Fig 2: Identification and validation of potential RBPs of TUG1.a An interaction network map showing 28 putative RBP candidates that could potentially bind to TUG1. A total of ten candidates were selected for further validation based on literature search, which were shown in green. b Subcellular localization of ten RBPs in TUG1 knockdown PDLSCs as determined by qRT-PCR measurement of nuclear and cytoplasmic RNA. GAPDH is the positive control for cytoplasm and U6 is the positive control for nucleus. c qRT-PCR analysis of the gene expression levels for the selected RBPs in TUG1 knockdown PDLSCs. d Summary of putative binding sites on Lin28A for TUG1 based on results generated from gene co-expression network and CLIP analysis. e Western blotting analysis of Lin28A levels in the four above mentioned experiment groups at day 0, 7, and 14 after the osteogenic induction. All experiments were performed in triplicate and results were expressed as means ±SD. *P < 0.05; **P < 0.01; NS not significant

Fig 3: Effects of Lin28A knockdown on the osteogenic differentiation of PDLSCs.a Transfection effects of small interference RNAs of Lin28A (si-Lin28A) were determined by qPCR and western blot. b TUG1 expression was measured by qPCR in PDLSCs transfected with si-Lin28A-2#. c–g The functions of siLin28A on the expression of key osteogenic markers of PDLSCs were measured by ALP staining, ALP activity, Alizarin Red staining, and qPCR after inducing different times. ALP staining (c) and Alizarin Red staining (e) were performed at the day 7 and 21 after the osteogenic induction on all four experiment groups. Quantification of ALP activity (d) and cellular calcium level (f) in all experiment groups were measured at different time points after the induction. g qRT-PCR analysis of ALP, OCN, and Runx2 mRNA levels in different experiment groups at day 0, 1, 3, 7, 10, and 14 after the osteogenic induction. h An illustrative view of the TUG1-Lin28A network and its role in the regulation of PDLSC osteogenesis. All experiments were performed in triplicate and results were expressed as means ±SD. *P < 0.05; **P < 0.01. Scale bar: 100 µm

Fig 4: Lin28A facilitated aerobic glycolysis by stabilizing SNHG14.a SNHG14 was downregulated in the sh-Lin28A group, re-expression of Lin28A reversed SNHG14 expression. Data are presented as the mean ± SD (n = 3 in each group). **P < 0.01 versus sh-Lin28A-NC group (empty vector); ##P < 0.01 versus sh-Lin28A + Lin28A-NC group. b RNA immunoprecipitation (RIP) showed an enrichment of SNHG14 in Lin28A immunoprecipitated samples. Data are presented as the mean ± SD (n = 3, each group). **P < 0.01 versus anti-normal IgG group, using Student’s t test. c Immunoblotting for the specific associations of Lin28A with biotinylated-SNHG14 or antisense RNA from streptavidin RNA pulldown assay. d RNA half-life measurement to detect the T1/2 of SNHG14 upon Lin28A depletion or re-expression. e Click-iT Nascent RNA capture kit was conducted to label and capture newly synthesized RNA, and nascent SNHG14 was measured using qRT-PCR. f ECAR was measured to detect the effect of Lin28A and SNHG14 on glycolysis. g, h Lactate production and glucose uptake were measured upon depletion of Lin28A and SNHG14. i Expression of PKM2 and GLUT1 by western blot upon depletion of Lin28A and SNHG14. j CCK-8 assay was conducted to investigate the effect of Lin28A and SNHG14 on proliferation. k Flow cytometry analysis to evaluate the effect of depleting Lin28A and SNHG14 on apoptosis. Data are presented as the mean ± SD (n = 3 in each group). *P < 0.05, **P < 0.01 versus sh-Lin28A-NC + sh-SNHG14-NC group (empty vector); #P < 0.05, ##P < 0.01 versus sh-Lin28A+sh-SNHG14-NC group; &P < 0.05, &&P < 0.01 versus sh-Lin28A-NC + sh-SNHG14 group. One-way analysis of variance was used for statistical analysis.

Fig 5: Lin28A was upregulated in glioma cell lines and tissues and knockdown of Lin28A inhibited glycolysis and proliferation.a Protein levels of Lin28A in normal brain tissues (NBTs), low-grade glioma tissues (WHO I-II), and high-grade glioma tissues (WHO III-IV). Data are presented as the mean ± SD (n = 3, each group). *P < 0.05, **P < 0.01 versus NBTs group. b Protein levels of Lin28A in normal human astrocytes (NHA), glioma cell lines (U87 and U251) cells. Data are presented as the mean ± SD (n = 3, each group). *P < 0.05, **P < 0.01 versus NHA group. c Extracellular acidification rate (ECAR) was measured to detect the effect of Lin28A depletion and re-expression of Lin28A in sh-Lin28A cells on glycolysis in glioma; glycolysis and glycolytic capacity were analyzed. d, e The lactate production and glucose uptake in response to Lin28A depletion and re-expression in U87 and U251 cells. f Expression of PKM2 and GLUT1 by western blot upon Lin28A depletion and re-expression. g CCK-8 assay was conducted to investigate the effect of Lin28A on proliferation. h Flow cytometry analysis to evaluate the effect of Lin28A on apoptosis. Data are presented as the mean ± SD (n = 3, each group). **P < 0.01 versus sh-Lin28A-NC group, ##P < 0.01 versus sh-Lin28A + Lin28A-NC group. One-way analysis of variance was used for statistical analysis.