The Suppressive Role Of Inhibitor Of Growth 4(ING4) In HER2-positive Breast Cancer Growth And Metastasis Through PTEN And Its Underlying Mechanism

Objective:To elucidate the suppressive role of ING4 in HER2-positive breast cancer growth and metastasis through PTEN and its underlying mechanism.Methods:(1)The total cellular protein derived from human HER2-positive breast cancer cell lines including BT474 and SKBR3 as well as a human normal mammary epithelial cell line MCF-10A(used as a control)was extracted,respectively.The expression of ING4 in human HER2-positive breast cancer cells was detected by Western blot analysis.(2)The pLenti6.3/ING4/IRES/GFP expressing human ING4 or blank control pLenti6.3/IRES/GFP lentiviral plasmid was cotransfected into 293T human embryonic kidney cells with helper packaging plasmids including pLP1,pLP2 and VSVG by Lipofectamine 2000,respectively.The lentivirus expressing ING4(LV-ING4)or blank lentivirus(LV)was consequently generated and the titre was then determined.(3)The BT474 and SKBR3 human HER2-positive breast cancer cells were infected with LV-ING4 or LV(control)at a multiplicity of infection(MOI)of 100 and selected with drug Blasticidin S(BSD),leading to the generation of BT474-ING4,SKBR3-ING4 and BT474-mock,SKBR3-mock HER2-positive breast cancer cells.The transgene efficiency in BT474 and SKBR3 breast cancer cells was analyzed according to GFP reporter by fluorescence microscopy and flow cytometry.The overexpression efficiency of ING4 in BT474 and SKBR3 cells was determined by qRT-PCR and Western blot analysis.(4)The effect of ING4 overexpression(BT474-ING4 vs BT474-mock and SKBR3-ING4 vs SKBR3-mock)on in vitro proliferation,cell cycle,migration and invasion of HER2-positive breast cancer cells was evaluated by Cell Counting Kit-8(CCK-8)assay,colony formation assay,propidium iodide(PI)cell cycle assay,Transwell migration and invasion assays and Tumor stem cell microspheres assay.(5)The effect of ING4 on expression of PTEN,p-AKT(Thr308),p-AKT(Ser473),AKT,NF-?B p65,NF-?B p50,p-JAK2(Tyr1007),JAK2,p-STAT3(Tyr705),STAT3,p-Src(Tyr416)and Src in HER2-positive breast cancer cells was analyzed by Western blot analysis.(6)The effect of ING4 on expression of IL-6 and IL-8 in HER2-positive breast cancer cells was analyzed by ELISA.(7)The effect of ING4 on level of PTEN and miR-21 was expressed by qRT-PCR analysis.Results:(1)Compared with a normal mammary epithelial cell line,the protein level of ING4 are upregulated in human HER2-positive breast cancer cells(P<0.05).(2)The stable ING4-overexpressed BT474-ING4 vs BT474-mock and SKBR3-ING4 vs SKBR3-mock transgenic cell lines were successfully established.The lentivirus-mediated ING4 overexpression remarkably inhibited proliferation,G1/S phase cell cycle transition,migration,invasion and stem cell microspheres assay in BT474 and SKBR3 cells(P<0.05).(3)Overexpression of ING4 significantly upregulated level of PTEN,downregulated level of miR-21,p-AKT(Thr308),p-AKT(Ser473),NF-?B p65,pJAK2(Tyr1007),pSTAT3(Tyr705)and pSrc(Tyr416)and downregulated level of IL-6 and IL-8 to suppresses the activation of AKT and NF-?B signal pathway(P<0.05).Conclusions:Upregulation of ING4 expression in HER2-positive breast cancer cells can regulate the expression of PTEN by regulation of miR-21/PTEN by inhibiting the NF-?B signaling pathway,leading to the inactivation of the AKT signaling pathway.This effect is an important molecular mechanism by which ING4 mediates PTEN to inhibit the growth and metastasis of HER2-positive breast cancer cells.