Fig 2: LINC00842 is a potential therapeutic target for PDAC.a Immunoprecipitation and immunoblot analysis of YY1, acetyl-PGC-1a, and FASN in PDAC and paired non-tumor tissues (n = 30) from one experiment. b Levels of LINC00842 RNA and YY1, FASN, and acetyl-PGC-1a proteins in PDAC and paired non-tumor tissues (n = 30). RNA levels were determined with qPCR while protein levels were determined with western blotting and quantified using Image J. Data are shown in box plots; the lines in the middle of the box are median and the upper and lower lines indicate 25th and 75th percentiles. The P values were determined by Wilcoxon test (two-tailed). c, d Spearman’s correlations between levels of LINC00842 and YY1, acetyl-PGC-1a or FASN (c) and between YY1, acetyl-PGC-1a, and FASN (d) in PDAC tissues (n = 30). e Timeline schematic for treatment of mice carrying xenograft in the pancreas with antisense oligonucleotide (ASO)-control (Ctrl), ASO-LINC00842, Orlistat or combination of ASO-LINC00842 and Orlistat. Arrows indicate different treatment time points. f, g Bioluminescence imaging (left panel), quantitative fluorescent intensities (upper right panel), and survival time (lower right panel) of mice with different treatment. Fluorescent intensity data represent mean ± SD from 5 mice in each group; The P values were determined by Student’s t-test (two-tailed). h Timeline schematic for treatment of mice carrying patient-derived tumor xenograft (PDX). Arrows indicate different treatment time points. i ASO-LINC00842 and Orlistat treatment significantly inhibited PDX growth in mice. Data in each time point represent mean ± SEM from 5 mice. **P < 0.01 and ***P < 0.001 of Student’s t-test (two-tailed. No adjustments were made for multiple comparisons). j–l LINC00842 RNA (j), acetyl-PGC-1a (k), and FASN (l) protein or activity levels in PDX of mice before and after treatment. Data are mean ± SD from 5 mice; The P values in (j–l) were determined by Student’s t-test (two-tailed). m Proposed acting model for LINC00842 that regulates metabolic reprogramming in PDAC.

Fig 3: Knock-down of YY1 in linc-MYH KO MuSCs Knock-down of Yy1 in proliferating MuSCs in vitro reduces Yy1 transcript abundance in linc-MYH KO and wild-type controls (WT scramble shRNA n = 3 biological replicates, WT Yy1 knock-down n = 3 biological replicates, Mann–Whitney test: significant *P = 0.05; linc-MYH KO scramble shRNA n =3 biological replicates, linc-MYH KO yy1 shRNA n = 3 biological replicates, Mann–Whitney test: significant *P = 0.05). Data are mean ± SEM.Knock-down of Yy1 in linc-MYH KO MuSCs compared with linc-MYH KO scramble shRNA controls results in lower confluence of proliferating MuSCs (n = 3/3 with 4 technical replicates/animal for each transfection, 95 time points, two-way ANOVA P = 0.0001) compared with their scramble controls. Similarly, knock-down of Yy1 in WT MuSCs compared with WT scramble shRNA results in lower confluence of proliferating MuSCs. (n = 3/3 with 4 technical replicates per animal for each transfection, 95 time points, two-way ANOVA P = 0.0001). Knock-down of Yy1 eliminated the increased confluence of linc-MYH KO MuSC observed in linc-MYH KO MuSCs compared to the respective wild-type control MuSCs.

Fig 4: Deletion of INO80 prevents linc-MYH-dependent changes in MuSC proliferation and gene expression A–DIsolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos/INO80-/- (Ino80 KO) and linc-MYH-/-/linc-MYH-/-/Pax7-Crepos/INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.EDifferences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT/6 linc-MYH KO/3 Ino80 KO/3 dKO-independent preparations of MuSCs).FStatistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT/6 linc-MYH KO/3 Ino80 KO/3 dKO-independent preparations of MuSCs, Student's t-test, two-tailed; *P < 0.05, ns = not significant). Data are mean ± SEM.G–IGene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared with Pax7-Crepos/INO80-/- MuSCs.J–LHeat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/Pax7-Crepos/INO80-/- dKO vs. Pax7-Crepos/INO80-/- MuSCs. The expression of upregulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos/INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).

Fig 5: YY1 regulates T-bet by binding to the promoter region and interacting with T-bet protein.(A) Sequence logo representing the consensus YY1 binding motif obtained from JASPAR database. (B) Luciferase activity of HEK293T cells cotransfected with different promoter constructs of T-bet and lentivirus containing YY1 shRNA (LV-YY1-shRNA) or empty vectors (LV-NC). Data presented as mean ± SEM (n = 3 independent experiments). ***P < 0.001 (Student’s t test). (C) ChIP assay for verifying direct interaction between T-bet gene and YY1 antibody. The illustration in the right panel represents the chromatin loci associated with YY1 for ChIP (data from 2 independent experiments). (D) Representative confocal images of the distribution of YY1 protein (immunofluorescence staining for green) and T-bet protein (immunofluorescence staining for red) in pTh17-polarized group in vitro (scale bars = 20 µm, data from 2 independent experiments). (E) Co-IP analysis to confirm in vivo interactions between YY1 and T-bet protein (data from 2 independent experiments).