Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma (ESCC), and marginal ZD is prevalent in humans. In rats, marked-ZD (3 mg Zn/kg diet) induces a proliferative esophagus with a 5-microRNA signature (miR-31, -223, -21, -146b, -146a) and promotes ESCC. Here we report that moderate and mild-ZD (6 and 12 mg Zn/kg diet) also induced esophageal hyperplasia, albeit less pronounced than induced by marked-ZD, with a 2-microRNA signature (miR-31, -146a). On exposure to an environmental carcinogen, ∼16% of moderate/mild-ZD rats developed ESCC, a cancer incidence significantly greater than for Zn-sufficient rats (0%) (P ≤ 0.05), but lower than marked-ZD rats (68%) (P < 0.001). Importantly, the high ESCC, marked-ZD esophagus had a 15-microRNA signature, resembling the human ESCC miRNAome, with miR-223, miR-21, and miR-31 as the top-up-regulated species. This signature discriminated it from the low ESCC, moderate/mild-ZD esophagus, with a 2-microRNA signature (miR-31, miR-223). Additionally, Fbxw7, Pdcd4, and Stk40 (tumor-suppressor targets of miR-223, -21, and -31) were downregulated in marked-ZD cohort. Bioinformatics analysis predicted functional relationships of the 3 tumor-suppressors with other cancer-related genes. Thus, microRNA dysregulation and ESCC progression depend on the extent of dietary Zn deficiency. Our findings suggest that even moderate ZD may promote esophageal cancer and dietary Zn has preventive properties against ESCC. Additionally, the deficiency-associated miR-223, miR-21, and miR-31 may be useful therapeutic targets in ESCC.

MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden followingN-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P= 0.038) and miR-31 gene knockout abrogated development of ESCC (P= 1 × 10−6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB–controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31–associated EGLN3/NF-κB–controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31−/−rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.

SignificanceProstate cancer in man is associated with Zn loss, citrate metabolite reduction, overexpression of the miR-183-96-182 cluster, and regulation of Zn homeostasis through Zn transporter suppression. Our mechanistic study shows that a low-Zn diet upregulates this miR cluster in Zn-deficient middle-aged rat prostate, with ZIP1 mRNA/protein downregulation and a citrate-oxidizing metabolic phenotype, linking citrate reduction directly to prostatic Zn loss. The findings of this study show that the transcriptional and metabolic signal pathways induced by Zn deficiency in rats and almost certainly in men are critical for the development of human and rat prostate cancer and provide a strong rationale for including Zn supplementation in clinical trials to reduce the prostate cancer burden in the human population.

Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression and addiction. Here we report the synthesis, characterization and in-vitro evaluation of novel fluorescent peptide-peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a μ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through μ-opioid receptor-mediated endocytosis. The Kd by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ~5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack μ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.