The Effect Of Bmi-1-siRNA On Radiosensitivity Of Human Lung Carcinoma A549Cell Line

1Objectiveto study Bmi-1gene mRNA and protein expression in A549cells after2,4,6Gy X-rays in different time points and to observe the effect of Bmi-1-siRNA on A549cellsproliferation and radiation sensitivity.2MethodWe respectively used real-time quantitative PCR technology and Western blot to testBmi-1mRNA and protein expression level in A549lung cancer cell lines after0,2,4,6GyX-rays in0,6,12,24,48and72h.we completed Bmi-1-siRNA restructuring plasmid withslow virus expression system pLP1/pLP2/VSVG/slow virus expression plasmid andstably transfected A549cell line. The stably transfected cells were sorted by flowcytometry. Bmi-1gene expression was measured by fluorescence real-time quantitativePCR technology and Western blot after RNAi. MTT method and the cells cloned formationmethod were detected the effect of Bmi-1-siRNA on proliferation and radiosensitivity ofthe cell line.3Result1) Bmi-1mRNA gene expression after the X-rays.Bmi-1mRNA in A549cell line was highly regulated after2,4,6Gy radiation.Theexpression of Bmi-1mRNA except for2Gy group during48h had statistical differencecompared with the untreated group.（-8.64≤t≤-2.90，0.001≤P≤0.04）.2) Bmi-1protein gene expression after the X-rays.After radiation, The expression of Bmi-1protein increased during48h, but decreasedafter48h,evently approached to the level of the untreated at72h,with statisticaldifference at most time points(-10.5<t<3.77,0.001<P<0.05).3) The inflence of RNA interference （RNAi）on Bmi-1gene expression onradiosensitivity of the A549cell line We successfully constructed A549/Bmi-1-siRNA1, A549/Bmi-1-siRNA2, A549/Bmi-1-siRNA3restructured plasmid,and transfected A549cell lines, flow cytometrysorted out the cell lines stably expressing GFP green fluorescence.Fluorescence real-timequantitative PCR technology detected that the A549/Bmi-1-siRNA3had the strongestinflence with relative expression0.27±0.02and interference rate64%,Obviously lowerthan the control group.A549/pSicoR-siRNA, pSicoR A549/-siRNA, A549/Bmi-1-siRNA2. A549/pSicoR-siRNA interference efficiency compared with the controlhad no obvious difference. Western blot protein imprinting detected interference efficiencyof A549, A549/pSicoR-siRNA, A549/Bmi-1-siRNA3showed that A549/Bmi-1-siRNA3protein expression was significantly lower than A549and A549/pSicoR-siRNAwith significant difference values compared with the two groups(P<0.05).MTT assay respectively detected cell proliferation of A549group, A549/pSicoR-siRNA,A549/Bmi-1-siRNA3, A549radiation group, A549/pSicoR-siRNA+combined withradiation group and A549/Bmi-1-siRNA3combined with radiation group cultured1d,2d,3d and4d after radiation. proliferation capacity of A549/Bmi-1-siRNA was obviouslyrestrained compared with the A549group between3d and4d (P <0.05). theproliferation capacity of A549radiation group raise gradually within3d and4d, butcompared with A549group cell proliferation capacity was restrained obviously (P <0.05).the proliferation capacity of A549/Bmi-1-siRNA3combined with radiation group wassignificantly inhibited and was significantly lower than A549/Bmi-1-siRNA3and A549radiation group (P <0.05). Clone formation assay found that A549group, A549/pSicoR-siRNA and A549/Bmi-1-siRNA3cells cloning formation rate and survival rategradually reduced with the increase of the radiation dose, A549/pSicoR-siRNA groupcloning formation rate had no obvious difference compared with A549group (P>0.05).A549/Bmi-1-siRNA3combined with radiation group cloning formation rate and survivalrate dropped significantly compared with A549group and the A549/pSicoR-siRNA after2,4and6Gy (P <0.05),simultaneously survival curve showed A549/Bmi-1-siRNA3obviously moved left and A549/Bmi-1-siRNA3significantly increased radiationsensitivity.4Conclusion1. Bmi-1mRNA expression in A549cell line after2~6Gy X-rays within48h isincreased. 2. Bmi-1protein in A549cell line begins to rise at6h after2~6Gy X ray, increasesbetween24h (in addition to4Gy)and decreases gradually,but are higher than the controlgroup between24h and48h. Bmi-1protein is relatively close to untreated group level in72h.3. The screened siRNA can effectively inhibit Bmi-1expression.Bmi-1-siRNA caninhibit A549lung cancer cell proliferation ability and improve the radiosensitivity of lungcancer.