Negative Regulation of ULK1 by microRNA-106a in Autophagy Induced by a Triple Drug Combination in Colorectal Cancer Cells In Vitro

Abstract

Colorectal cancer (CRC) is among the top three most deadly cancers worldwide. The survival rate for this disease has not been reduced despite the treatments, the reason why the search for therapeutic alternatives continues to be a priority issue in oncology. In this research work, we tested our successful pharmacological combination of three drugs, metformin, doxorubicin, and sodium oxamate (triple therapy, or TT), as an autophagy inducer. Firstly, we employed western blot (WB) assays, where we observed that after 8 h of stimulation with TT, the proteins Unc-51 like autophagy activating kinase 1(ULK1), becline-1, autophagy related 1 protein (Atg4), and LC3 increased in the CRC cell lines HCT116 and SW480 in contrast to monotherapy with doxorubicin. The overexpression of these proteins indicated the beginning of autophagy flow through the activation of ULK1 and the hyperlipidation of LC3 at the beginning of this process. Moreover, we confirm that ULK1 is a bona fide target of hsa-miR-106a-5p (referred to from here on as miR-106a) in HCT116. We also observed through the GFP-LC3 fusion protein that in the presence of miR-106a, the accumulation of autophagy vesicles in cells stimulated with TT is inhibited. These results show that the TT triggered autophagy to modulate miR-106a/ULK1 expression, probably affecting different cellular pathways involved in cellular proliferation, survivance, metabolic maintenance, and cell death. Therefore, considering the importance of autophagy in cancer biology, the study of miRNAs that regulate autophagy in cancer will allow a better understanding of malignant tumors and lead to the development of new disease markers and therapeutic strategies.