Research Of Radiosensitising Enhancement Of Retinoblastoma (Rb94) To A549 Cells In Vivo

OBJECTIVE:A NH2-terminal truncated RB protein of 94 kDa (Rb94), lacking 112-amino acid residues, has been found to have greater efficacy than Rb110 in tumor suppression. Former studies have showed that Rb94 could enhance radiosensitivity by inhibiting the expression of SphKl, enhancing the expression of SphK2, suppressoring cell proliferation, promoting apoptosis and regulating cell cycle in A549 cell line after transfection. To construct a mouse xenograft model of Rb94 gene transfering A549 cells, and to investigate the Rb94's inhibition in mice tumors. To observe the effect of Rb94 on the expression of SphK and the functions of SphK in the regulation of cell proliferation and apoptosis in cancer cells. To research the possible mechanisms of radiosensitising enhancement, thus to offer theorical proof for Rb94 enhancement radiosensitivity in A549 cells.METHODS:PIRES and pIRES-Rb94, which preserved by the laboratory of QiLu Hospital, were transformed into competent E.coli. respectively. To pick the positive colony, and then to extract the plasmid after amplification. After identification with XhoI, plasmid pIRES and pIRES-Rb94 were amplified by Genscript Company for ulterior study. 5×106 cells suspension were subcutaneously injected into flanks of every nude mouse. Then the inoculated mice were devided into 4 groups after tumors arriving 180mm3: treated with PBS, lipofectamine, pIRES empty vector and pIRES-Rb94 respectively. Then to give halves of the mice with X-ray. The tumor inhibiting rate was observed and the expression of Rb94 gene was detected by Real time RT-PCR and Western blot. The expression of SphK was determined by real-time RT-PCR and immunofluorescence(IF). The number of apoptosis cells was measured by Hoechst staining. Expression of hTERT and Bcl-2mRNA were detected by real-time RT-PCR. Cell cycle distribution was measured by Immunohistology(IH).RESULTS:Treatment of pIRES-RB94, which inhibited ratio was 51.99%, had statistically differences compared with no treatment, liposome treatment and pIRES treatment(P<0.01). There were no statistical differences between no treatment, liposome treatment and pIRES treatment. The inhibited ratio of tumor in combination group was 95.84%, and statistically significant differences on the inhibition ratios were found between the combination group and the other groups(P<0.01). Real-time RT-PCR and Western blot analysis showed that Rb94 gene could be effectively expressed in the treatment of pIRES-RB94. The expression of SphKl was down-regulated, while the expression of SphK2 was up-regulated. Hoechst staining analysis demonstrated that the number of apoptosis cells was increased. The expression of hTERT and Bcl-2 mRNA were down-regulated by real-time RT-PCR. Immunohistology analysis showed that the number of cells of G2/M phases was increased in treatment of pIRES-RB94.CONCLUSION:The mouse xenograft model has been successfully constructed and analysis suggest that Rb94 has significant effects for inoculated tumor in nude mice. The tumor inhibition rate of combination group seems to be higher than that of the other groups. The possible mechanisms of radiosensitising enhancement of Rb94 gene are about down-regulation of SphKl,up-regulation of SphK2,promoting apoptosis, G2/M phase blocking and down-regulation of hTERT and Bcl-2 mRNA expression.