MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.
Keywords: 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose; ABCG2, ATP-binding cassette subfamily G member 2; ACN, acetonitrile; Au/Uv, absorbance unit of ultraviolet-visible spectroscopy; BCRP, breast cancer resistant protein; BERA, bioengineered miRNA agent; Bioengineered RNA; CI, combination index; CPT, cisplatin; Cancer; Chemosensitivity; DOX, doxorubicin; E. coli, Escherichia coli; ESI, electrospray ionization; FPLC, fast protein liquid chromatography; Fa, fraction affected; GLUT1; GLUT1, glucose transporter protein type 1; HCC, hepatocellular carcinoma; HPLC, high-performance liquid chromatography; IS, internal standard; KRB, Krebs–Ringer bicarbonate; LAT1; LAT1, large neutral amino acid transporter 1; LC–MS/MS, liquid chromatography–tandem mass spectroscopy; MCT4, monocarboxylate transporter 4; MRE, miRNA response elements; MRM, multiple reaction monitoring; MiR-328; OS, osteosarcoma; PAGE, polyacrylamide gel electrophoresis; PTEN, phosphatase and tensin homolog; PVDF, Polyvinylidene fluoride; RAGE, receptor for advanced glycosylation end products; RT-qPCR, reverse transcription quantitative real-time polymerase chain reaction; SLC2A1, 7A5, 16A3, solute carrier family 2 member 1, family 7 member 5, family 16 member 3; WT, wild type; hBERA, humanized bioengineered miRNA agent; hsa, Homo sapiens; htRNASer, human seryl-tRNA; mTOR, mammalian target of rapamycin; miR or miRNA, microRNA; ncRNA, noncoding RNAs; nt, nucleotide.
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