThe length of decorin gene was amplificated from the plasmid of pBluescript by PCR,the product of PCR and eukaryotic expression vector was connected after both was cut by enzyme,the product of connection was identified by both enzyme cutting,PCR and sequencing. We confirmed that the decorin gene fragment was correctly inserted into eukaryotic expression vector pcDNA3.1. This established the experimental basis for future research on function of decorin.2. Successfully set up HepG2 cell line of stable transfected decorin.The HepG2 of hepatoma carcinoma cell was cultured in DMEM and cells in exponential phase of growth were chosen to test. The plasmid of pcDNA3.1-decorin was transfected into HepG2 cell. decorin mRNA was detected by RT-PCR and decorin protein was detected by immunohistochemistry and western blot. Comparing with pcDNA3.1-HepG2, the mRNA expression of pcDNA3.1-dec-HepG2 was obviously increased by RT-PCR method and the protein expression of pcDNA3.1-dec-HepG2 was obviously increased by the methods of immunohistochemistry and western blot. The results indicated that the plasmid of pcDNA3.1-decorin was transfected into HepG2 hepatoma carcinoma cell.This supplied experimental evidence for the further essay to study the effect of decorin on target cells.3. decorin can inhibit cell growth curve of HepG2.Differnt groups cells were inoculated into 96-well plates at the amount of 1×10~4/well, then we detected the cell condition of proliferation on 7 successive days,finally drew the cell growth curve according MTT method.Comparing with pcDNA3.1-HepG2, the growth of pcDNA3.1-dec -HepG2 was slow .The results indicated that decorin can inhibit the growth of the HepG2 cells.4. decorin can inhibit the shift of G0/G1 phase to S phase of HepG2.After pcDNA3.1-dec-HepG2 cells and pcDNA3.1-HepG2 cells were cultured for 5 days,all cells were washed by using PBS,then cells were stayed overnight in iced alcohol,and then added to PI,PBS,RNase,finally cell cycle was detected by Flow cytometry. Comparing with pcDNA3.1-HepG2, the G0/G1 phase of the cell cycle of pcDNA3.1-dec-HepG2 obviosly increased and the S phase of the cell cycle obviously decreased. The results indicated that decorin can inhibit the shift of G0/G1 phase to S phase of HepG2 cells.5. decorin can increase the enzyme contents of the caspase-3 and caspase-8 .After pcDNA3.1-dec-HepG2 cells and pcDNA3.1-HepG2 cells were cultured for 5 days,all cells was detected by using caspase-3 and caspase-8 Activity Assay Kit.Comparing with pcDNA3.1-HepG2, caspase-3 and caspase-8 enzyme contents of pcDNA3.1-dec-HepG2 obviously increased by activity detection of caspase-3 and caspase-8. The results indicated that decorin can increase the apopotosis of HepG2 cells.6. decorin can increase the mRNA expression and protein of P21WAF1/CIP1After pcDNA3.1-dec-HepG2 cells and pcDNA3.1-HepG2 cells were cultured for 5 days, the mRNA expression and protein of P21WAF1/CIP1 was detected by RT-PCR and western blot method. Comparing with pcDNA3.1-HepG2, mRNA and protein expression of P21WAF1/CIP1 of pcDNA3.1-dec-HepG2 obviously increased by the method of RT-PCR and western blot .The results indicated that decorin can increase the mRNA and protein expression of P21WAF1/CIP1 of HepG2 cells.7. decorin can decrease the mRNA expression of TGF-β1.After pcDNA3.1-dec-HepG2 cells and pcDNA3.1-HepG2 cells were cultured for 5 days, the mRNA expression of TGF-β1 was detected by RT-PCR method.Comparing with pcDNA3.1-HepG2, mRNA expression of TGF-β1 of pcDNA3.1-dec-HepG2 obviosly decreased by the method of RT-PCR. The results indicated that decorin can decrease the mRNA expression of TGF-β1 of HepG2.The research firstly constructed eukaryotic expression vector pcDNA3.1-decorin, then successfully set up HepG2 cell line of stable transfected decorin,at last we confirmed decorin can inhibit the growth of HepG2. Our results indicated that decorin can increase the enzyme contents of the caspase-3 and caspase-8,decrease the mRNA expression of TGF-β1,increase the mRNA expression and protein of P21WAF1/CIP1 and inhibit the shift of G0/G1 phase to S phase of HepG2 cells.