| Objective POLι gene coding region containing2147bp, the entire gene have five conserved motif. The encoding product——DNA polymerase ι(Polι)of715amino acid residues, has the lowest fidelity in Y-family DNA polymerases found now, and is one of the main DNA polymerase members involved in TLS. This experiment studies effect of Poligene on growth, proliferation and chemo-radiosenstivity of non-small cell lung cancer cell H1299,and provides Polι gene as a new target for cancer gene therapy with experimental basis.Methods In this study, by constructing Polι overexpression vector pcDNA-Polι, Polι gene interference vector shRNA-Polι and empty vector shRNA-NC, and setting pcDNA3.1vector as control group,liposome-mediated transfection of non-small cell lung cancer H1299cells (1) detect effect of over-expressing and silencing Polιgene on mRNA and protein expression of Poligene in lung cancer H1299cells by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot;(2) assay absorbance valuesof each group by MTT and calculate the cell viability of control group (non-transfected) and each transfected cells;(3) assay cell survival rate of H1299cells treated with different concentrations of cisplatin after transfected with shRNA-Polιand shRNA-NC by MTT,and calculate cisplatin concentration in whch survival rate of H1299cells transfected with shRNA-Polι is50%;(4)With the10μg/ml concentrations of cisplatin before each H1299cells transfected group after24h, assay Polι impact on H1299platinum drugs tolerance by using the MTT;(5) By different doses (0,2,4,6,8and10Gy) of60Coy rays in each group of H1299cells after transfection, detect the impact of Pohon the H1299radiosensitivity by cell colony formation.And"Multi-target single hit survival model" (SF=1-(1-e-D/D0)N)) was used for dealing with data, then built the cell surviving curve.Cell cycle and apoptosis percentage were detected with flow cytometry..Results (1) RT-PCR and Western blot analysis showed, mRNA and protein expression levels of cell lines transfected pcDNA-Polιis higher than the control group (pcDNA3.1)(P<0.05); mRNA and protein expression levels of cell lines transfected with shRNA-Polιis lower than the control group (shRNA-NC)(P<0.05);(2) MTT assay showed that, cell proliferation rate of Polι overexpression (pcDNA-Polι) cell lines is higher than its control group (pcDNA3.1)(P<0.05), while cell proliferation of Polι genes downregulated (shRNA-Polι) cell lines are less than the control group,(P<0.05);(3) With the drug concentration increased, compared with the control group and shRNA-NC transfected group, shRNA-Polι transfected cells relative survival rate is lower (P<0.05);(4) Tolerance to cisplatin of Polι over-expressing cell lines relative to that of the control group (pcDNA3.1vector transfected cells) increase (P<0.05), and sensitivity to cisplatin of cell lines with low expression Polιelative to their control group (shRNA-NC vector-transfected cells) increase (P<0.05);(5) shRNA-Polι transfected with the control group (shRNA-NC-transfected group) compared to its colony formation was inhibited (P <0.05); pcDNA-Polι transfected with the control group (pcDNA3.1transfected group) compared enhanced colony formation (P<0.05);(6) Mean lethal dose(D0),Dq,N and SF2value of shRNA-Polιtransfection group (2.266,0.1533,1.070and0.4352, respectively) were lower than the control group (shRNA-NC transfected group)(3.149,0.3001,1.10and0.5640, respectively)(P<0.05),;mean lethal dose (D0),Dq, N and SF2value of pcDNA-Polιtransfected group (3.702,2.064,1.746and0.7825, respectively) is higher than that of the control group (pcDNA3.1transfected group)(respectively2.97,0.4382,1.159and0.5625)(P<0.05).Conclusions Overexpression of Polιpromotes proliferation, increases cisplatin and radiation resistance of the H1299cell; suppressing expression of Polιcan slow proliferation, increase sensitivity of the cells to cisplatin and radiation of the H1299cell.It may be an important strategy for increasing chemo-radiosenstivity of esophageal squamous cancer cells through inhibiting Polι overexpression... |
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