Background. MCOLN1 (mucolipin subfamily, member 1) was first identified as an autophagic regulator, which was essential for efficient fusion of both autophagosomes and late endosomes with lysosomes. This study is aimed at investigating the role of MCOLN1 in the development of pancreatic ductal adenocarcinoma (PDAC). Methods. Immunohistochemistry (IHC) assay was conducted to evaluate the expression level of MCOLN1 in 82 human PDAC tumor tissues. Overall survival (OS) and recurrence-free survival (RFS) analysis was performed to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. The expression level of related genes was measured by RT-PCR (reverse transcription polymerase chain reaction) and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis in vivo. Results. The results of IHC showed that a high level of MCOLN1 expression was associated with the poor clinical characteristics of PDAC patients. OS and RFS were significantly worse in patients with high MCOLN1 expression. Silencing of MCOLN1 dramatically blocked the proliferation of PDAC cells. Mechanism studies confirmed that knockdown of MCOLN1 decreased the expression of Ki67 and PCNA (proliferating cell nuclear antigen), two markers of cell proliferation. In vivo, MCOILN1 depletion reduced the formation and growth of tumors in mice. Conclusion. The high level of MCOLN1 expression was associated with poor clinical outcomes of PDAC patients. MCOLN1 ablation could inhibit PDAC proliferation of both in vitro and in vivo, which provide a new insight and novel therapeutic target for the treatment of PDAC.

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Drug discovery from plants usually focuses on small molecules rather than such biological macromolecules as RNAs. Although plant transfer RNA (tRNA)-derived fragment (tRF) has been associated with the developmental and defense mechanisms in plants, its regulatory role in mammals remains unclear. By employing a novel reverse small interfering RNA (siRNA) screening strategy, we show that a tRF mimic (antisense derived from the 5 0 end of tRNA His(GUG) of Chinese yew) exhibits comparable anti-cancer activity with that of taxol on ovarian cancer A2780 cells, with a 16-fold lower dosage than that of taxol. A dual-luciferase reporter assay revealed that tRF-T11 directly targets the 3 0 UTR of oncogene TRPA1 mRNA. Furthermore, an Argonaute-RNA immunoprecipitation (AGO-RIP) assay demonstrated that tRF-T11 can interact with AGO2 to suppress TRPA1 via an RNAi pathway. This study uncovers a new role of plant-derived tRFs in regulating endogenous genes. This holds great promise for exploiting novel RNA drugs derived from nature and sheds light on the discovery of unknown molecular targets of therapeutics.

Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC–MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC–MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

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