Sensitivity Alteration To CDDP And Its Mechanism Of HepG2 Expressing HBx

Hepatocellular carcinoma(HCC) is now generally accepted as a consequence of multiple factors, and HBV infection is the major one of them. Epidemiological study showed that the incidence rate of HCC in patients with HBV was 200 times higher than those who were not infected. Although it is not exactly understood how HBV induces HCC, with the development of molecular virology, the relationship between HCC and HBV X gene (HBx), and its protein pX was more and more revealed.HBV has a genome of incomplete double strain DNA circle. The longer strain includes four open reading frames(ORF). S gene encodes pre-S 1, pre-S2 and S protein, c gene encodes pre-C and C protein, p gene encodes the polymerase and the x gene encodes pX. HBx is the smallest ORF of HBV genome and distributes between 1374-1836nt, encodes a protein of 154aa which can regulate the transcription of many genes.It has been proved that in nulear, pX binds to many trans-acting factors such as TBP, TFII A, TFIIB, TFII D,bzip etc, functioning as a transcription regulator. While in the plasma, pX can activate several signal pathways such as Ras-Raf-MAP kinase cascades to interact with the transcription factors. Signal transduction pathways can often induce the alteration of cell cycle, mitosis and differentiation and are generally believed to contribute to the occurrence and development of HCC. In this experiment, HepG2 cells stably expressing pX were employed to demonstrate in which way can pX influence the cells' sensitivity to CDDP and protect the infected cells from death. METHODS1 Cloning of HBx-ORF The fragment of HBx-ORF aquired from the plasmaid pCPIO by PCR was ligated into a T-A vector. Transform the competent E.coli JM 109 with the reconstructed plasmid. The positive clones were identified bythe blue-white trial and restriction enzyme digestion.2 Sequencing The plasmids in the positive clones were sequenced and compared with the HBV genome sequence in pCP10.3 Construction of expression vector The fragment of HBx-ORF was cut from the sequenced T-A vector and ligated into the expression vector pCDNAS. The reconstructed plasmid was transformed into competent E.coli JM109 and positive clones were identified by blue-white trial, and restriction enzyme digestion.4 Transfection and positive cell clone selection Transfect the human hepatoma cell line HepG2 with the identified vector pCDNA3-HBx by liposome. Add G418 into the medium to select the cell clones that were resistant to it. Identify the clones containing HBx-ORF by RT-PCR.5 Comparing the expression of p-ERKl/2 in the positive and negative clones by Western blot.6 Comparing the sensitivity of HBx positive and negative clones to CDDP by MTT Plant the HBx+ HepG2, mock vector transfected HepG2 and liposome transfected HepG2 into 96-well plate and incubate with 2 u g/ml CDDP for 24h. Test the inhibitory rate of differtent cell groups with the method of MTT.7 After blocking the activation of ERK1/2, detect the sensitivity of HBx+ HepG2 to CDDP Incubate the HBx+ HepG2 first with 100 u mol/L PD98059 for 2h to block the activation of ERK1/2. Then incubate the cells with 2 u g/ml CDDP for 24h, Test the inhibitory rate with the method of MTT.RESULTS1 Cloning of HBx-ORF By PCR, a fragment of 482bp was got from pCP10. Digeste the reconstructed T-A vector with two restriction enzymes HindIII and EcoRI , two bands of 471bp and 3015bp were got as expected.2 Sequencing The fragment in the reconstructed T-A vector was sequenced and showed that it had a 100% homology with the HBx-ORF sequence in pCPlO.3 The identification of expression vector Digestion of pCDNA3-HBx with the same restriction enzymes got 471bp and 5.4kb bands, just as expected.4 Selection and Identification of the transfected cell After 4~5 weeks of selection in G418, 3~5 cell clones resistant to G418 were got both in pCDNA3-HBx group and mock vector group. RT-PCR showed 482bp bands in 2 clones in pCDNA3-HBx group which proved that it contained the HBx-ORF.5 The le...