Fig 2: Repression of MTDH expression enhances prostate cancer cell sensitivity to cisplatin. (A and B) Compared with control group C, MTDH messenger RNA and protein expression were significantly lower in the intervention group D (*P<0.01). (C and D) An apoptosis assay, (E) an MTT assay for cell viability and (F and G) a Transwell chamber invasion assay with hematoxylin staining (magnification, x400) were also performed. The results suggested that intervention group D had a higher apoptotic rate, lower cell viability and weaker cell invasive ability than the control group C (*P<0.05). MTDH, metadherin; FITC, fluorescein isothiocyanate.

Fig 3: Astrocyte elevated gene 1 (AEG-1) is a direct target gene of miR-628-5p in gastric cancer (GC) cells and can be positively regulated by DLGAP1-AS1. (A) The expression of miR-628-5p in SNU-1 and AGS cells was analyzed after transient transfection with either the miR-628-5p mimic or negative control miRNA mimic (miR-NC). (B–D) The proliferation and apoptosis of miR-628-5p–overexpressing SNU-1 and AGS cells were measured by the CCK-8 assay and flow-cytometric analysis (propidium iodide, PI), respectively. (E, F) Transwell migration and invasion assays were conducted to assess the migration and invasiveness of SNU-1 and AGS cells that were transfected with either the miR-628-5p mimic or miR-NC. (G) The predicted miR-628-5p–binding sequences in the 3'-UTR of AEG-1 mRNA. Mutated sequences in the 3'-UTR of AEG-1 mRNA are highlighted in red. (H) Either wild-type (wt)-AEG-1 or mutant (Mut)-AEG-1 plus either the miR-628-5p mimic or miR-NC were transfected into SNU-1 and AGS cells. Following 48 h of incubation, luciferase activity was determined using a dual-luciferase reporter assay system. (I) The relative expression of AEG-1 mRNA in 63 pairs of GC tissue samples and corresponding adjacent non-tumor tissue samples was measured by RT-qPCR. (J) The expression correlation between miR-628-5p and AEG-1 mRNA in the 63 GC tissues was evaluated via Spearman correlation analysis. r = -0.5365, P < 0.0001. (K, L) The changes in AEG-1 mRNA and protein expression in SNU-1 and AGS cells after miR-628-5p overexpression were assessed via RT-qPCR and Western blotting. (M, N) RT-qPCR and Western blotting were conducted to measure the expression of AEG-1 mRNA and protein expression in SNU-1 and AGS cells after small interfering RNA (siRNA) for silencing DLGAP1-AS1 expression (si-DLGAP1-AS1) or negative control siRNA (si-NC) transfection. *P < 0.05 and **P < 0.01.

Fig 4: Miat binds Mtdh to regulate microRNAs involved in stem cell maintenance, tumorigenesis, and treatment resistance. (A) RNA immunoprecipitation confirmed the association of Mtdh and Miat in stem-like MB cells. (B) Western blot of Mtdh and ß-actin in stem-like MB cells in two cell Mtdh-/- cell lines generated by CRISPR and Mtdh+/+ control. (C) Neurosphere formation in Mtdh-/- stem-like MB cells and Mtdh+/+ controls (n = 3). (D and E) Expression of genes associated with cancer stem cells (D) or stem cell differentiation (E) in Mtdh-/- stem-like MB cells and Mtdh+/+ controls (n = 3). (F) Cell viability as measured by MTS assay 48 h after increasing doses of IR (0, 1, 2, 3, 4, 6, 8, 10 Gy in Mtdh+/+ and Mtdh-/- stem-like MB cells; n = 4 in each group). (G) Heat map of differentially expressed small RNAs from small RNA-seq in stem-like MB cells generated with Cas9 and indicated sgRNAs targeting either Miat or nontargeting control Gal4-4 (Left) and Mtdh+/+ and Mtdh-/- stem-like MB cells (Right). (H) Correlation of magnitude and direction of change in 11 small RNAs that were significantly altered in sgMiat and Mtdh-/- stem-like MB cells compared to controls. Top, Venn diagram indicating the overlap in small RNAs altered by sgMiat or Mtdh loss; P < 10-6 by the Fisher exact test. (I) Expression of miRNAs in stem-like MB cells with indicated sgRNAs targeting either Miat with Cas9 or nontargeting control (Gal4-4) and Mtdh-/- stem-like MB cells. ***P value < 0.001; **P value < 0.01; *P value < 0.05 via Student t test.

Fig 5: LOC102553417 silencing suppresses MTDH expression via competitively binding to miR-30e. Alignment of the binding site between (A) LOC102553417, miR-30e and the designed MUT sequence, and (B) miR-30e, MTDH and the designed MUT sequence. Binding of (C) miR-30e to LOC102553417 and (D) miR-30e to MTDH were assessed via dual-luciferase reporter assay. (E) Binding of miR-30e to LOC102553417 was assessed via RNA binding protein immunoprecipitation. mRNA expression levels of (F) miR-30e and (G) MTDH were assessed via RT-qPCR. (H) Representative western blotting images of MTDH, Akt, p-Akt, p53, caspase-3 and cleaved caspase-3 protein bands. (I) Statistical analysis of MTDH, Akt, p-Akt, p53, caspase-3 and cleaved caspase-3 protein expression levels assessed via western blotting. Results are expressed as the mean ± SD (n=3). **P<0.01; ***P<0.001; ns, no significant difference; WT, wild-type; MUT, mutant; NC, negative control; Ago2, Argonaute RNA-induced silencing complex catalytic component 2; p, phosphorylated; miR, microRNA; MTDH, metadherin; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; KD, knockdown.