MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM.

Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex.

Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM.

MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance.

Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm³ VS 895.8±72.44 mm³ VS 987.3±68.49 mm³). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression.

In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment.