| Objectives:1.To detect HMGB1 mRNA,HMGB1 and their surface receptors in U266 cells of multiple myeloma;2.To:investigate the effect of exogenous HMGB1 on MM cell proliferation;3 To analyze whether exogenous HMGB1 can activate the NF-κB pathway;4.To analyze whether exogenous HMGB1 can stimulating MM cell proliferation by activating the NF-k B pathway;5.To clarify the changes of MM cell proliferation and apoptosis after the HMGB1 inhibitor sodium n-butyrate was used to inhitbit the expression of HMGB1 in U266 cell.Methods:1.In vitro culture of multiple myeloma U266 cell lines,Western Blot was used to detect the expression of HMGB1 protein and major HMGB1 receptors located on cell surface,namely Receptor of Advanced Glycation End products and toll-like receptors.,RT-PCR was used to detect the expression of HMGB1mRNA.2.After 24h and 48h pretreatment of U266 cell lines with different concentrations of exogenous HMGB1(0,50,100,150,200,250,500ng/ml),CKK8 assay was used to detect the effect of different concentrations of exogenous HMGB1 on MM cell proliferation.3.In vitro culture of multiple myeloma U266 cell lines,HMGB1 was used to stimulate MM cells for 0,5,10,30 and 60min respectively.Nuclear protein of NF-κB was detected by western blot.4.Multiple myeloma U266 cell lines were cultured in vitro and preincubated with different concentrations of PDTC(0,50,100,200 uM)to inhibit the activation of NF-κB signaling pathway.HMGB1 was used to stimulate cells.The number of U266 cells was detected by CCK8 24h later to evaluate whether HMGB1 could still promote the proliferation of MM cells after the activation of NF-κB signaling pathway inhibited by PDTC.5.Multiple myeloma U266 cell lines were cultured in vitro with different concentrations of HMGB1 inhibitor sodium butyrate(OmM,0.625mm,1.25mM,2.5mM,5mM,10mM,20mM,40mM)for 24h and 48h respectively.The expression of HMGB1 in U266 cell lines was detected by Western Blot,and the optimal inhibition time and concentration of sodium butyrate were determined(48h,1.25mm).The U266 cell lines were divided into the control group and the experimental group.The control group was cultured normally,and sodium butyrate(1.25mM,48h)was added into the experimental group to inhibit the expression of HMGB1.CCK8 method and flow cytometry were used to detect the control group and the experimental group to evaluate the relationship between HMGB1 expression and multiple myeloma cell proliferation and apoptosis.Results:1.In U266 cell lines,the expression of HMGB1 protein,the main receptors of HMGB1,namely RAGE and toll-like receptors,were detected to be expressed by Western Blot,and HMGB1mRNA was detected to be expressed by RT-PCR.2.The OD value of U266 cells increased in the experimental group stimulated by HMGB1 for 24h(HMGB1 concentration:50,100,150,200,250,500 ng/ml)compared with the control group(HMGB1 concentration:0 ng/ml)[(0.365±0.000)vs(0.319±0.006);(0.362±0.007),vs(0.31910.006);(0.360±0.002)vs(0.319±0.006);(0.361±0.003)vs(0.319±0.006);(0.351±0.003)vs(0.319±0.006);(0.366±0.005)(0.319±0.006)],the difference has statistic significance(P<0.05).The OD value of U266 cells increased in the experimental group stimulated by HMGB1 for 48h(HMGB1 concentration:50,100,150,200,250,500 ng/ml)compared with the control group(HMGB1 concentration:0 ng/ml),(P<0.05)[(0.523±0.007)vs(0.50910.009);(0.522±0.002)vs(0.509±0.009);(0.534±0.004)vs(0.509±0.009);(0.538±0.004)vs(0.50910.009);(0.534±0.010)vs(0.509±0.009);(0.60110.009)vs(0.50910.009)],the difference has statistic significance(P<0.05).3.Western blot showed that after HMGB1 stimulated MM cells,the expression level of nuclear NF-κB increased.Compared with the control group(adding exogenous HMGB1 for 0mim),NF-κB/GAPDH.increased significantly in the experimental group(adding exogenous HMGB1 for 60min)[(0.83±0.02)vs(0.41±0.05)(P<0.01).4.CCK8 method shows:compared with control group(PDTC concentration:Oμmol/L),the OD value of experimental group(PDTC:50uM、100uM、200uM)significantly decreased(P<0.05)after the NF-κB pathway was inhitbited by PDTC[(1.59±0.17)vs(0.05±0.00);(1.59±0.17)vs(0.05±0.01);(1.59±0.17)vs(0.05±0.01)],indicating that HMGB1 can promote MM cell proliferation through the activation of NF-κB pathways.5.Western Blot showed that the optimal inhibition concentration and time of HMGB1 inhibitor Sodium butyrate were 1.25mm and 48h.Therefore,sodium butyrate was used to inhibit U266 cells for 48h in the subsequent experimental group,while no treatment was done in the control group.CCK8 assay showed that compared with the control group,the cell growth in the experimental group was significantly decreased(P<0.01)[(0.35±0.00)vs(0.49±0.01)],and the apoptosis rate was significantly increased by flow cytometry(P<0.01)[(3.75±0.11)%vs(2.55±0.27)%],the difference has statistic significance.Conclusion1.HMGB1 protein and HMGB1 major receptors,namely RAGE and toll-like receptors,can be expressed in multiple myeloma U266 cell line by western blot..HMGB1mRNA was detected to be expressed by RT-PCR.2.Stimulation of U266 cells by exogenous HMGB1 can promote the proliferation of tumor cells.3.After exogenous HMGB1 stimulated MM cells,the expression level of nuclear NF-κB increased.Compared with the control group(adding exogenous HMGB1 for Omin),NF-κB/GAPDH increased significantly in the experimental group(adding exogenous HMGB1 for 60min)(P<0.01).4.Exogenous HMGB1 can promotes MM cell proliferation through activating NF-κB pathway.5.Compared with the control group,after inhibiting the endogenous HMGB1 expression of U266 cell line by sodium butyrate,the cell proliferation of experimental group decreased and the apoptosis rate increased significantly. |
PDF Full Text Request