Long Non-coding RNA MEG3 Induce Apoptosis By Regulating NF-?B Signalings In Human Breast Cancer MCF-7 Cells

Objective:To construct the eukaryotic expression vector pc DNA3.1-MEG3 of Lnc RNA MEG3.To screen out the cell lines which stably express MEG3.To explore the apoptosis effect of MEG3 on breast cancer MCF-7 cells and its possible mechanisms.Methods: A complete exon sequence based on the sequence of MEG3 in the Gen Bank was designed.Then the MEG3 fragments was amplified by RT-PCR. The MEG3 fragments was inserted into the eukaryotic expression vector pc DNA3.1 to construct recombinant plasmid pc DNA3.1-MEG3. PCR,enzyme digestion analysis and Sequencing identification were used to confirm the product. The MCF-7 cells which stably express MEG3 were selected by G418 and confirmed by RT-PCR.MTT was performed to detect the proliferation effect of MEG3 on MCF-7 cell.Hoechst33342 staining and flow cytometry were used to detect the apoptosis effect of MEG3 on MCF-7 cells. TNF? that the agonist of NF-?B signaling pathway and TW37 which is the inhibitor of Bcl-2 were used to treat the The MCF-7 cells which stablely express of MEG3.Western blot was used to detect the expression of NF-?B P65?NF-?B P50?Bcl-2 and Caspase3 protein.Results: A fragment about 1600 bp was amplified from human breast tissue.The magnitude of molecular weight is consistent with the expect size of the MEG3.The recombinant plasmid was sequenced by DNA program,and the results showed that the nucleic acid sequence of the MEG3 which inserted into the plasmid was correct.After transfecting pc DNA3.1-MEG3 into MCF-7 cells by liposome and screening using G418, and the RT-PCR results showing that the MCF-7 cells which transfected by pc DNA3.1-MEG3 can stably express MEG3.The results from MTT assay showing that the cell viability of stable transfection MCF-7 cells(MCF-7-M cells) is decreased compared with either MCF-7cells or transfection empty carrier MCF-7 cells(MCF-7-C cells)(p<0.01),and the cell viability of MCF-7-M cells which pretreated with TNF? and TW37 is lower than MCF-7-M cells which just pretreated with TNF?(p<0.01).The results from Hoechst33258 staining assay and flow cytometry showing that the apoptosis rate is higher in MCF-7-M cells compared with either MCF-7 cells or MCF-7-C cells( p < 0.01),the apoptosis rate is lower in MCF-7-M cells which pretreated with TNF?compared with MCF-7-M cells(p<0.01).and the apoptosis rate is higher in MCF-7-M cells which pretreated with TNF? and TW37 compared with MCF-7-M cells which pretreated with TNF?(p<0.01).The results from western blot showing that the phosphorylation of I-?B? is decreased in MCF-7-M cells,and TNF? could induce I-?B? phosphorylation. The expression of NF-?B P65, NF-?B P50 and Bcl-2 are decreased(p<0.01), and Caspase3 is increased( p < 0.01)in MCF-7-M cells compared with MCF-7 cells and MCF-7-C cells.TNF? induces the expression of NF-?B P65, NF-?B P50 and Bcl-2 proteins,apromotes the nuclear migration of NF-?B P65 and NF-?B P50, and inhibits the expression of Caspase3 in MCF-7-M cells. TW37 inhibits the expression of Bcl-2 and promotes the expression of Caspase3 in MCF-7-M cells.Conclusions:(1) The expression vector of lnc RNA MEG3 has been Constructed, and the cell line stably expressing MEG3 was eatablished.(2)Lnc RNA MEG3 induces apoptosis by inhibiting phosphorylation of I-?B? protein and nuclear translocation of NF-?B p65 and NF-?B P50 in MCF-7 cells. At the same time,it could down-regulate the expression of Bcl-2 and up-regulate the expression of Caspase3 in MCF-7cells.