Abstract
Biomarkers and effective therapeutic agents to improve the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) are urgently required. We aimed to analyze the prognostic value and mechanistic action of miR-93 in PDAC. Correlation of miR-93 tumor levels from 83 PDAC patients and overall survival (OS) was analyzed by Kaplan–Meier. MiR-93 depletion in PANC-1 and MIA PaCa-2 cells was achieved by CRISPR/Cas9 and miR-93 overexpression in HPDE cells by retroviral transduction. Cell proliferation, migration and invasion, cell cycle analysis, and in vivo tumor xenografts in nude mice were assessed. Proteomic analysis by mass spectrometry and western-blot was also performed. Finally, miR-93 direct binding to candidate mRNA targets was evaluated by luciferase reporter assays. High miR-93 tumor levels are significantly correlated with a worst prognosis in PDAC patients. MiR-93 abolition altered pancreatic cancer cells phenotype inducing a significant increase in cell size and a significant decrease in cell invasion and proliferation accompanied by a G2/M arrest. In vivo, lack of miR-93 significantly impaired xenograft tumor growth. Conversely, miR-93 overexpression induced a pro-tumorigenic behavior by significantly increasing cell proliferation, migration, and invasion. Proteomic analysis unveiled a large group of deregulated proteins, mainly related to G2/M phase, microtubule dynamics, and cytoskeletal remodeling. CRMP2, MAPRE1, and YES1 were confirmed as direct targets of miR-93. MiR-93 exerts oncogenic functions by targeting multiple genes involved in microtubule dynamics at different levels, thus affecting the normal cell division rate. MiR-93 or its direct targets (CRMP2, MAPRE1, or YES1) are new potential therapeutic targets for PDAC.
Highlights
MicroRNAs are short non-codingRNAs of 18–25 bp in length that regulate gene expression at the post-transcriptional level by binding to the mRNA and causing its degradation or translational repression[1]
To confirm the most relevant results of the proteomic analysis, we selected a group of proteins significantly dysregulated (CRMP2, ITGA2, MAD2L1, CDK1, CHMP4B, MAPRE1, and YES1) that are involved in cell cycle, mainly G2/M or cytokinesis, cell adhesion, cell migration, or microtubule organization, and we evaluated their levels in human pancreatic ductal epithelial (HPDE) and PANC-1 models by Western Blot (Supplementary Fig. 6)
In the present study, we have deciphered an oncogenic role of miR-93 in the context of Pancreatic ductal adenocarcinoma (PDAC) by modulating constitutively its expression using two different approaches: a loss-of-function approach generated by CRISPR– Cas[9] technology to abrogate miR-93 expression in two pancreatic cancer cellular models and a gain-of-function approach generated by retroviral transduction to stably overexpress miR-93 in a normal pancreatic cellular model
Summary
RNAs of 18–25 bp in length that regulate gene expression at the post-transcriptional level by binding to the mRNA and causing its degradation or translational repression[1]. By exerting this function, miRs modulate a multitude of processes, such as development, cell differentiation, Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths, as it is one of the most lethal epithelial malignancies with a very poor prognosis as over 80% of cases are diagnosed at an advanced stage of the disease and survival for these patients is less than one year[7,8]. A better understanding of the molecular basis of pancreatic carcinogenesis is urgently required for the identification of more effective therapeutic targets
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