Knockdown Of Pyruvate Kinase M Inhibits Cell Growth By Reducing NF-?B Activity In Triple-negative Breast Cancer Cells

A common feature of cancer is the adaptation of cellular metabolism to satisfy the demands of inappropriate growth,survival,and proliferation,this is marked by the Warburg Effect,which is characterized by hyperactive aerobic glycolysis.One attractive approach to targeting therapy on cancer has been to exploit the differential expression of isoenzymes that are present throughout the glycolytic pathway,the most studied among which is the isoforms of the pyruvate kinase M(PKM).But the relative importance of PKM isoforms in tumor growth has been controversial.Herein,we innovatively proposed co-expression of PKM1 and PKM2 and investigated their possible effect in TNBC cell lines.Provide us a new theoretical basis for the diagnosis and treatment of TNBC.Methods?.Verifying the expression of PKM in TNBC tissues and cells 1.IHC was employed to analyze PKM expression in TNBC tissues.2.Western blot and RT-PCR were employed to analyze the expression levels of PKM in TNBC cell lines.?.Confirming the significance of PKM in TNBC development1.Western blot and RT-PCR were employed to analyze the knockdown efficiency of PKM in lentivirus stably transfected cell lines.2.MTT and Colony formation assays were performed to detect the effect of knockdown of PKM on cell growth.3.Flow cytometry was employed to detect the effect of knockdown of PKM on cell cycle and apoptosis.4.Transwell assay analysis knockdown of PKM on cell migration.?.To analyze the effect of PKM on glycometabolism in TNBC and mammary epithelial cellsMTT assay was employed to detect the effect of 2-DG on TNBC and mammary epithelial cells growth.?.Verifying the signal-regulated pathway of PKM/NF-?B in TNBC cells1.Luciferase reporter assay and Western blot confirmed the effect of knockdown PKM on NF-?B in TNBC cells.2.RT-PCR and agarose gel electrophoresis were employed to analyze the knockdown of PKM to regulate the target of NF-?B pathway.Results?.Verify the expression of PKM in TNBC tissues and cell lines1.PKM is highly expressed in TNBC tissues.2.PKM is highly expressed in TNBC cell lines while low expressed in mammary epithelial cells.?.Confirming the significance of PKM in TNBC development1.The stable transfected cell line PKM constructed with lentivirus were highly knockdown efficient.2.Knockdown of PKM can inhibit the proliferation of TNBC cells.3.Knockdown of PKM causes cell cycle arrest and induces apoptosis.4.Knockdown of PKM inhibit the migration and invasion of TNBC cells.?.2-Deoxy-D-glucose inhibits proliferation of TNBC cells? Confirming the regulation mechanism of PKM on NF-?B signaling pathway1.knockdown of PKM significantly suppressed NF-?B activity by reducing the phosphorylation of p65 at serine 536.2.Knockdown of PKM decreased the expression of NF-?B target genes.Conclusions 1.PKM is highly expressed in TNBC tissues and cells.2.Knockdown of PKM inhibits proliferation and migration of TNBC cells and causing cell cycle arrest.3.Knockdown of PKM significantly suppressed NF-?B activity and decreased the expression of NF-?B target genes.