The Effect Of P21^Ras Domain Of Mitofusin2on Its Anti-proliferation Fuction In Breast Cancer Cells

Objectives Mitochondrial fusion protein2(mitofusin2, Mfn2) is a novel cancersuppress gene for many malignant tumors. Our previous studies showed that theexpression level of Mfn2is lower in breast cancer cells than that in normal breast cells,and over expression of it can inhibit proliferation of breast cancer cells. In this study, weaimed to study the importance of P21Rasdomainof Mfn2protein on its anti-proliferationfunction in human breast cancer cells.Methods Four groups were designed: group Mfn2(transfected with pEGFP-Mfn2plasmids containing the full length of open reading frame (ORF) of Mfn2), group Mfn2-△P21Ras(transfected with pEGFP-Mfn2-△P21Rasplasmids containg the Mfn2ORFwithout P21Rascoding sequence), group N1(transfected with empty pEGFP-N1plasmids)and group Mock (non transfection group).1. Construct eukaryotic expression vectorscontaining full-length ORF of Mfn2and Mfn2ORF without p21Rascoding sequence,respectively. The sequences of Mfn2ORF and Mfn2-△P21Rasconstructed in plasmidswere verified by DNA sequencing assay. The plasmids were respectively transfected intohuman breast cancer cell MCF-7. Fluorescence microscopy was used to observe greenfluorescence of EGFP to acsess transfection efficiency.2. The mRNA and protein levelsof Mfn2were detected by real-time fluorescence quantitative PCR (FQ-PCR) andimmunocelularchemistry respectirely.3. MTT was used to determine the proliferationactivity of MCF-7cells in each group, and Flow Cytometry was used to analysis the cellcycle of MCF-7cells in each group.4. The mRNA and protein levels of Cyclin E weredetected by real-time fluorescence quantitative PCR (FQ-PCR) andimmunohistochemistry.5. Western blot was used to analysis the expression level ofphosphorylated ERK1/2protein, an important molecule in Ras signaling pathway in eachgroup.Results1. The green fluorescence of EGFP was observed in Mfn2, Mfn2-△P21RasandN1groups by fluorescence microscopy, but not in group Mock. This showed that thepEGFP-plasmids were successfully transfected into MCF-7cells in the three groups andthe transfection efficiency were comparable among the three groups.2. The mRNA andprotein levels of Mfn2were markedly increased in group Mfn2and group Mfn2-△P21Ras, compared with those in group N1and group Mock (F=22.702, P<0.05). There were nodifference between group Mfn2and group Mfn2-△P21Ras. This indicated that both theexogenous Mfn2and Mfn2-△P21Raswere transcribed and translated in MCF-7cells.3.Cell proliferation rate of group Mfn2-△P21Raswas similar to that of group N1and groupMock, which is significantly higher than that of group Mfn2(P<0.05). The MCF-7cellsof group Mfn2were most arrested in G0/G1phase, whereas the cells in G0/G1phase ofgroup Mfn2-△P21Raswere decreased significantly. The cell cycle distribution of groupMfn2-△P21Raswas similar to that of group N1and group Mock (P<0.05). These showedthat Mfn2-△P21Raslost the anti-proliferation function in MCF-7cells.4. There was nosignificant difference of Cyclin E mRNA levels among the four groups. Whereas theprotein levels of Cyclin E in group Mfn2-△P21Raswere similar to those of group N1andgroup Mock, which is markedly higher than those of group Mfn2(F=3.367, P>0.05).This suggested that the anti-proliferation function of Mfn2was probabaly due to thedownregulation of Cyclin E protein expression. However, Mfn2-△P21Raslost thisfunction.5. The protein level of p-ERK1/2of group Mfn2-△P21Ras, which wascomparable to that in group N1and group Mock, was markedly higher than that of groupMfn2(F=18.130, P<0.05). This indicated that Mfn2plays a role in ERK1/2proteinphosphorylation, whereas Mfn2-△P21Raslost this function.Conclusions1The overexpression of Mfn2gene can block the cycle cyle in G0/G1phase and then inhibit MCF-7cell proliferation. These probably induced by impairedERK1/2protein phosphorylation and Cyclin E protein expression, which is a downstreamof Ras-ERK1/2pathway.2Although the exogenous Mfn2-△P21Raswas transcribed andtranslated in MCF-7cells, Mfn2-△P21Rasprotein failed to inhibit the expression, and toinhibite the phosphoration of ERK1/2protein and Cyclin E expression, and then failed toblock cell cycle and cell proliferation. These data suggested that the P21Rasbindingdomain of Mfn2is important for its anti-profiferation function in breast cancer cells.