Screening Of Prothrombotic State-related Genes From Human Hepatic CDNA Library And Their Bioinformatic Analyses And Pilot Study Of Expression

Prothrombotic states(PTS) caused by homeostatic maladjustment of fibrinolysis and coagulation is related with changes of coagulative factors, anticoagulant components and fibrinolysis. Liver plays important roles in the development of PTS. To investigate the mechanism of and the roles of liver in PTS, Fang Dingzhi et al. constructed subtracted cDNA library for differentially expressed genes in rat liver of PTS. To screen prothrombotic state-related cDNA sequences from human hepatic cDNA library and to explore the mechanism of and the roles of liver in PTS, human hepatic cDNA library was screened by probes of the target DNA sequences of positive cDNA clones of differentially expressed genes from rat liver of PTS.The target DNA sequences of positive cDNA clones were amplified by PCR. The PCR products were cleaned, mixed and used as probes to screen a human hepatic cDNA library. After the first, second and third screening, positive A TripIEx lysates were transduced into E.coli strain BM25.8 and then converted to pTripIExs. The positive circularized plasmids were identified by double enzyme digestion. The cDNA fragments of the positive plasmids were sequenced and analyzed by bioinformatics (blastn). 4 PTS-related cDNA sequences were identifiedfrom human hepatic cDNA library. For 3 of them, their products of expression were respectively fibrinogen gamma polypeptide, liver fibrinogen-related gene-1 mRNA, and chromosome 10 open reading frame 104 mRNA. The fourth sequence had homology with carbamoyl-phosphate synthetase 1 gene.To explore the roles of liver fibrinogen-related gene-1 (LFIREl), and chromosome 10 open reading frame 104 genes, we measured their mRNA levels in the hepatoma cell line, HepG2, in response to varying glucose concentrations. HepG2 cells were maintained in Dulbecco's minimal essential medium (DMEM) containing 10% fetal calf serum, 1 × 105IU/L penicillin, 100mg/L streptomycin, and 1000umol/L L-glutamine, in a humidified 5% CO2 incubator at 37, in media of different glucose concentration, llmmol/L(Gll group), 33mmol/L(G33 group), 11mmol/L glucose with 22 mmol/L mannitol(G11+M22 group). After HepG2 cells were cultured for 48h, total RNAs were isolated and used to perform RT-PCR. The mRNA products of LFIREl increased in G11 group, G33 group and G11+M22 group compared with non-treated group. But it was not statistically significant. The mRNA product of chromosome 10 open reading frame 104 significantly increased in G11+M22 group compared with other groups.In conclusion, our data demonstrated that the expression changes of fibrinogen gamma polypeptide gene, liver fibrinogen-related gene-1, chromosome 10 open reading frame 104 gene and the gene having homology with carbamoyl-phosphate synthetase 1 may associate with PTS. The mRNA level changes stimulated by increased glucose of liver fibrinogen-related gene-1, and chromosome 10 open reading frame 104 need further investigation.