Circ_0041150 inhibits proliferation of pancreatic adenocarcinoma cells by regulating triglyceride accumulation via the miR-1178-3p/AADAC axis

Figures

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  Fig. 1. The expression patterns of AADAC, miR-1178-3p, and circ_0041150 in PAAD tissues. A In the GEPIA database, box plot analysis was utilized to identify the differential expression of AADAC mRNA between 179 PAAD and 171 normal tissues, with color labels as red box for PAAD tissues and gray box for normal tissues. B qRT-PCR was employed to observe a significant difference in AADAC mRNA expression between PAAD and adjacent tissues (n = 40). C qRT-PCR was used to detect the altered expression of miR-1178-3p in PAAD tissues compared to adjacent ones (n = 40). D The differential expression of RPH3AL mRNA, the host gene of circ_0041150, between 179 PAAD and 171 normal tissues, was also reported in the GEPIA database. E qRT-PCR demonstrated the altered expression of circ_0041150 in PAAD tissues relative to adjacent tissues (n = 40). F-H Finally, Pearson’s correlation tests were carried out to analyze the correlations among the expression levels of circ_0041150 and miR-1178-3p (F), circ_0041150 and AADAC mRNA (G), and miR-1178-3p and AADAC mRNA (H) in PAAD tissues (n = 40). Statistical significance was assessed using two-tailed unpaired Student’s t-test. *P < 0.05 and **P < 0.01 vs. control

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  Fig. 2. Analysis of circ_0041150 expression and its impact on PAAD cell proliferation. A The chromosomal location and structure of circ_0041150 were depicted. B The expression patterns of circ_0041150 in HPDE6-C7 and PAAD cell lines (Capan-2, BxPC-3, AsPC-1, PANC-1) were examined by qRT-PCR (n = 4). C, D The stability of circ_0041150, compared to its host gene RPH3AL mRNA, was analyzed by qRT-PCR after RNase R digestion (n = 4). E, F Its subcellular localization was assessed via qRT-PCR (n = 4). G BxPC-3 cells were transfected with sh-circ_0041150#1 or sh-circ_0041150#2, and circ_0041150 expression was detected by qRT-PCR (n = 4). H, I Cell proliferation in BxPC-3 cells with circ_0041150 knockdown was evaluated by CCK-8 and EdU incorporation assays (n = 4). J PANC-1 cells were transfected with p-circ_0041150, and circ_0041150 expression was detected by qRT-PCR (n = 4). K, L Cell proliferation in PANC-1 cells overexpressing circ_0041150 was assessed by CCK-8 and EdU incorporation assays (n = 4). Statistical significance was assessed using two-tailed unpaired Student’s t-test or two-way ANOVA. *P < 0.05 and **P < 0.01 vs. control

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  Fig. 3. Effects of circ_0041150 on TG accumulation of PAAD cells. A Flutamide hydrolase activity was assayed in BxPC-3 cells following circ_0041150 knockdown (n = 4). B, C Lipid deposition, induced by FFA, was visualized in BxPC-3 cells with knocked-down circ_0041150 via BODIPY 493/503 staining using fluorescence microscopy (40×; scale bar = 20 μm) (n = 4). D Triglyceride (TG) content was quantified in BxPC-3 cells post-knockdown. E Flutamide hydrolase activity was assayed in PANC-1 cells following circ_0041150 overexpression (n = 4). F, G FFA-induced lipid deposition was visualized in PANC-1 cells with overexpressed circ_0041150 via BODIPY 493/503 staining using fluorescence microscopy (40×; scale bar = 20 μm) (n = 4). H TG content was quantified in PANC-1 cells post-overexpression (n = 4). Statistical significance was assessed using two-tailed unpaired Student’s t-test or two-way ANOVA. *P < 0.05 and **P < 0.01 vs. control

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  Fig. 4. Circ_0041150 targets and regulates miR-1178-3p in PAAD cells. A, B qRT-PCR analysis compared the expression levels of miR-1178-3p, miR-1184, miR-188-3p, and miR-766 in BxPC-3 and PANC-1 cells transfected with sh-circ_0041150#1 or p-circ_0041150 to controls (n = 4). C Bioinformatics analysis predicted binding site sharing between circ_0041150 and miR-1178-3p, with wild-type and mutated sequences highlighted, with mutation site in red. D, E FISH analysis confirmed co-localization of circ_0041150 (red) and miR-1178-3p (green) in BxPC-3 (D) and PANC-1 (E) cells, with nuclei counterstained with DAPI (blue) (20×; scale bar = 10 μm). F, G Luciferase reporter assays demonstrated altered activity in BxPC-3 (F) and PANC-1 (G) cells transfected with wild-type (WT) or mutant (MUT) circ_0041150 reporters alongside miR-1178-3p mimic or control mimic (n = 4). H, I RNA pull-down assays followed by qPCR detected circ_0041150 enrichment in BxPC-3 (H) and PANC-1 (I) cell lysates using biotin-labeled miR-1178-3p or control probes (n = 4). Controls include: (1) bio-NC probe (negative control, a non-specific biotin-labeled miRNA probe with no homology to miR-1178-3p); (2) bio-miR-1178-3p probe (MUT)…

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  Fig. 5. AADAC is a direct target of miR-1178-3p in PAAD cells. A Potential binding site overlap between the 3’ UTR of AADAC and miR-1178-3p was revealed by bioinformatics analysis. B, C Luciferase activity was assessed in reporter plasmids containing wild-type and mutant AADAC 3’ UTR sequences co-transfected with miR-1178-3p mimic or negative control in BxPC-3 (B) and PANC-1 (C) cells (n = 4). D, E AADAC mRNA levels were quantified in pulldown products from cell lysates of BxPC-3 (D) and PANC-1 (E) cells incubated with biotin-labeled miR-1178-3p or control probes (n = 4). F, G AADAC mRNA levels were detected in BxPC-3 (F) and PANC-1 (G) cells transfected with miR-1178-3p mimic or control mimic (n = 4). H, I miR-1178-3p and AADAC mRNA expressions were detected in BxPC-3 cells transfected with miR-1178-3p mimic or control mimic (n = 4). J Following the relevant transfections, AADAC protein expression levels in BxPC-3 cells were evaluated by Western blot (n = 3). Lane 1, control mimic; Lane 2, miR-1178-3p mimic. K, L miR-1178-3p and AADAC mRNA expressions were also detected in PANC-1 cells transfected with miR-1178-3p inhibitor or miRNA inhibitor-NC (n = 4). M Following the relevant…

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  Fig. 6. Effects of miR-1178-3p inhibition or AADAC overexpression on BxPC-3 cell proliferation and TG accumulation inhibited by circ_0041150 knockdown. A, B Following the relevant transfections, cell proliferation in BxPC-3 cells was assessed using CCK-8 (A) and EdU incorporation (B) assays (n = 4). C-F Following the relevant transfections, lipid droplet (C, D) via BODIPY 493/503 staining using fluorescence microscopy (20×; scale bar = 20 μm), TG content (E) and flutamide hydrolase activity (F) levels in BxPC-3 cells were detected using the relevant commercial kits (n = 4). G, H Following the relevant transfections, AADAC mRNA and protein expression levels in BxPC-3 cells were evaluated by qRT-PCR (G) and Western blot (H), respectively (n = 3). Lane 1, sh-NC; Lane 2, miRNA inhibitor-NC; Lane 3, vector control; Lane 4, sh-circ_0041150 + miRNA inhibitor-NC; Lane 5, miR-1178-3p inhibitor + sh-NC; Lane 6, sh-circ_0041150 + miR-1178-3p inhibitor; Lane 7, p-AADAC + sh-NC; Lane 8, sh-circ_0041150 + p-AADAC. Statistical significance was assessed using two-way ANOVA.*P < 0.05, **P < 0.01 vs. sh-NC; #P < 0.05,##P < 0.01 vs. miRNA inhibitor-NC; ΔΔP < 0.01 vs. vector control

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  Fig. 7. A schematic model summarizes the study results. In the cytoplasm, circ_0041150 acts as a sponge for miR-1178-3p, preventing its binding to the 3’ UTR of AADAC mRNA, thus increasing AADAC expression, inhibiting TG accumulation and reducing PAAD cell proliferation. Conversely, without circ_0041150, miR-1178-3p binds to AADAC mRNA, inhibiting its expression, promoting TG accumulation and enhancing PAAD cell proliferation