Research Of Radiosensitising Enhancement Of Retinoblastoma (Rb94) In Lung Adenocarcinoma Cell Line(A549)

OBJECTIVE:To amplify the Rb94 gene fragment and analyze the DNA sequence, and then to construct eukaryofic expression plasmids pIRES-Rb94. To investigate the effect of Rb94 on the expression of SphK and the fuctions of SphK in the regulation of cell proliferation and apoptosis in cancer cells. And to study and research the possible mechanisms of radiosensitising enhancement of retinoblastoma gene (Rb94 ) in lung adenocarcinoma cell line (A549 ) .Thus, to offer theorical proof for Rb94 enhancement radiosensitivity of human lung adenocarcinoma A549 cells.METHODS:The primer of Rb94 was designed and composed at the base of the Rb94 sequence. Total RNA was extracted from cultured A549 cells using Trizol reagent and Rb94 gene was cloned by the reverse transcription-polymerase chain reaction (RT-PCR). The resulting PCR fragment of Rb94 was purified from agarose gels using . DNA Agarose Gel Purification Kit and was linked to pMD19-T simple vector by T4 DNA ligase.The recombinant clone vector named pMD19-T-Rb94 was identified by automated sequencing analysis and proceeded to be transformed into competent E. coli. To construct the human resulting expression vector, the Rb94gene was amplified from recombinant plasmid vector pMD19-T-Rb94 in which the full-length Rb94 gene had been inserted . The resulting PCR fragment for subcloning was purified from agarose gels according to the manufacturer's instructions. The eukaryofie expression plasmid plRKS-Rb94 was constructed correctly and transfected into A549 cells by lipofectamine 2000. Then stably transfected pIRES-Rb94 cell line was selected through G418 for 3 weeks, The Rb94-positive colonies were selected and detected by Real time RT-PCR and Western blotting and the effect on the expression of SphK was determined. The growth curve, the population doubling time was tested by cell counting. The radiosensitivity of A549 cells was determined by clonogenic assay; The expression of hTERT and Bcl-2 mRNA were detected by real-time RT-PCR. Cell cycle distribution and apoptotic percentage were measured by flow cytometry.RESULTS:The recombination expression vector contained Rb94 gene was successfully constructed and a new cell line with stable and high expression of Rb94 was established. Real time RT-PCR and Western Blotting analysis demonstrated that Rb94 gene could be effectively expressed in A549 cells. Moreover, the expression of SphK1 was down-regulated and the expression of SphK2 was up-regulated. The population doubling time increased (31.5h∶39.5h; t=5.15, P<0.01). The expression of hTERT and Bcl-2 mRNA were down-regulated (0.02∶1.00; t=18.99, P<0.01; 0.01∶1.00; t=13.73, P<0.01) The number of cells of G₂/M phases increased (35.91%∶4.53%; t=36.78, P=0.00),whereas the cell number of G₀/G₁ phases and S phases decreased.pIRES-Rb94 significantly (47.02%∶74.07%; t=11.71, P=0.00å’Œ17.07%∶22.32%; t=2.30, P<0.05) .Enhanced radiosensitivity of A549 cells with an sensitization enhancement of ratio of 1.30.Conclusion:The pIRES-Rb94 expression plasmid was successfully constructed and could be effectively transfected into A549 cells. After transfection, Rb94 can efficiently inhibit the expression of SphK1, while enhance the expression of SphK2 in A549 cell line. These results suggest pRb94 possesses significant radiosensitizing effect on A549 cells by G₂/M phase blocking and down-regulation of hTERT and Bcl-2 mRNA expression. And Rb94 may inhibit the proliferative ability by changing the cell cycle pattern.