Effects Of Bortezomizomi Combined With Siramesine On The Expression Of Transcription Factor TFEB In Multiple Myeloma RPMI8226 And U266 Cell Lines

Objective:We use Bortezomib and Siramesine alone and combined application of the two drugs in multiple myeloma cell lines RPMI8226 and U266 cells,and observe the changes of the two cell lines in the cytoplasm and nucleus of TFEB,to explore the effects of two drugs on the change of the content of TFEB.To provide the experimental basis for the clinical treatment of multiple myeloma.Methods:Multiple myeloma RPMI8226 and U266 cells were divided into blank control group,siramesine 0.5umol/L group,bortezomib 6nmol/L group and combination group,Cell Counting Kit-8(CCK-8)method was used to detect the inhibitory effect of two drugs on cell proliferation,real-time quantitative PCR(RT-PCR)and Western blot to detect expression of TFEBmRNA and protein.The difference between P < 0.05 was statistically significant.Results:1.Different time treatment with bortezomib or siramesine monotherapy and two drug combination(6h,12 h,24h),compared with the control group,the growth of RPMI8226 and U266 cells was significantly inhibited,and this inhibition was time dependent,the difference was statistically significant(P < 0.05).2.Effects of sirmaesine and bortezomib on the expression of TFEBmRNA and protein in RPMI8226 cells and U266 cells in multiple myeloma2.1 Real time quantitative analysis showed that the expression of TFEBmRNA in cytoplasm of RPMI8226 cells and U266 cells could be down regulated by the sirmaesine and bortezomib,and the expression of TFEBmRNA in the nuclei of RPMI8226 cells and U266 cells could be upregulated.Compared with the control group,the expression of TFEBmRNA in the cytoplasm of RPMI8226 cells and U266 cells decreased after the treatment of 24 h,and the expression of TFEBmRNA in nucleus increased,the difference was statistically significant(P < 0.05).2.2 Detection by Western Blot showed that sirmaesine and bortezomib could down regulate the expression of TFEB protein in the cytoplasm of RPMI8226 cells and U266 cells,and up regulate the expression of TFEB protein in RPMI8226 cells and U266 cells.Compared with the control group,the expression of TFEB protein decreased in the cytoplasm of RPMI8226 cells and U266 cells after the treatment of 24 h,and the expression of TFEB protein in the nucleus increased.The difference was statistically significant(P < 0.05).3.Effects of bortezomib combined with sirmaesine on the expression of TFEBmRNA and protein in RPMI8226 cells and U266 cells in multiple myeloma3.1 Real time quantitative analysis showed that the combination of the sirmaesine and bortezomib could down regulate the expression of TFEBmRNA in the cytoplasm of RPMI8226 cells and U266 cells,and up regulate the expression of TFEBmRNA in the nuclei of RPMI8226 cells and U266 cells.Siramesine combined with bortezomib treated cells after 24 h and the single application of bortezomib used alone or siramesine compared to reduce the expression of TFEBmRNA RPMI8226 cells and U266 cells in the cytoplasm,increased expression of TFEBmRNA in the nucleus,the difference was statistically significant(P < 0.05).3.2 The Western Blot revealed that the application of bortezomib combined with siramesine could down regulate the expression of TFEB protein in the cytoplasm of RPMI8226 cells and U266 cells,and up regulate the expression of TFEB protein in RPMI8226 cells and U266 cells.Compared with the monotherapy group,the expression of TFEB protein decreased in the cytoplasm of RPMI8226 cells and U266 cells after the treatment of 24 h with the combination of bortezomib and siramesine,and the expression of TFEB protein in the nucleus increased.The difference was statistically significant(P < 0.05).Conclusion:1.Bortezomib,siramesine and the combination of these two drugs can significantly inhibit the proliferation of multiple myeloma cells,and this inhibition is time-dependent in a certain range.2.Both the expression of TFEBmRNA and protein in cytoplasm and the expression level of TFEBmRNA and protein in the nucleus were reduced by the application of siramesine and bortezomib alone.3.The combination of bortezomib and siramesine could reduce the expression level of TFEBmRNA and protein in the cytoplasm and up regulate the expression level of TFEBmRNA and protein in the nucleus compared with the bortezomib or siramesine alone group.