The Effect Of MiR-302b On Regulating The Osteogenic Differentiation Of Multiple Myeloma Microenvironment By DKK1

Background and Objective: Multiple myeloma(MM)is characterized by malignant clonal proliferation of plasma cells in the bone marrow and MM is one of the most common hematological malignancies.In recent years,more and more MM patients have bone pain as the first symptom.Bone-related diseases such as bone pain and pathological fractures caused by osteolytic destruction of MM have attracted the attention of the orthopedics community.Osteogenesis-osteoclast coupling imbalance in MM bone marrow microenvironment is the main cause of MM bone destruction.DKK1 is a secreted protein that binds to the Wnt receptor LRP5 on the cell membrane and inhibits the Wnt/?-catenin signaling pathway related to osteogenic differentiation.miRNA is a type of small non-coding RNA that can regulate a variety of biological processes including proliferation and apoptosis.The basis of previous research found that the expression level of miR-302 b in bone marrow samples of MM patients was significantly lower than that of the control group.This study will explore the specific regulatory role of miR-302 b and DKK1 in the process of osteogenic differentiation of MM cells at the cellular and molecular levels;explore the correlation between miR-302 b and DKK1 on MM bone destruction in animal experiments,and established a brand-new gene knockout mouse model to provide new ideas for the treatment of MM bone destruction.Methods: 1.Using RT-PCR to detect the expression of miR-302 b in MM cell lines and MM bone marrow samples,and compare with the control group.2.Culture multiple myeloma cell lines RPMI-8226 and MM1.S in vitro,and transfect miR-302 b mimic,inhibitor and control sequence(mimic,mimic NC,inhibitor and inhibitor NC)into RPMI-8226 In MM1.S cells,RT-PCR was used to detect the expression level of miR-302 b in each group of cells after transfection.3.The MTT method was used to detect the cell proliferation ability of each group after transfection,flow cytometry was used to detect the apoptosis of each group,and Western Blot was used to detect the expression level of apoptotic protein in each group.4.Using bioinformatics to predict miR-302 b target genes,and use dual luciferase reporter gene experiments to verify.5.Using Western Blot to detect the expression level of DKK1 in MM cells after transfection of miR-302 b compounds in each group.6.Co-transfect the miR-302 b compound and DKK1 plasmid into the MM cell line,and use Western Blot to detect the expression level of DKK1 in each group of MM cells.7.Mi R-302 b compound was transfected into MM cells,and co-culture the transfected MM cells with osteoblast precursor cells MC3T3-E1,and induce osteogenic differentiation of the co-cultured MC3T3-E1 cells.ELISA was used to detect the secretion level of DKK1 in the upper culture medium of each group of MC3T3-E1 cells,RT-PCR was used to detect the expression level of Collagen I in each group of MC3T3-E1 cells,and Alizarin Red staining was used to detect the osteogenic differentiation of each group of MC3T3-E1 cells.8.Mi R-302 b compound and DKK1 plasmid was co-transfected into MM cell line,and inject the transfected groups of MM cells into the mouse femur to establish a mouse model of multiple myeloma bone destruction.Micro CT was used to detect the extent of bone destruction in each group of mice.9.Mi R-302 b compound and DKK1 plasmid was co-transfected into MM cell line,and co-culture the transfected MM cells with MC3T3-E1 cells.Western Blot was used to detect the expression of Wnt/?-catenin signaling pathway related proteins in MC3T3-E1 cells of each group after co-culture.Results: 1.The RT-PCR test results show that,compared with the control group,the expression of miR-302 b in MM cell lines and MM bone marrow samples were significantly decreased.2.RT-PCR results showed that after miR-302 b mimic(mimetic)transfection,the expression of miR-302 b in RPMI-8226 and MM1.S cells were significantly higher than mimic-NC(negative control group);After miR-302 b inhibitor(inhibitor)transfection,the expression of miR-302 b in the two MM cell lines was significantly lower than that of inhibitor-NC(negative control group).3.The results of the MTT experiment showed that compared with the negative control group,the proliferation ability of MM cells in the miR-302 b mimic group was reduced;and the cell proliferation ability of the miR-302 b inhibitor group was significantly higher than that of the control group.4.The results of flow cytometry and Western Blot of apoptotic protein showed that compared with the control group,the up-regulation of miR-302 b can promote the apoptosis of MM cells and increase the expression level of Bax.5.The results of bioinformatics and dual luciferase reporter gene experiments showed that compared with the control group,the luciferase activity of the experimental group cotransfected with miR-302 b mimic and wild-type DKK1 reporter gene plasmid in the MM cell line was significantly decreased,DKK1 is the direct target gene of miR-302 b.6.Western Blot results show that compared with the negative control group,upregulation of miR-302 b can lead to a significant decrease in the expression level of DKK1 in MM cells.In addition,in MM cells co-transfected with miR-302 b synthesis and DKK1 plasmid,overexpression of DKK1 can reduce the inhibitory effect of miR-302 b.7.In the co-cultivation experiment,the ELISA results showed that compared with the control group,the secretion level of DKK1 in the miR-302 b overexpression group was significantly down-regulated.Alizarin red staining results show that MM cells can inhibit the osteogenic differentiation of MC3T3-E1 cells in the co-culture system,and overexpression of miR-302 b can reduce the inhibition of MM cells on the osteogenic differentiation of MC3T3-E1 cells.8.Western Blot and RT-PCR results of Wnt/?-catenin signaling pathway related proteins in MC3T3-E1 cells of each group after coculture showed that the miR-302 b overexpression group(miR-302 b mimic + DKK1ctrl)and the control group(Compared with miR-302 b mimic NC+DKK1 ctrl),the expressions of LRP6,Wnt3 a and ?-catenin are up-regulated.Overexpression of miR-302 b in MM cells can inhibit the secretion of DKK1 by MM cells,thereby activating Wnt/? in MC3T3-E1 cells-catenin signaling pathway.9.Micro CT results of multiple myeloma bone destruction mouse models show that DKK1 can promote bone destruction mediated by multiple myeloma,and miR-302 b can inhibit the expression of DKK1,thereby reducing the osteolysis of multiple myeloma Bone destruction.Conclusion: 1.The expression of miR-302 b is lower than control group in multiple myeloma.Up-regulating miR-302 b inhibits the proliferation of multiple myeloma cells and promote their apoptosis.2.The overexpression of miR-302 b in MM cells alleviated the inhibition of MM cells on the osteogenic differentiation of MC3T3-E1 cells.3.miR-302 b reduces the osteolytic destruction of multiple myeloma in NOD/SCID mice by inhibiting the secretion of DKK1 in MM cells.