Construction Of The Eukaryotic Expression Vector Of HIL-24 And Its Synergistic Effects With RhDCN On HepG2 Cells

Objectives To construct a eukaryotic expression vector of human interleukin-24 (hIL-24) and to investigate its synergistic effects with human DCN on proliferation, apoptosis and cell cycle of HepG2 cells.Methods 1.RT-PCR was performed to obtain IL-24 cDNA from human peripheral blood mononuclear cells (PBMC).The eukaryotic expression vector pcDNA3.1(+)-IL-24 was constructed with DNA recombination technique and was identified by PCR, restriction enzyme digestion and DNA sequencing.2.The pcDNA3.1(+)-IL-24 and pcDNA3.1(+)-DCN were transfected into HepG2 cells by liposome transfection. RT-PCR was performed to determine mRNA expression of IL-24 and DCN in HepG2 cells.3.Cellular growth, morphological changes and apoptosis were observed under inverted microscope at 48h after transfection.4. The proliferation-inhibiting effects of IL-24 and DCN on HepG2 cells, respectively and jointly, were observed with MTT assay at 24h,48h and 72h after transfection.5.Apoptosis and cell-cycle of transfected HepG2 cells were analyzed by flow cytometry at 48h after transfection.Results 1.IL-24 cDNA was cloned from PBMC with RT-PCR.The results of PCR,restriction enzyme digestion and DNA sequencing confirmed that the hIL-24 gene fragment had subcloned into the eukaryotic vector pcDNA3.1(+) correctly.2.The mRNA of IL-24 and DCN were detected in the transfected HepG2 cells in the corresponding groups with RT-PCR.3.The typical changes of apoptotic morphology, such as the decrease in cellular volume, cellular density and light refraction of cytoplasm, cell shrinkage, cell blebbing and formation of apoptotic bodies, can be observed under inverted microscope at 48h after transfection.4.The results of MTT assay showed that at 48h posttransfection, the rate of proliferation-inhibiting in IL-24 group and DCN group were 14.64±2.36(%) and 17.97±5.17(%), respec-tively, which were higher significantly than 6.29±2.90(%) of empty plasmid group(p<0.01).The proliferation-inhibiting rate in the group cotransfected with IL-24 and DCN was 31.88±6.57 (%), showing significant difference with other groups(p<0.01).The similar results can be seen at 72h posttransfection. The rate of proliferation-inhibiting in the group of combination IL-24 and DCN was 36.83±3.76 (%), displaying remarkable difference with those of the IL-24 group and DCN group,18.51±3.40 (%) and 20.77±2.39 (%), respectively.5.Compared to the early apoptosis rate of 20.01±1.08(%) and 22.20±0.91(%) in the groups transfected with IL-24 and DCN singly, a remarkable apoptosis,32.56±0.90(%),can be seen in the group treated with IL-24 and DCN(P<0.01).6.The result of cell cycle analysis revealed that, compared to the control groups, the proportion of cells was higher in the phase of G2/M in the IL-24 group (11.24±0.35%), with that being higher in the phase of G0/G1 in the DCN group(77.93±0.67%).The proportions of cells in the phases of G2/M increased to 71.36±0.60% and that of G0/G1 increased to 10.39±0.67% in the group co-transfected with IL-24 and DCN,showing statistically significant differences with other groups(P<0.01).Conclusions l.The recombinant eukaryotic expression vector pcDNA3.1(+)-IL-24 was constructed successfully.2.The exogenous gene IL-24 and DCN were transferred to HepG2 cells successfully by liposome transfection.3.Both IL-24 and DCN display growth-inhibiting and apoptosis-inducing activity on HepG2 cells, and combinatorial treatment of HepG2 cells with the two genes can even exert stronger synergistic inhibitory effect in comparison to single therapies.4.IL-24 and DCN can induce cell circle arrest on HepG2 cells, occurred in the phase of G2/M and G0/G1, respectively. Promoting effect of cell cycle arrest in the phase of both G2/M and G0/G1 can be seen in the combinatorial treatment of HepG2 cells with IL-24 and DCN.