CircSTX6 promotes pancreatic ductal adenocarcinoma progression by sponging miR-449b-5p and interacting with CUL2

Figures

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  Fig. 1. Identification and characterization of circSTX6 in PDAC cells and tissues. a Clustered heatmap of the differentially expressed circRNAs in PDAC tissues from five T4 PDAC patients with long survival time (Group B) and tissues from five T4 PDAC patients with short survival time (Group A). b Clustered heatmap of the differentially expressed circRNAs in three paired PDAC tissues (Group T) and matched noncancerous tissues (Group N). c Clustered heatmap of the intersection of two sequencing results. d Relative expression of three circRNA candidates (circSTX6, hsa_circ_0007665, hsa_circ_0001411) in 31 pairs of T4 PDAC tissues and corresponding noncancerous tissues. Kaplan–Meier analyses of the correlation between circRNA expression and overall survival were constructed. e Relative expression of circSTX6 in 97 pairs of PDAC tissues and corresponding noncancerous tissues. Kaplan–Meier analyses of the correlation between circSTX6 expression and overall survival were performed. f Schematic illustration of the genomic location and back splicing of circSTX6, with the splicing site validated by Sanger sequencing. g The existence of circSTX6 and STX6 were detected by qRT–PCR and agarose gel…

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  Fig. 2. CircSTX6 promotes the proliferation, migration and invasion of PDAC cells in vitro. a-b qRT–PCR analysis of circSTX6 and linear STX6 expression in PANC-1 and CFPAC-1 cells transfected with circSTX6 siRNAs and circSTX6 plasmid. c-f Colony formation assays were performed in PDAC cells transfected with cirSTX6 siRNAs or plasmid. g-h EdU assays of PANC-1 cells were performed to evaluate cell proliferation. Original magnification 400 ×. Scale bar = 50 μm. i-j The growth curves of cells were evaluated by CCK-8 assays after knocking down and overexpressing circSTX6 in PANC-1 cells. k-l Transwell assays in circSTX6-knockdown and circSTX6-overexpressing PANC-1 cells. Original magnification 200 ×. Scale bar = 100 μm. m-n Wound healing assays were used to investigate the migratory ability of circSTX6-overexpressing and circSTX6-knockdown PANC-1 cells. Original magnification 200 ×. Scale bar = 100 μm. (Values are expressed as the means ± SDs; *P < 0.05, **P < 0.01 and ***P < 0.001)

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  Fig. 3. CircSTX6 facilitates the tumorigenesis and metastasis of PDAC cells in vivo. a Representative picture of subcutaneous xenograft tumors (n = 6 for each group). b-c Curves of tumor volumes and weights show negative effects of circSTX6 knockdown on the formation of subcutaneous xenograft tumors. d HE and Ki-67 IHC staining of xenograft tumors. Original magnification 400×. Scale bar = 50 μm. H-scores of the Ki67 staining. e Representative images and analysis of luminescence intensity in tail vein tumor metastasis mouse models (n = 6 for each group). f Representative images and HE staining of metastatic nodules in the lungs and livers of mice. Original magnification 100×. Scale bar = 200 μm. g The numbers of lung and liver metastatic nodules were measured. (Values are expressed as the means ± SDs; *P < 0.05, **P < 0.01 and ***P < 0.001)

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  Fig. 4. CircSTX6 serves as a sponge for miR-449b-5p. a Clustered heatmap of the differentially expressed miRNAs in PDAC tissues from five T4 PDAC patients with long survival time and tissues from five T4 PDAC patients with short survival time. b Venn diagram showing the overlap of the target miRNAs of circSTX6 predicted by miRanda, PITA, RNAhybrid, TargetScan and the results of RNA-seq. c. Table of target miRNAs selected for circSTX6. d The efficiency of the circSTX6 probe in PDAC cells was validated using qRT–PCR after the RNA pull-down assay. A random oligo probe served as a negative control. e-f The expression levels of 23 miRNA candidates were detected in the RNAs pulled down by circSTX6 and oligo probes. g-h Anti-AGO2 RIP was performed to detect circSTX6 and miR-449b-5p in PDAC cells. i A schematic of the wild-type (WT) and mutant (MUT) circSTX6 luciferase reporter vectors. j-k A luciferase reporter assay was used to confirm the interaction between circSTX6 and miR-449b-5p. l The colocalization of circSTX6 and miR-449b-5p in PDAC cells was detected using a FISH assay. The circSTX6 probe was labeled with Cy3 (red), the miR-449b-5p probe was labeled with FAM (green), and nuclei were…

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  Fig. 5. MiR-449b-5p suppresses the proliferation, migration and invasion of PDAC cells in vitro. a The construction of PDAC cells transfected with miR-449b-5p mimics and miR-449b-5p inhibitor. b-d CCK-8 and colony formation assays of PDAC cells were performed to evaluate cell proliferation. e-h Transwell and wound healing assays were used to investigate the migratory and invasive abilities of PDAC cells transfected with miR-449b-5p mimics and miR-449b-5p inhibitor. (e-h, original magnification 200 ×. Scale bar = 100 μm. Values are expressed as the means ± SDs; *P < 0.05, **P < 0.01 and ***P < 0.001)

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  Fig. 6. MYH9 is a direct target of miR-449b-5p. a Clustered heatmap of the differentially expressed mRNAs in PDAC tissues from five T4 PDAC patients with long survival time and tissues from five T4 PDAC patients with short survival time. b Venn diagram shows the overlap of the target mRNAs of miR-449b-5p predicted by miRDB, miWalk and the results of RNA-seq. c Table of target mRNAs selected for miR-449b-5p. d A schematic of the wild-type (WT) and mutant (MUT) COL12A1 or MYH9 luciferase reporter vectors. e-f A luciferase reporter assay was used to confirm the interaction between COL12A1 and miR-449b-5p. g-h A luciferase reporter assay was used to confirm the interaction between MYH9 and miR-449b-5p. i-l The expression levels of COL12A1 and MYH9 in PDAC tissues and matched noncancerous tissues were detected by qRT–PCR. Kaplan–Meier analyses were used to illuminate the correlation between the levels of COL12A1 and MYH9 and the survival time of PDAC patients. The median expression value of COL12A1 and MYH9 was used as the cutoff (n = 49). Log-rank tests were used to determine the statistical significance. m-n Representative COL12A1 and MYH9 IHC images of a tissue microarray containing 97…

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  Fig. 7. MYH9 is indirectly regulated by circSTX6. a-c The expression levels of MYH9 mRNA (a-b) and protein (c) were significantly upregulated in circSTX6-overexpressing and downregulated in circSTX6 knockdown PDAC cells, and these effects could be reversed by the transfection of miR-449b-5p mimics or inhibitor. d Correlation analysis showed a negative relationship between the levels of miR-449b-5p and MYH9. e Correlation analysis showed a positive correlation between the levels of circSTX6 and MYH9. f MYH9 knockdown and overexpression cell lines were constructed. g-j MYH9 knockdown suppressed and MYH9 overexpression promoted the migratory and invasive abilities of PDAC cells, and these effects could be rescued by the transfection of circSTX6 plasmid or siRNA. Original magnification 200 ×. Scale bar = 100 μm. k-o The results of colony formation and CCK-8 assays revealed that MYH9 knockdown suppressed and MYH9 overexpression promoted proliferative abilities of PDAC cells, and these effects could be reversed by the transfection of circSTX6 plasmid or siRNA. (Values are expressed as the means ± SDs; *P < 0.05, **P < 0.01 and ***P < 0.001)

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  Fig. 8. CircSTX6 physically interacts with CUL2 and participates in the regulation of HIF1A. a CircSTX6 and oligo probes were incubated with proteins extracted from PDAC cells for RNA pull-down assays. Proteins pulled down were used for silver staining, and a specific band appeared between 75 and 100 kDa (arrow). b Levels of CUL2 protein were detected in the proteins pulled down by circSTX6 and oligo probes. c RIP assays were performed using antibodies against CUL2 and IgG. d-f Levels of CUL2 mRNA and protein in circSTX6-overexpressing and circSTX6-knockdown PDAC cells were quantified by qRT–PCR and western blotting. β-actin served as the internal control. g The levels of interacting proteins immunoprecipitated by CUL2 were detected in circSTX6-overexpressing PDAC cells. h CUL2 was truncated (1–186 aa, 187–372 aa, 373–568 aa, and 569–744 aa) to identify the specific fragment of CUL2 that bound to circSTX6. RIP assays were performed to detect the enrichment of circSTX6 in cells transfected with full-length and truncated flag-tagged constructs. i HIF1A was first immunoprecipitated, and the ubiquitination level of HIF1A was detected in PDAC cells transfected with the circSTX6 plasmid. j The…

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  Fig. 9. HIF1A promotes MYH9 transcription. a-b. The expression level of HIF1A in PDAC tissues and matched noncancerous tissues was detected by qRT–PCR. Kaplan–Meier analysis was used to illuminate the correlation between the level of HIF1A and the survival time of PDAC patients. The median expression value of HIF1A was used as the cutoff (n = 49). Log-rank tests were used to determine the statistical significance. c. HIF1A knockdown and overexpression cell lines were constructed. d. GEPIA analysis showed a positive relationship between MYH9 and HIF1A in PDAC tissues. e. Correlation analysis showed a positive relationship between MYH9 and HIF1A in 97 PDAC tissues. 18 S served as the internal control. f. MYH9 mRNA expression was significantly downregulated in HIF1A-knockdown PDAC cells and upregulated in HIF1A-overexpressing PDAC cells. g. CHIP assays revealed that HIF1A could bind to two fragments (-1194 – -1185, -561 – -552) of the MYH9 promoter. h-j. Luciferase reporter assays were used to confirm the interaction between HIF1A and two fragments (-1194 – -1185, -561 – -552) of the MYH9 promoter. k. Schematic diagram illustrating the mechanism by which circSTX6 promotes PDAC proliferation…