BAG3 regulates stability of IL-8 mRNA via interplay between HuR and miR-4312 in PDACs

Abstract

Bcl-2 associated athanogene 3 (BAG3) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and its high expression appears to be a poor prognostic factor for patients with PDAC. In this study, we show that BAG3 knockdown significantly decreases migration and invasion of PDACs via reduction of interleukine-8 (IL-8) production. BAG3 knockdown regulates IL-8 expression at the posttranscriptional levels via interplay between recruitment of RNA-binding protein HuR and miR-4312. HuR binds to the cis-elements located in the 3'-untranslational region (UTR) of the IL-8 transcript to stabilize it, whereas miR-4312-containing miRNA-induced silencing complex (miRISC) is recruited to the adjacent seed element to destabilize it. The binding of HuR prevents the recruitment of Argonaute (Ago2), overriding miR-4312-mediated translation inhibition of IL-8. BAG3 knockdown decreases cytoplasmic distribution of HuR via increasing its phosphorylation at Ser202, therefore compromising its recruitment while promoting recruitment of miR-4312 containing miRISC to IL-8 transcript. Furthermore, our data indicate that only phosphorylated Ago2 at Ser387 interacts with IL-8 transcript. BAG3 knockdown increases phosphorylation of Ago2 at Ser387, thereby further promoting loading of miR-4312 containing miRISC to IL-8 transcript. Taken together, we propose that BAG3 promotes invasion by stabilizing IL-8 transcript via HuR recruitment, and subsequently suppressing the loading of miR-4312 containing miRISC in PDACs. Our results reveal a novel pathway linking BAG3 expression to enhanced PDAC metastasis, thus making BAG3 a potential target for intervention in pancreatic cancer.

Fig. 1. Knockdown of BAG3 inhibits migration and invasion of PDACs via reduction of IL-8 production.

a BAG3 was knocked down using CAS9 system, and BAG3 expression was confirmed by western blot. b–c the migration and invasion of control or BAG3 knockdown (KD) cells was evaluated by a Matrigel-uncoated (b) and coated (c) transwell assay, respectively. d Cytokine release by control or BAG3 knockdown PDACs was analyzed using cytokine antibody microarray. e–f BxPC3 cells were exposed to the indicated antibodies, cell migration and invasion was evaluated by a Matrigel-uncoated (e) and coated (f) transwell assay, respectively. g–h SW1990 cells were exposed to the indicated antibodies, cell migration, and invasion was evaluated by a Matrigel-uncoated (g) and coated (h) transwell assay, respectively. N.S., not significant; *, P < 0.01

Fig. 2. BAG3 knockdown destabilizes IL-8 mRNA via its 3′-UTR.

a IL-8 mRNA was analyzed using real-time RT-PCR. b Nascent RNA was labeled and isolated, newly synthesized IL-8 mRNA was analyzed using qRT-PCR. c–d Actinomycin (d) was added for the indicated period to block RNA synthesis, and IL-8 mRNA was analyzed using qRT-PCR in BxPC3 (c) or SW1990 d cells. e–f Amanitin was added for the indicated period to block RNA synthesis, and IL-8 mRNA was analyzed using qRT-PCR in BxPC3 (e) or SW1990 (f) cells. g–h Relative IL-8 mRNA expression at 4 h of Actinomycin D (g) or Amanitin (h) was normalized by that of 2 h. I, BxPC3 cells were transfected with the indicated luciferase reporter vector and a Renilla reporter vector. Luciferase activity was measured 2 days after transfection and Renilla activity was measured to normalize luciferase activity. N.S., not significant; *, P < 0.01

Fig. 3. BAG3 knockdown destabilizes IL-8 mRNA via increasing HuR phosphorylation at Ser202.

a–b RIP was performed using the indicated antibodies, enrichment of IL-8 mRNA was measured by qRT-PCR in BxPC3 (a) and SW1990 (b) cells. c HuR protein levels were measured using western blot. d RIP was performed using the antibody against BAG3, enrichment of IL-8 was analyzed using qRT-PCR. e Cell lysates were immunoprecipitated by pan-phospho Ser/Thr antibody, western blot was then performed to analyze HuR phosphorylation. f Cell lysates were fractioned to nuclear (NF) and cytoplasmic (CF) fraction, western blot was performed using both fractions. g BxPC3 cells were infected with lentivirus containing shRNA against HuR (shHuR), HuR knockdown efficiency was confirmed by western blot. h BxPC3 cells were infected with lentivirus containing shHuR, IL-8 mRNA expression was analyzed using qRT-PCR. i Luciferase containing IL-8 3′-UTR was transfected to the indicated cells, luciferase activity was analyzed. j–k Control or BAG3 KD BxPC3 cells were transfected with wild type (WT), mutation at Ser202 to alanine (S202A) or to aspartic acid (S202D) HuR, HuR expression was confirmed by western blot j, IL-8 mRNA was analyzed using qRT-PCR k. l BxPC3 cells were co-transfected with the indicated HuR construct and luciferase construct containing IL-8 3′-UTR, luciferase activity was analyzed. N.S., not significant; *, P < 0.01

Fig. 4. BAG3 knockdown destabilizes IL-8 mRNA via miRISC complex.

a RIP was performed using Ago2 antibody, enrichment of IL-8 mRNA was analyzed using qRT-PCR. b Ago2 protein expression was analyzed using western blot. c BxPC3 cells were infected with lentivirus containing Ago2 (shAgo2), knockdown efficiency wax confirmed by western blot analysis. d–e BxPC3 cells were infected with lentivirus containing shAgo2, IL-8 mRNA was analyzed using qRT-PCR. f–g The indicated BxPC3 cells were transfected with luciferase containing IL-8 3′-UTR, luciferase activity was analyzed. h–i Control or BAG3 KD BxPC3 cells were transfected with Ago2, Ago2 expression was confirmed by western blot (h), IL-8 mRNA was analyzed using qRT-PCR i. j BxPC3 cells were co-transfected with Ago2 and luciferase construct containing IL-8 3′-UTR, luciferase activity was analyzed. N.S., not significant; *, P < 0.01

Fig. 5. BAG3 knockdown destabilizes IL-8 mRNA via interplay between miRISC complex and HuR.

a BxPC3 were infected with lentivirus containing shAgo2, HuR protein in whole-cell lysates (WCL) as well cytoplasmic fraction (CF) was analyzed using western blot analysis. b RIP was performed using HuR antibody, enrichment of IL-8 mRNA in the indicated cells was analyzed using qRT-PCR. c–d control or BAG3 KD BxPC3 cells were infected with lentivirus containing scrmble or shHuR, Ago2 expression was analyzed using western blot c, enrichment of IL-8 mRNA by Ago2 was analyzed using RIP followed by qRT-PCR d. e–f control or BAG3 KD BxPC3 cells were transfected with WT, S202A, or S202D HuR, Ago2 expression was analyzed using western blot e enrichment of IL-8 mRNA by Ago2 was analyzed using RIP followed by qRT-PCR f. g Immunoprecipitation was performed using pan-phospho Ser/Thr antibody, Ago2 phosphorylation was then analyzed by western blot. h–i BxPC3 cells were transfected with wild type (WT), mutation at Ser387 to alanine (S387A), or to aspartic acid (S387D) Ago2. HuR expression was analyzed in both whole-cell lysate and cytoplasmic fraction using western blot h, and enrichment of IL-8 mRNA by HuR was analyzed using RIP followed by qRT-PCR i. j BxPC3 cells were transfected with the indicated Ago2 construct tagged with Flag, enrichment of IL-8 mRNA by Ago2 was measured using RIP followed by qRT-PCR. k control or BAG3 KD cells were infected with lentivirus containing shHuR, then transfected with the indicated Ago2 construct. Enrichment of IL-8 mRNA by Ago2 was analyzed by RIP followed by qRT-PCR. l–m BxPC3 cells were co-transfected with the indicated construct and luciferase containing IL-8 3′-UTR, luciferase activity was analyzed post 48 h transfection. N.S., not significant; *, P < 0.01

Fig. 6. miR-4312 competes with HuR to interact with IL-8 mRNA and involves in suppression of IL-8 expression by BAG3 knockdown in PDACs.

a Schematic illustration of HuR and miR-4436b-5p and miR-4312 potential binding sites on IL-8 mRNA. b–c BxPC3 cells were co-transfected with miR-4312 or miR-4436b-5p mimic (b) or antagomir (c) and luciferase containing IL-8 3′-UTR, luciferase activity was measured after 48 h of transfection. d control or BAG3 KD BxPC3 cells were transfected with luciferase construct containing wild-type (WT) or potential miRs binding site mutant IL-8 3′-UTR, luciferase activity was measured. e BxPC3 cells were transfected with miR-4312 or miR-4436b-5p mimic, enrichment of IL-8 mRNA by HuR was analyzed using RIP followed by qRT-PCR. f–g miR-4312 f and miR-4436-5p (g) expression levels were analyzed using qRT-PCR. h–i, miR-4312 (h) and miR-4436-5p (i) containing miRISC formation was analyzed using RIP followed by qRT-PCR. N.S., not significant; *, P < 0.01

Fig. 7. Correlation of BAG3 and IL-8 expression in pancreatic cancer tissues.

a–e BAG3, IL-8, and HuR protein levels were investigated using western blot, miR-4312 levels were measured using qRT-PCR in fresh pancreatic cancer tissues. Scatter plots showing the correlation between BAG3 and IL-8 (Pearson’s coefficient test r = 0.671, P < 0.001) (a) miR-4312 and IL-8 (Pearson’s coefficient test r = −0.398, P < 0.01) (b), HuR and IL-8 (Pearson’s coefficient test r = − 0.087, P > 0.05) (c), as well as the correlation between BAG3 and miR-4312 (Pearson’s coefficient test r = −0.189, P > 0.05) (d), BAG3 and HuR (Pearson’s coefficient test r = −0.029, P > 0.05) (e) in pancreatic cancer tissues. Pearson’s coefficient tests were performed to assess statistical significance

Fig. 8

Schematic representation of destabilization of IL-8 mRNA by BAG3 knockdown in PDACs