Effects Of Knockdown Of NUPR1 Gene On The Proliferation And Apoptosis Of Multiple Myeloma Cells And Its Mechanism

Objective:To construct nuclear protein 1-short hairpin RNA(NUPR1-shRNA)expressing lentiviral vector targeting NUPR1 gene and investigate the effect of NUPR1 gene silencing on the proliferation and apoptosis of human multiple myeloma U266 and RPMI8226 cells and its related mechanisms.Methods:The normal human bone cells were used as control and the mRNA expression level of NUPR1 in multiple myeloma cell lines(U266 and RPMI8226)and MM patient specimens were detected by quantitative real-time PCR(qRT-PCR).Then the NUPR1 gene target shRNA plasmid was constructed and screen for the best target for transfected into U266 and RPMI8226 cells.The transfection efficiency was detected by flow cytometry.The interference effect of NUPR1 gene was detected by qRT-PCR and Western blotting.The cell proliferation was analyzed by CCK-8 assay.The cell apoptosis and cell cycle were determined by flow cytometry.Quantitative RT-PCR and Western blot were used to detect the expression of proliferation and apoptosis-related genes and proteins.Results:The mRNA expression levels of NUPR1 in MM cell lines(U266 and RPMI8226)and MM patient specimens were higher than that in normal bone cells.NUPR1-shRNA-expressing lentiviral vector was successfully constructed,and flow cytometry results shows that the transfection efficiency of both cell line reached over 80%.Quantitative RT-PCR and Western blot showed that NUPR1-shRNA2 lentiviral vector most effectively and as the best target.With NUPR1 gene interference,cell proliferation were significantly inhibited in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group.Compared with the control group,the apoptosis rate of U266 and RPMI8226 in shNUPRl group were significantly increased(P<0.05).Flow cytometer indicated that the U266 and RPMI8226 cells in shRNA group were arrested in G0/G1 phase(P<0.01).Compared with the control group,qRT-PCR and Western blot showed that the levels of Bcl-2 and PCNA were significantly down-regulated,and the level of PTEN was significantly increased in shNUPRl group.Meanwhile,the expression of cleaved caspase-3,cleaved caspase-8 and cleaved caspase-9 protein were significantly higher in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group.Conclusions:The mRNA expression levels of NUPR1 in U266 and RPMI8226 cells and MM patient specimens were higher than that in normal bone cells.Down-regulate the expression of NUPR1 in U266 and RPMI8226 cells can inhibit cell proliferation and promote its apoptosis.The mechanism may be that knocking down NUPR1 can activate mitochondrial apoptosis pathway and regulate PCNA and PTEN expression.It is suggested that NUPR1 is an oncogene in MM,which may be a potential target for MM treatment.