Coronel-Hernández Jossimar, Salgado-García Rebeca, Cantú-De León David, Jacobo-Herrera Nadia, Millan-Catalan Oliver, Delgado-Waldo Izamary, Campos-Parra Alma D, Rodríguez-Morales Miguel, Delgado-Buenrostro Norma L, Pérez-Plasencia Carlos
Laboratorio de Genómica Funcional, Unidad de Biomedicina, FES-Iztacala, UNAM, Tlalnepantla, Mexico.
Laboratorio de Genómica, Instituto Nacional de Cancerología, Tlalpan, Mexico.
Front Oncol. 2021 May 26;11:594200. doi: 10.3389/fonc.2021.594200. eCollection 2021.
Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide in both sexes. Current therapies include surgery, chemotherapy, and targeted therapy; however, prolonged exposure to chemical agents induces toxicity in patients and drug resistance. So, we implemented a therapeutic strategy based on the combination of doxorubicin, metformin, and sodium oxamate called triple therapy (Tt). We found that Tt significantly reduced proliferation by inhibiting the mTOR/AKT pathway and promoted apoptosis and autophagy in CRC derived cells compared with doxorubicin. Several autophagy genes were assessed by western blot; ULK1, ATG4, and LC3 II were overexpressed by Tt. Interestingly, ULK1 was the only one autophagy-related protein gradually overexpressed during Tt administration. Thus, we assumed that there was a post-transcriptional mechanism mediating by microRNAs that regulate UKL1 expression during autophagy activation. Through bioinformatics approaches, we ascertained that ULK1 could be targeted by mir-26a, which is overexpressed in advanced stages of CRC. experiments revealed that overexpression of mir-26a decreased significantly ULK1, mRNA, and protein expression. Contrariwise, the Tt recovered ULK1 expression by mir-26a decrease. Due to triple therapy repressed mir-26a expression, we hypothesized this drug combination could be involved in mir-26a transcription regulation. Consequently, we analyzed the mir-26a promoter sequence and found two HIF-1α transcription factor recognition sites. We developed two different HIF-1α stabilization models. Both showed mir-26a overexpression and ULK1 reduction in hypoxic conditions. Immunoprecipitation experiments were performed and HIF-1α enrichment was observed in mir-26a promoter. Surprisingly, Tt diminished HIF-1α detection and restored ULK1 mRNA expression. These results reveal an important regulation mechanism controlled by the signaling that activates HIF-1α and that in turn regulates mir-26a transcription.
结直肠癌（CRC）是全球范围内男女癌症相关死亡的第三大主要原因。目前的治疗方法包括手术、化疗和靶向治疗；然而，长期接触化学药物会在患者体内引发毒性和耐药性。因此，我们实施了一种基于阿霉素、二甲双胍和草酸钠联合使用的治疗策略，称为三联疗法（Tt）。我们发现，与阿霉素相比，Tt通过抑制mTOR/AKT途径显著降低了细胞增殖，并促进了CRC衍生细胞中的凋亡和自噬。通过蛋白质印迹法评估了几种自噬基因；Tt使ULK1、ATG4和LC3 II过表达。有趣的是，ULK1是在Tt给药期间唯一逐渐过表达的自噬相关蛋白。因此，我们推测存在一种由微小RNA介导的转录后机制，在自噬激活过程中调节UKL1的表达。通过生物信息学方法，我们确定ULK1可能是mir-26a的靶点，mir-26a在CRC晚期过表达。实验表明，mir-26a的过表达显著降低了ULK1的mRNA和蛋白表达。相反，Tt通过降低mir-26a使ULK1表达恢复。由于三联疗法抑制了mir-26a的表达，我们推测这种药物组合可能参与了mir-26a的转录调控。因此，我们分析了mir-26a启动子序列，发现了两个缺氧诱导因子-1α（HIF-1α）转录因子识别位点。我们建立了两种不同的HIF-1α稳定模型。两者均显示在缺氧条件下mir-26a过表达和ULK1减少。进行了免疫沉淀实验，在mir-26a启动子中观察到HIF-1α富集。令人惊讶的是，Tt减少了HIF-1α的检测并恢复了ULK1 mRNA表达。这些结果揭示了一种由激活HIF-1α的信号控制的重要调控机制，进而调节mir-26a的转录。
