| Objectives Breast cancer is a malignant tumor that serious impact on women’s health.In recent years, the incidence of breast cancer increased significantly. Mitofusin2(Mfn2),a newly discovered anti-oncogene, can inhibit the proliferation of a variety of tumor cellsincluding breast cancer. This study aims to study the importance of the442ndserinephosphorylation of Mfn2protein mediated by protein kinase A (PKA) on its anti-proliferation function in breast cancer cells. This work will provide theoretical basis forclinical applications.Methods Using normal human breast tissue cDNA as a template and the pEGFP-N1asa vector, gene expression plasmids were constructed respectively: pEGFP-Mfn2plasmidscontaining the full-length open reading frame (ORF) of Mfn2and pEGFP-Mfn2-PKA(△)plasmids containing Mfn2ORF without the DNA sequence encoding the442ndserine.Four groups were designed: Mfn2-PKA(△) group (transfected with pEGFP-Mfn2-PKA(△)), Mfn2group (transfected with pEGFP-Mfn2), N1group (transfected withpEGFP-N1) and Mock group (untransfected). The plasmids were transfected into MCF-7cells with liposome. After48h, the transfection efficiency was detected by laser confocalmicroscopy. The mRNA and protein expression levels of Mfn2were detected by real-time quantitative PCR and immunocytochemistry respectively, in order to conformwhether exogenous Mfn2was transcribed and translated in the MCF-7cells. Cellproliferation rate was assessed within72h post-transfection by cell counting and MTT.After transfection48h, cell cycle distribution was analyzed by flow cytometry. Thephosphorylation level of ERK1/2in serum-stimulated was detected by western blot.Results The full-length ORF of Mfn2gene (2274bp) and the Mfn2-PKA(△) gene(2271bp) from normal human breast tissue were amplified. Then gene expressionplasmids of pEGFP-Mfn2and pEGFP-Mfn2-PKA(△) were constructed respectively.The sequence was matched with the ORF sequence in GenBank by DNA-sequencing. At48h post-transfection the green fluorescent of EGFP protein were observed in Mfn2-PKA(△) group, Mfn2group and N1group by the laser confocal microscopy. Thisindicated that the plasmids were successfully transfected into MCF-7cells and thetransfection efficiencies were about60%. Mfn2protein was localized in the cytoplasm. The mRNA and protein levels of Mfn2in Mfn2-PKA(△) and Mfn2group weresignificantly higher than those in N1and Mock group (F=21.052, P<0.05). There was nosignificant difference between Mfn2-PKA(△) and Mfn2group (P>0.05). The cellproliferation ability of Mfn2-PKA(△) group was similar to that of N1and Mock groups(P>0.05), whereas significantly lower than that of Mfn2group (P<0.05). Most of MCF-7cells of Mfn2group were arrested in G1phase, whereas the cells in G1phase of Mfn2-PKA(△) group were decreased significantly (P<0.05). Cell cycle distribution of Mfn2-PKA(△) group was similar to that of N1and Mock group (P>0.05). The ERK1/2proteinlevel were similar in the four groups, whereas the p-ERK1/2protein level of Mfn2-PKA(△) group was similar to that of N1and Mock groups (P>0.05), which wassignificantly higher than that of Mfn2group (F=19.632, P<0.05).Conclusions Exogenous Mfn2and Mfn2-PKA(△) were expressed in MCF-7cells atboth mRNA and protein levels. Over expression of Mfn2inhibited the proliferation ofMCF-7cells, and the cell cycle was blocked in G0/G1phase. Whereas Mfn2-PKA(△)protein failed to inhibit the MCF-2cell proliferation. The phosphorylation level ofERK1/2, which is a key protein in the Ras-ERK1/2signaling pathway, was significantlydecreased, but not Mfn2-PKA(△). These data suggested that the442ndphosphorylationof Mfn2protein is necessary for its anti-proliferation function in breast cancer cells. Ras-ERK1/2signaling pathway is probably a downstream pathway of phosphorylated Mfn2. |
PDF Full Text Request