Antimir-21 Causes Autophagy in the K562 Cell Line and Sensitizes Cells to the Effect of Doxorubicin and Etoposide
Authors: Seca Hugo , Lima Raquel T. , Lopes-Rodrigues Vanessa , Guimaraes Jose E. , Almeida Gabriela M. , Vasconcelos M. Helena 
Published: October 2019

Abstract
Abstract 4908

Background:
microRNAs (miRs) are short non-coding RNAs that regulate gene expression by binding to targeted mRNAs and causing their degradation or inhibition of translation. Some miRs have been described to play a role in disease pathogenesis and response to therapy in cancer [Lee and Dutta (2009), Annu Rev Pathol 4:199–227]. Other miRs have been associated with cancer drug resistance. For example, miR-21 has been shown to be involved in chemoresistance in a variety of cancer cells, namely leukemia cells [Li et al. (2010), Hematology 15:215–21] and to target several genes known to be involved in apoptosis, such as Pdcd4 [Lu et al. (2008), Oncogene 27:4373–79], FasL [Sayed et al. (2010), J Biol Chem 285:20281–90] and PTEN [Meng et al. (2007), Gastroenterology 133:647–58]. A recent report showed that miR-21 targets and upregulates Bcl-2 [Dong et al. (2011), Arch Med Res 42:8–14] by a non-canonical mechanisms, as demonstrated for other miRs [Lu et al. (2010), Cardiovasc Res 86:410–20]. Given that miR-21 regulates Bcl-2 expression and it has been well established that Bcl-2 has a role in the control of cellular autophagy, the aim of this study was to investigate if antimiR-21 is involved in autophagy in a Chronic Myeloid Leukemia cell line (K562) and if the influence of antimiR-21 in drug sensitivity was associated with autophagy.

Methods:
K562 cells were transfected with antimiR-21 (or control antimiR) and further treated with etoposide, doxorubicin or with an appropriate control. Additional experiments were carried out by treating cells with 3-MA (inhibitor of autophagy) or serum starvation (inducer of autophagy). RT-qPCR was used to confirm downregulation of miR-21 levels. Cellular proliferation (BrdU assay), cell cycle (flow cytometry following PI labeling) and programmed cell death (TUNEL assay) were analyzed. The expression levels of proteins involved in apoptosis (Bcl-2, PARP) and in autophagy (Beclin-1, Vps34 and LC3B) were investigated (Western Blot). Cellular sensitization to the effects of doxorubicin or etoposide was confirmed by counting viable cell number (Trypan blue). Monodansylcadaverine (MDC) was used to assess the presence of autophagic vacuoles by immunofluorescence microscopy. The presence of autophagosomes was analysed by transmission electron microscopy (TEM).

Results:
miR-21 expression was downregulated with antimiR-21. Downregulation of miR-21 caused a decrease in viable cell number, no alterations in the cell cycle profile, decreased cellular proliferation and increased programmed cell death. Although Bcl-2 expression was downregulated, PARP cleavage was not affected, indicating that the mechanism of cell death was not apoptotic. The expression levels of autophagy-related proteins such as Beclin-1, Vps34 and LC3B-II was increased and autophagic vacuoles typical of MDC staining were observed. The presence of autophagic vacuoles was further confirmed by TEM. Downregulation of miR-21 with antimiR-21 sensitized K562 cells to the effects of doxorubicin (by 26% comparing to treatment with doxorubicin alone and by 22% comparing to treatment with antimiR-21 alone) and of etoposide (by 23% comparing to treatment with etoposide alone and by 38% comparing to treatment with antimiR-21 alone). This effect was reversed by treatment with the inhibitor of autophagy, 3-MA. Moreover, treatment with 3-MA also reverted the sensitivity of cells to the drugs when used alone (without antimiR-21), indicating that these drugs induced autophagy. In agreement with this, serum starvation, which induces autophagy, also sensitized cells to the effect of these drugs.

Conclusion:
miR-21 downregulation caused autophagy and sensitized K562 chronic myeloid leukemia cells to doxorubicin and etoposide. Serum starvation sensitized cells to death induced by these drugs, indicating that autophagy is involved in the cell death induced by these drugs. In addition, it was further confirmed that autophagy was involved in antimiR-21 induced sensitization to the drugs, by reverting sensitization of cells to these drugs with an inhibitor of autophagy (3-MA).

Acknowledgments:
Fundação Calouste Gulbenkian for financial support. FCT for grants to HS (SFRH/BD/47428/2008) and RTL (SFRH/BPD/68787/2010). GMA is supported by FCT and the European Social Fund. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT.

Disclosures:
No relevant conflicts of interest to declare.