Effect On Proliferation Of Breast Cancer MCF-7Cells By Stable Transfection Of Recombinant14-3-3σgene

ObjectiveTo identify eukaryotic expression vector of pEGFP-GV142-SFN.Theeukaryotic expression vector were transfected into breast cancer cell MCF-7（Breast cancer cell line MCF-7）,screened the cell line stably expressing thegene of14-3-3σ,further observed the possible mechanism of MCF-7cellproliferation.To find the new therapy method of breast cancer,at the same timeprovide experimental basis for biological evaluation of14-3-3σ and genetherapy of breast cancer.Methods1, The recombinant plasmids pEGFP-GV142-SFN were identified bydouble-endonuclease and sequencing,the sequencing was accomplished byShanghai Jikai biotechnology company. Recombinant plasmid mediated bylipofection was transfected into human breast cancer MCF-7cells, the MCF-7cell line stably expressing14-3-3Sigma was screened by G418monoclonal.2, The experiment was divided into three groups: pEGFP-GV142-SFNgroup, pEGFP-N1group and blank control group. RT-PCR method was used todetect the14-3-3σ gene mRNA expression, Western blot method was used todetect the14-3-3σ protein expression, MTT method was used to observe thestate of cell proliferation. Results1, The results of double-endonuclease and sequencing:A774bp band（2Lane1）was obtained by double-endonuclease and the sequence resultswere fully accordant with that published in GeneBank. and sequence alignment（NM₀06142.3）was100%homology, no mutation.2, The establishment of stable transfection of breast cancer MCF-7,pEGFP-GV142-SFN experimental group, pEGFP-N1control group gave outgreen fluorescence after transfected, G418（700g/mL） was used to screenpositive cell clones, amplified in breast cancer MCF-7cell line culture isthe stable expression of14-3-3σ, named MCF-7-GV142cells.3, MCF-7cells in three groups Western blot the results showed that: theexperimental group of14-3-3σ was significantly higher than the other twogroups in the protein expression, there was significant difference （P <0.01）; while the negative control group, blank control group14-3-3σexpression was no significant difference （P>0.1）.4, MCF-7cells in three groups RT-PCR results show that: the14-3-3σmRNA expression in experimental group was significantly higher than theother two groups, there was significant difference （P <0.01）; while thenegative control group and blank control groupof intracellular14-3-3σmRNA showed no significant differencebetween the amount （P>0.1）.5, MCF-7cells in three groups were MTT test results show that: theproliferation of cells in the experimental group in logarithmic time wassignificantly lower than other groups （P <0.01）, and the inhibition effect withthe extension of time and more obvious; but no obvious differences between theproliferation of negative control group and blank control group cells inlogarithmic time （P>0.1）.Conclusions1, the analysis proved that the recombinant14-3-3σ gene expression vector pEGFP-GV142was successfully identified;2, G418stably expressed14-3-3σgene in breast cancer MCF-7;3, recombinant eukaryotic expression vector of pEGFP-GV142-SFNenhanced the expression of mRNA and protein in breast cancer MCF-7of14-3-3σ gene.4, the recombinant eukaryotic expression vector pEGFP-GV142-SFNinhibit the proliferationcan significantly of breast cancer MCF-7.