Fig 2: MiR-22-3p, miR-9-5p, and miR-218-5p control the expression of multiple folate-metabolizing enzymes in human NCSLC cells. (A) SHMT1 was identified as a putative target for miR-218-5p, and SHMT1, MTHFD1L and MTHFD2 for miR-9-5p, as well as MTHFR and MTHFD2 for miR-22-3p. Underlined are the seed sequences of individual miRNAs that interact with corresponding MREs via complimentary base pairing. SHMT1: serine hydroxymethyltransferase 1, MTHFD2: methylenetetrahydrofolate dehydrogenase 2, MTHFR: methylenetetrahydrofolate reductase, MTHFD1L: methylenetetrahydrofolate dehydrogenase 1 like. (B) Dual luciferase reporter assay validated the interactions between individual pairs of miRNAs and target genes. Values are mean ± SD (n = 5/group). (C) The protein levels of SHMT1, MTHFD1L, MTHFD2, and MTHFR were determined in A549 and H1975 cells by Western blot analyses. Cells were treated with 15 nmol/L of hBERA/miRNAs, control RNA or vehicle for 72 h. GAPDH was used as a loading control for data normalization. Values are mean ± SD (n = 3/group). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with respective control RNA treatment (one- or two-way ANOVA with Bonferroni post hoc tests).

Fig 3: Antifolate effects of miR-22-3p, miR-9-5p, and miR-218-5p in human NSCLC cells. (A) Schematic representation of the folate cycle and chemical structures of folate metabolites. DHFR, dihydrofolate reductase; ALDH1L1, 10-formyltetrahydrofolate dehydrogenase; MTR, methionine synthase; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; SHMT1, cytosolic form of serine hydroxymethyltransferase; MTHFR, methylene tetrahydrofolate reductase; SHMT2, mitochondrial form of serine hydroxymethyltransferase; MTHFD2, mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase; MTHFD2L, methylenetetrahydrofolate dehydrogenase 2-like protein; ALDH1L2, aldehyde dehydrogenase 1 family, member L2, the mitochondrial isoform of ALDH1L1; MTHFD1L, mitochondrial monofunctional C1-tetrahydrofolate synthase; TYMS, thymidylate synthase; DHF, dihydrofolate; THF, tetrahydrofolate; Ser, serine; Gly, glycine; Met, methionine; Hcy, homocysteine. (B) Intracellular folate metabolite (Folic acid, DHF, THF, 5-methyl-THF, 5,10-methenyl-THF, and 5-formyl-THF) contents were determined in A549 and H1975 cells by selective LC–MS/MS method after cells were treated with 15 nmol/L of hBERA/miRNAs, control RNA or vehicle for 72 h. Values are mean ± SD (n = 3/group). (C) Schematic diagram of the incorporation of one-carbon unit from serine to folate metabolites. (D) Intracellular 5-methyl-THF (M+3) and 5,10-methenyl-THF (M+2) were quantified by LC–MS/MS method after replacing serine with isotope-labeled serine (13C, D, 15N) in cell culture medium. Cells were treated with 15 nmol/L of bioengineered miRNAs, control RNA or vehicle for 72 h. Values are mean ± SD (n = 3/group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and #P < 0.0001, compared with control RNA (two-way ANOVA with Bonferroni post hoc tests).