Construction of recombinant vectors carrying antisense RNA to dualˉtarget BAGˉ1 and Bclˉ2 and its effect on proliferation of SGCˉ7901 cell / 国际检验医学杂志

ABSTRACT

Objective To construct the recombinant co-expression vector carrying antisense RNA to dual-target BAG-1 and Bcl-2 genes and recombinant single expression vector carrying antisense RNA to target BAG-1 and Bcl-2 gene respectively,then to pre-liminarily investigate their effect on the proliferation of gastric cancer cell SGC-7901 in order to lay the foundation for further study the effect of this recombinant vector on the tumor cells.Methods RT-PCR was used to amplify the full length of BAG-1 and Bcl-2 cDNA from total RNA of gastric cancer cell line SGC-7901.The BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were insert-ed into pMD18-T simple vector respectively.The pMD18-T-BAG-1 was digested with BamH Ⅰ and Cla Ⅰ and the pMD18-T-Bcl-2 was digested with EcoR Ⅰ and Nhe Ⅰ.Then the BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were inserted into the mcs1 and mcs2 of the eukaryotic co-expression vector pVITRO2 in the antisense orientation respectively.The construction of the single expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 was confirmed by restriction endonuclease treatment and se-quence identification.Then the Bcl-2 cDNA fragment was inserted into the mcs2 of the recombinant vector pVITRO2-AsBAG-1 in the antisense orientation to construct the co-expression vector pVITRO2-AsBAG-1-Bcl-2,and the recombinant vector was also iden-tified by restriction endonucleases digestion and sequence identification.Then the recombinant vector was transfected into SGC-7901 cell respectively.The proliferation of the cell was determined by the MTT assay.The level change of BAG-1 and Bcl-2 mRNA in SGC-7901 cell was detected by the semi quantitative RT-PCR and the change situation of cell cycle was detected by the flow cytom-etry(FCM).Results The restriction endonucleases digestion and sequencing identification indicated that the eukaryotic co-expres-sion vector pVITRO2-AsBAG-1-Bcl-2 and the single gene expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 were con-structed successfully.The MTT assay method demonstrated that compared with the control group,the recombinant vector could in-hibit the proliferation of the cells in time dependent manner,and the inhibiting effect was most notably in the 72 h transfection group(P 0. 05);the FCM detection results showed that the apoptosis rate of the recombinant vector groups was significantly higher than that of the control group and the pVITRO2 group with statistical difference(P <0.01),and the co-expression vector group was more nota-bly than single expression vector groups(P <0.01).In addition,the effect of the co-expression recombinant vector group was more significant than that of the single expression co-expression vector(P <0.01 ),the apoptosis rate was increased from 0.57% to 15.75%.Conclusion The co-expression recombinant vector pVITRO2-AsBAG-1-Bcl-2 and single expression vector pVITRO2-As-BAG-1 and pVITRO2-AsBcl-2 are successfully constructed and they can inhibit the proliferation of SGC-7901 cell and induce cell apoptosis,moreover the pVITRO2-AsBAG-1-Bcl-2 vector is most notably.