The Construction Of A Lentiviral Vector Overexpression Of HJURP Gene And Its Effect On The Biological Behavior Of Human Liver Cancer Cell Line HepG2

Objective:To construct and identify the over-expression lentiviral vector for HJURP gene and then investigate the effect of overexpression of HJURP gene on the biological behaviors of human hepatocellular carcinoma cell line HepG 2.Methods:HJURP gene was cloned through gene recombination technique,and inserted into the retroviral expression vector GV358.The recombinant retroviral expression vector was identified by restriction endonuclease digestion and RT-PCR,then the recombinant vector was infected into the packaging cell line 293T.Western bolt and RT-PCR were used to detect the expression of the mRNA and protein of HJURP gene.Results:Restriction endonuclease digestions revealed that the HJURP gene was cloned into the retroviral expression vector successfully,and the sequences were identified by DNA sequencing.The HJURP gene of the recombinant lentiviral vector was successfully packed into 293T cells.The recombinant lentivirus was harvested from 293T cells,and the titer of concentrated virus was 2E+8.Conclusions:The lentiviruses which carrying with HJURP and their genen sequence were made correct.Objective:To investigate the effect of overexpression of HJURP gene on the biological behaviors of human hepatocellular carcinoma cell line HepG 2.Methods:The plasmid for HJURP overexpression was constructed and then the HepG2 cells were infected with the lentiviral packaging plasmid.The cell proliferation was dectected by MTT method.The cell aggression and migration was dectected by Transwell assay.The mRNA and protein levels was measured by RT-PCR and Western blot.Cell cycle distribution was detected by flow cytomtry.An animal model of the human hepatoma cancer xenograft was established.RT-PCR and Western blot was performed to dectect the mRNA and protein expression level of HJURP in tumor.Results:Compared with control group NC group and blank group,LV-HJURP mRNA and protein were increased significantly in HepG2.Cell growth and cell migration also cell aggression were promoted.Cell number in G0/G1 phase was decreased and that in S and G2/M phase was increased.Protein and mRNA in tumor of LV-HJURP were also increased.Conclusion:The stable cell line with the overexpression of HJURP has been constructed ssuccessfully.The results showed the HJURP overexpression promoted cell proliferation in HepG2 cell lines and increase the probability that HJURP gene has effect on the tumor malignant behaviors of hepatoma carcinoma cells.