Whether The Abnormal Expression Of INPP4B Is Associated With Cell Proliferation And Clinical Outcome In Multiple Myeloma

Objective: 1.To investigate the expression of the inositol polyphosphate-4-phosphatase,type-II(INPP4B)in multiple myeloma(MM)cell lines and bone marrow samples of new diagnosed MM patients.To investigate the expression of extramedullary plamacytoma.2.To investigate the relationship between the expression level of INPP4 B and the clinical prognosis.3.To investigate the relationship between the overexpression of INPP4 B and the proliferation of MM cell lines.The expression level of PI3K/Akt signaling pathway related proteins.To investigate the potential mechanism of the effect of INPP4 B to MM cells proliferation.Methods: 1.The expression level of INPP4 B in new diagnosed MM patient samples: qPCR was used to assess the expression level of normal bone marrow plasma cells and 28 new diagnosed MM patient samples malignant plasma cells between January 2011 and January 2016.2.The expression of INPP4 B of extramedullary plasmacytoma tissues: IHC(immunohistochemical)was used to assess the expression level of INPP4 B in tissues of extramedullary plasmacytoma between January 2010 and December 2015.Breast cancer tissues was used as positive and negative controls.3.The expression of INPP4 B protein and m RNA in MM cell lines: Western and qPCR were used to assess the protein and m RNA expression of INPP4 B in seven MM cell lines,LP-1,OPM-2,RPMI 8226,U266,H929,MM1.R,MM1.S.4.To achieve the overexpression of INPP4B: The infection efficiency of U266 and H929 was low.We use the LP-1、OPM2 and RPMI 8226 cells to achieve the overexpression of INPP4 B.we receive the virus of NEG and INPP4 B through HEK cells,then use the virus to infect the LP-1、OPM2 and RPMI 8226 cells and achieve the overexpression of INPP4 B.5.The expression of INPP4 B protein and m RNA after the virus infection: Western and qPCR were used to assess the protein and m RNA expression of INPP4 B in LP-1、OPM2 and RPMI 8226 and assess the success of the overexpression of INPP4 B in myeloma cell lines.6.The effect of the INPP4 B overexpression on the proliferation of MM cells: CCK8 assay was used to investigate the change of the cell proliferation after the overexpression of INPP4 B or NEG.7.The change of MM cell cycle after the INPP4 B overexpression: Flow cytometry was used to assess the cell cycle change of LP-1、OPM2 and RPMI 8226 after the overexpression of INPP4 B.8.The mechanism of INPP4 B in MM: Western blotting was used to assess the expression level of the PI3K/Akt signaling pathway-related protein such as Akt,p-Akt et al and cell cycle related proteins Cyclin D1.9.The datas were managed by SPSS 17.0.Results: 1.qPCR results: Compared with normal human bone marrow plasma cells,the INPP4 B expression level of 26 patients was lower between 28 patients which only 2 patients were high.The median was used to be cut-off,the lower expression level predict a worse prognosis.PFS and OS all have statistical significance.2.IHC results: Seven extramedullary plasmacytoma patients expressed INPP4 B reversible focal positive.Thirty-five extramedullary plasmacytoma patients express no INPP4 B.3.qPCR results: Compared with normal human bone marrow plasma cells,the INPP4 B expression level of 7 MM cell lines was lower.4.Western Blotting and qPCR results: The protein and m RNA expression of INPP4 B in 3 MM cell lines,LP-1,OPM-2 and RPMI 8226 were overexpressed.5.CCK8 results: The LP-1,OPM-2 and RPMI 8226 cell proliferation after the overexpression of INPP4 B were higher than NEG and control.6.Flow cytometry results: The cell cycle arrest of LP-1 、 OPM2 and RPMI 8226 after the overexpression of INPP4 B in G1 phase.7.Western Blotting results: The expression level of PI3K/Akt signaling pathway-related akt-p473 protein and cell cycle-related protein Cyclin D1 are up-regulated.Conclusion: The expression level of INPP4 B in majority of MM patients are lower normal donor human.The expression level of INPP4 B in 7 MM cell lines are lower.The overexpression of INPP4 B in MM inhibit the cell proliferation via down-regulating the expression level of akt-p473 protein.The overexpression of INPP4 B in MM induce the MM cell G1 phase arrest via regulating the down-regulating of cell cycle-related protein Cyclin D1.