Abstract
BackgroundMany patients diagnosed with oesophageal adenocarcinoma (OAC) present with advanced disease and approximately half present with metastatic disease. Patients with localised disease, who are managed with curative intent, frequently undergo neoadjuvant chemoradiotherapy. Unfortunately, ~ 70% of patients have little or no response to chemoradiotherapy. We previously identified miR-330-5p as being the most significantly downregulated microRNA in the pre-treatment OAC tumours of non-responders to treatment, but that loss of miR-330-5p had a limited impact on sensitivity to chemotherapy and radiation in vitro. Here, we further examined the impact of miR-330-5p loss on OAC biology.MethodsmiR-330-5p was suppressed in OE33 OAC cells following stable transfection of a vector-driven anti-sense RNA. Whole transcriptome digital RNA-Seq was employed to identify miR-330-5p regulated genes, and qPCR was used for validation. Protein expression was assessed by protein array, Western blotting and zymography. Invasive potential was measured using a transwell assay system. Tumour xenograft growth profile studies were performed in immunocompromised CD1 mice.ResultsIn OE33 cells, suppression of miR-330-5p significantly altered expression of 42 genes, and several secreted proteases. MMP1 gene expression and protein secretion was significantly enhanced with miR-330-5p suppression. This corresponded to enhanced collagen invasion in vitro. In vivo, OE33-derived tumour xenografts with miR-330-5p suppression grew faster than controls.ConclusionsLoss of miR-330-5p expression in OAC tumours may influence tumour cell invasive capacity, tumour growth and therapeutic sensitivity via alterations to the tumour microenvironment.
Highlights
Many patients diagnosed with oesophageal adenocarcinoma (OAC) present with advanced disease and approximately half present with metastatic disease
Identifying the gene targets of miR-330-5p Endogenous miR-330-5p expression in OE33 OAC cells was silenced using the miRZIP-330-5p vector, which produces an anti-sense miR-330-5p that irreversibly binds to endogenous miR-330-5p, thereby inhibiting its function
Validating MMP1 as a target of miR-330-5p The digital RNA-seq (DGE) analysis identified a 5-fold increase in MMP1 and a 2.5-fold increase in MMP7 in the miRZIP-330-5p single clone (SC), which was validated via qPCR (Fig. 1)
Summary
Many patients diagnosed with oesophageal adenocarcinoma (OAC) present with advanced disease and approximately half present with metastatic disease. Patients with localised disease, who are managed with curative intent, frequently undergo neoadjuvant chemoradiotherapy. We previously identified miR-330-5p as being the most significantly downregulated microRNA in the pre-treatment OAC tumours of non-responders to treatment, but that loss of miR-330-5p had a limited impact on sensitivity to chemotherapy and radiation in vitro. More than half of patients diagnosed with oesophageal cancer will not survive more than a year and UK incidence rates are one of the highest in Europe [1]. Tumours are predominated by two histological subtypes, squamous cell carcinoma (SCC) and adenocarcinoma (OAC). OAC develops from the premalignant chronic acid reflux disease Barrett’s oesophagus (BO) [4]. OAC is a disease of stepwise progression from non-dysplastic BO, to dysplastic BO and adenocarcinoma. The biological drivers of OAC include chronic inflammation, disrupted cell adhesion, hypoxia and genomic instability [6,7,8,9]
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