ING4 Inhibits Migration And Invasion Of Breast Cancer Cells Through Reversing Epithelial-mesenchymal Transition(EMT)

Objective: This study aimed to investigate the effect of lentivirus-mediated overexpression of inhibitor of growth 4(ING4) on migration and invasion of breast cancer cells and its underlying mechanism.Methods:(1) The coding sequence(CDS) fragment encoding humanized ING4 was amplified by PCR using the pAdTrack-CMV/ING4 plasmid as a template and using a primer pair specific for ING4(ING4-F: 5’-gaa gct agc gcc acc atg gct gct ggg atg tat ttg-3’; ING4-R: 5’-ata ggc gcg ccc tat ttc ttc ttc cgt tct tg-3’).(2) The ING4 CDS fragment was subsequently inserted into a green fluorescent protein(GFP)-labeled pLenti6.3/IRES/GFP lentiviral plasmid between NheI and Sgs I restriction enzyme sites and identified by PCR, double enzyme digestion and DNA sequencing to construct a recombinant lentiviral plasmid pLenti6.3/ING4/IRES/GFP.(3) The p Lenti6.3/ING4/IRES/GFP or blank control pLenti6.3/IRES/GFP lentiviral plasmid was then cotransfected into 293 T human embryonic kidney cells with helper packaging plasmids including pLP1, pLP2 and VSVG by Lipofectamine2000, respectively. The lentivirus expressing ING4(LV-ING4) or blank lentivirus(LV) was consequently generated and concentrated by ultracentrifugation and the titre was then determined according to GFP expression by limiting dilution analysis.(4) After an optimal dose infection of the MDA-MB-468 human triple negative breast cancer cells with 10 multiplicity of infection(MOI) LV-ING4 or LV followed by selection with 10 μg/ml of Blasticidin S(BSD), the MDA-MB-468-LV-ING4(termed MDA-MB-468-ING4) and MDA-MB-468-LV(termed MDA-MB-468-Mock) transfectants were obtained. The lentivirus-mediated transgene efficiency was detected by fluorescence microscopic and flow cytometric analysis of GFP expression. The lentivirus-mediated ING4 transgene expression in MDA-MB-468 breast cancer cells was further determined by RT-PCR and Western blot, respectively.(5) The migratory and invasive ability of MDA-MB-468-ING4 and MDA-MB-468-Mock breast cancer cells was evaluated by Transwell chamber migration and invasion assay. The effect of lentiviral-mediated ING4 overexpression on migration and invasion of MDA-MB-468 triple negative breast cancer cells was analyzed.(6) The expression of epithelial-mesenchymal transition(EMT)-associated proteins such as E-cadherin, N-cadherin, Snail1, Snail2, Zeb1, Zeb2, Twist1 and Twist2 in MDA-MB-468-ING4 and MDA-MB-468-Mock breast cancer cells was analyzed by Western blot. The molecular mechanism for ING4-elicited inhibition of MDA-MB-468 triple negative breast cancer cell migration and invasion was then elucidated.Results:(1) The recombinant lenitivirus expressing humanized ING4 and marker GFP was successfully prepared.(2) The lentivirus-mediated ING4-transgenic MDA-MB-468 human triple negative breast cancer cell line was successfully constructed.(3) The lentivirus-mediated ING4 overexpression remarkably suppressed migration(relative migratory ability, 45.7% of MDA-MB-468-Mock) and invasion(relative invasive ability, 36.8% of MDA-MB-468-Mock) of MDA-MB-468 breast cancer cells.(4) ING4 markedly downregulated Snail1 and Snail2 EMT-inducing transcription factors(EMT-TFs) and N-cadherin mesenchymal marker as well as upregulated E-cadherin epithelial marker in MDA-MB-468 breast cancer cells. However, it has almost no effect on the expression of other EMT-TFs including Zeb1, Zeb2, Twist1 and Twist2.Conclusion: Lentivirus-mediated ING4 overexpression can suppress breast cancer metastasis through reversal of EMT possibly via downregulation of Snail1 and Snail2 EMT-TFs.