MiR-221/222 Is Involved In Glucocorticoid Resistance Of Multiple Myeloma Through Inhibiting Autophagy

Objective: To investigate whether mi R-221/222 exerts an inhibitory effect on autophagy through regulating the expression level of autophagy-related gene ATG4 A, which further mediates the glucocorticoid resistance in multiple myeloma cells.Methods: Real time RT-PCR analysis was used to analyze the expression level of mi R-221/222 in multiple MM cell lines and CD138+ cells of bone marrow from MM patients and healthy individuals; To detect the expression extent of ATG4 A in protein and m RNA levels, western-blotting and real time RT-PCR were also used; mi R-221/222 mimics was transfected into MM cell lines MM.1S with transient transfection method, forming an over-expressed system of mi R-221/222. Then, the expression level of ATG4 A in this over-expressed system was analyzed with western-blotting?real time RT-PCR. To detect the change of autophagy level before and after transfecting mi R-221/222 mimics into MM cells, under the effect of dexamethasone, protein P62?LC3B-I and LC3B-II were detected by western-blotting; Autophangy was also detected by transmission electron microscopy. The different effects of dexamethasone on cell death, before and after adding the autophagy inhibitor into MM cells and over-expressing or down-expressing mi R-221/222 in MM cells, were detected by CCK-8.Result: As compared with normal controls,bone marrow CD138+ cells from MM patients expressed high level of mi R-222, whereas the expression level of mi R-221 was decreased. The expression of mi R-221/222 and ATG4 A is negatively related in MM cell lines. Meanwhile, mi R-221/222 can inhibite the protein and m RNA expression of ATG4 A in MM cells. As compared with MM.1R cell, the basal expression level of mi R-221/222 in MM.1S cell was low, and dexamethasone could lead to an significant decline of mi R-221/222 in MM.1S cell, which is sensitive to dexamethasone, and a mild decline in MM.1R cell, which is resistante to dexamethasone. Moreover, over-expressed mi R-221/222 could suppress the death of glucocorticoid- sensitive cells, while down-regulated expression of mi R-221/222 in glucocorticoid- resistant cells could also lead to cell death. In a word, dexamethasone could induce autophagy of MM cells, and the level of autophagy happened in glucocorticoid-sensitive cell lines was apparently higher than in glucocorticoidresistant cell lines. Inhibition of autophagy could reduce dexamethasone –induced cell death, and mi R-221/222 just could suppress the occurrence of autophagy.Conclusion: By means of suppressing the expression of autophagy-related gene ATG4 A in MM cells, mi R-221/222 could inhibit the excessive activation of autophagy, accordingly, reducing glucocorticoid-induced autophagic cell death and involving in drug resistance of MM cells.