Fig 2: DAZAP1 inhibits cell proliferation of ESCC cells. (A) Relative DAZAP1 expression in 86 pairs of ESCC tissues and normal esophageal tissues (Jiangsu set and Shandong set). Significantly down-regulated DAZAP1 expression was observed in ESCC tissues compared with normal esophageal samples in both patient sets. (B) ESCC patients with high DAZAP1 expression exhibited significantly prolonged survival. (C, D) Silencing DAZAP1 expression (siDAZ1-1 and siDAZ1-2) promoted cell proliferation. However, ectopic DAZAP1 expression inhibited KYSE450 and KYSE510 cell growth. Cell numbers were counted at 24h, 48h and 72h after transfection. (E, F) Colony formation assays. On the 14th day after transfection of DAZAP1 siRNAs or expression constructs, colony number in each well was counted. The difference between two groups was calculated using Student's t test (assuming Gaussian distributions) or Wilcoxon Signed Rank Test (not assuming Gaussian distributions). All results of the mean of triplicate assays with standard deviation are presented. **P < 0.01; ***P < 0.001.

Fig 3: MiR-10b promotes autophagy by silencing DAZAP1 expression in ESCC. (A) Venn diagram of potential candidate target genes of miR-10b by integrating the results of the algorithms TargetScan, PICTAR, Micro-RNA and MiRDB. (B) qRT-PCR validation of the nine potential target genes of miR-10b in KYSE450 and KYSE510 cells transfected with either miR-10b mimics or NC RNA. (C) miR-10b could significantly inhibit DAZAP1 protein and mRNA expression in ESCC celllines. (D) Schematic constructions of pGL3-DAZAP1 and pGL3-Mut10b. (E) pGL3-DAZAP1 and pGL3-Mut10b were co-transfected into KYSE450 and KYSE510 cells with miR-10b mimics or NC RNA. Luciferase activity was detected at 48h after transfection and normalized relative to the Renilla luciferase expression. Inhibition effects of miR-10b mimics on pGL3-DAZAP1 or pGL3-Mut10b were showed. (F, G) Immunoblot results of extracts from non-starved or starved KYSE450 and KYSE510 cells. Silencing DAZAP1 expression (siDAZ1-1 and siDAZ1-2) increased starvation-induced conversion of LC3B-I to LC3B-II and accelerated rapamycin-induced SQSTM1 degradation in both ESCC celllines. Over-expressed DAZAP1 suppressed conversion of LC3B-I to LC3B-II and down-regulation of SQSTM1 in KYSE450 and KYSE510 cells. (H) DAZAP1 inhibited starvation-induced GFP-LC3 LC3B+ autophagosomes formation in both ESCC celllines. The number of LC3 punctae in cells of each group was calculated from 3 random fields, and at least 30 cells were chosen. Autophagy was assessed under non-starved (STV-) or starved (STV+) conditions. The difference between two groups was calculated using Student's t test (assuming Gaussian distributions) or Wilcoxon Signed Rank Test (not assuming Gaussian distributions). All results of the mean of triplicate assays with standard deviation are presented. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: DAZAP1 regulates alternative splicing of TSC2 mRNA. (A) RNAseq of KYSE510 cells transfected with siRNAs (siDAZ1-1 and siDAZ1-2) or NC RNA was performed to identify endogenous splicing events controlled by DAZAP1. MATS analyses indicate that dysregulated DAZAP1 led to six hundred and thirteen alternative splicing events, including exon skipping (SE, n = 352), intron retention (RI, n = 22), alternative 5' splice site (A5SS, n = 34), alternative 3' splice site (A3SS, n = 36), and mutually exclusive exon (MXE, n = 102). (B) Venn diagram of the overlapped genes between the alternative splicing (AS) genes and autophagy genes. (C) Decreased inclusion of TSC2 exon26 in mature mRNA of KYSE510 cells transfected with siRNAs (siDAZ1-1 and siDAZ1-2). (D) A 2706bp DNA fragment including human TSC2 exons 25, 26 and 27 as well as introns 25 and 26 was cloned into the pcDNA3.1 vector (pcDNA3.1-TSC2-minigene). (E) Different alternative splicing patterns of TSC2 pre-mRNA were detected using RT-PCR in KYSE450 and KYSE510 cells after silencing DAZAP1 or over-expressed DAZAP1.

Fig 5: DAZAP1 inhibits oncogenic autophagy via the TSC2/RHEB/mTOR signal pathway. (A) KYSE450 and KYSE510 cells were transfected with NC RNA, miR-10b mimics, or DAZAP1 siRNAs (siDAZ1-1 and siDAZ1-2). After 48h, cell lysates were analyzed by western blotting. GAPDH levels were measured as loading controls. (B) KYSE450 and KYSE510 cells were transfected with pcDNA3.1 or pcDNA-DAZAP1. After 48h, cell lysates were analyzed by western blotting. GAPDH levels were measured as loading controls. (C) Model for regulation of alternative splicing of TSC2 mRNA and oncogenic autophagy by DAZAP1 under conditions of nutrient sufficiency.