Exosomal miR-199a-3p Secreted From Cancer-Associated Adipocytes Promotes Pancreatic Cancer Progression

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Recent studies indicated that cancer-associated adipocytes (CAAs) play crucial roles in tumor progression; however, the precise mechanism remains unknown. Here, we analyzed specific exosomal microRNAs (miRNA) signatures derived from pancreatic CAAs to investigate their role in cancer progression.

Methods: CAAs were generated by co-culturing human adipocytes with human pancreatic cancer cells, and exosomes were isolated from the CAA-conditioned medium (CAA-CM). Small RNA-seq analysis was used to identify differentially expressed miRNAs in these exosomes. The effects of miRNAs on cell proliferation, migration/invasion, and drug sensitivity were examined. Luciferase reporter assays, real-time polymerase chain reaction, and western blotting were performed to investigate the molecular mechanisms of the miRNAs. The clinical relevance of the miRNAs was investigated using publicly available data and our cohort of patients with PDAC.

Results: miR-199a-3p expression was significantly increased in CAA-CM-derived exosomes. CAA-derived exosomes transferred miR-199a-3p to pancreatic cancer cells. Transfection with miR-199a-3p increased the proliferation, invasion, migration, and drug resistance of pancreatic cancer cells by downregulating SOCS7, increasing STAT3 phosphorylation, and upregulating SAA1 expression. High tissue miR-199a-3p expression is correlated with poor prognosis in patients with PDAC. Liquid biopsies revealed that exosomal miR-199a-3p could accurately differentiate patients with PDAC from healthy controls. Multivariate survival analysis indicated that miR-199a is an independent prognostic factor for PDAC.

Conclusion: miR-199a-3p in CAA-derived exosomes contributes to the malignant transformation of pancreatic cancer via the SOCS7/STAT3/SAA1 pathway, suggesting its potential as a biomarker and therapeutic target for PDAC.

Keywords: biomarker; cancer‐associated adipocytes; miR‐199a‐3p; pancreatic ductal adenocarcinoma.

FIGURE 1

Identification of differentially expressed miRNAs in CAA‐CM‐derived exosomes using next‐generation sequencing analysis. (A–C) Characterization of serum exosomes. (A) Nanoparticle tracking analysis of the size distribution and number of exosomes derived from the cancer‐associated adipocytes‐conditioned medium (CAA‐CM) isolated via ultracentrifugation. (B) Representative transmission electron microscopy images of CAA‐CM‐derived exosomes. Scale bar, 100 nm. (C) Western blotting analysis of exosome markers CD63, Alix, and tsg101 in CAA‐CM‐derived exosomes and exosome‐depleted supernatants of Panc‐1 cells. (D) Representative fluorescent microscopy images of the internalization of fluorescently labeled exosomes in Panc‐1 cells. Scale bar, 50 μm. (E) Hierarchical clustering analysis of differentially expressed miRNAs in CAA‐CM‐ and adipocytes‐CM (A‐CM)‐derived exosomes. The heatmap shows the top 54 differentially expressed miRNAs between the two groups. Each row represents a single miRNA and each column represents an individual sample. (F) Volcano plot of the miRNAs of 6 samples (3 CAA‐CMs and 3 A‐CMs). The abscissa represents the log₂ (fold change) of the miRNA expression, and the ordinate represents the logarithmic transformation of the p‐value gained using the t‐test. (G) RT‐qPCR analysis of miR‐199a‐3p expression in adipocyte‐derived exosomes (A‐exosomes) or CAA‐derived exosomes (CAA‐exosomes). *p < 0.01. (H) RT‐qPCR analysis of miR‐6126 expression in A‐exosomes or CAA‐exosomes. (I) RT‐PCR analysis of miR‐199a‐3p expression in Panc‐1 cells cultured in the control medium, A‐CM, or CAA‐CM for 48 h. *p < 0.01.

FIGURE 2

MiR‐199a‐3p promotes the growth, migration/invasion, and drug resistance of pancreatic cancer cells. (A) Growth curve of Panc‐1 cells transfected with miR‐199a‐3p mimics (Panc‐1‐miR‐199a‐3p) and scramble siRNA (Panc‐1‐miNC), as analyzed using a WST‐8 assay (n = 6). (B) Migration area of Panc‐1‐miR‐199a‐3p‐ or Panc‐1‐miNC‐transfected cells, as assessed using a wound healing assay. (C) Migration area was quantified using Tscratch software (n = 6). Scale bars, 100 μm. (D) Invasion capability of Panc‐1‐miR‐199a‐3p‐ or Panc‐1‐miNC‐transfected cells, as assessed using a Transwell invasion assay. (E) Cell migration was quantified by counting the number of migrating cells in six randomly chosen visual fields (n = 6). Scale bar, 200 μm. (F) Panc‐1‐miR‐199a‐3p‐ or Panc‐1‐miNC‐transfected cells were treated with different concentrations of gemcitabine for 72 h. Viable cells were quantified using a WST assay (n = 6). *p < 0.05.

FIGURE 3

MiR‐199a‐3p suppresses SOCS7 to increase STAT3 activation and induce SAA1 expression in pancreatic cancer cells. (A, B) SOCS7 is a downstream target gene of miR‐199a‐3p. (A) The wild‐type (WT) SOCS7 3′‐untranslated region (UTR) has a putative binding site for miR‐199a‐3p (position 3449–3455) predicted using the Target Scan database. SOCS7 3′‐UTR‐mutant type (MT) contains mutated sequences in the putative binding site of miR‐199a‐3p (shown in red). The indicated sequences (SOCS7 3′‐UTR‐WT and SOCS7 3′‐UTR‐MT) were cloned in the psiCHECK2 vector. (B) Panc‐1 cells were co‐transfected with the control miRNA mimics and psiCHECK2 vectors containing the WT or MT sequences. Luciferase activities in these cells were measured using a Dual‐Luciferase Reporter Assay System (n = 3). *p < 0.05. (C–E) Relative mRNA levels of various genes in Panc‐1, PK‐1, and PK‐45H cells transfected with miR‐199a‐3p mimics or control miRNA were determined via RT‐qPCR: (C) miR‐199a‐3p, (D) SOCS7, and (E) SAA1. n = 6. *p < 0.05. (F) The expression of SOCS7, STAT3, p‐STAT3 (Tyr705), and SAA1 in Panc‐1, PK‐1, and PK‐45H cells were examined using western blot analysis 24 h after transfection with miR‐199a‐3p mimics or control miRNAs.

FIGURE 4

MiR‐199a‐3p expression is negatively correlated with survival in patients with PDAC. (A) Kaplan–Meier survival curves showing the overall survival of the low‐miR‐199a‐3p and high‐miR‐199a‐3p groups from the GDC TCGA Pancreatic Cancer (PAAD) dataset. The fragments per kilobase million with upper quantile (FPKM–UQ) values normalized using RNA‐seq counts were used to perform survival analyses. The cutoff value was set at 50,000 FPKM–UQ based on the median miRNA expression. Cases with ≥ 50,000 and < 50,000 FPKM–UQ were designated the high and low expression groups, respectively. (B) Receiver operating characteristic (ROC) curve analysis for discriminating between healthy individuals and patients with pancreatic ductal adenocarcinoma (PDAC). (C) Comparison of miR‐199a‐3p expression levels between healthy individuals (n = 10) and patients with PDAC (n = 61). (D) Comparison of miR‐199a‐3p expression levels in healthy individuals (n = 10) and patients with stages I + II (n = 12), III (n = 13), and IV (n = 36) PDAC. (E, F) Kaplan–Meier survival curves for (E) OS and (F) PFS in the low‐miR‐199a‐3p and high‐miR‐199a‐3p groups of patients with stage III/IV PDAC who received chemotherapy (n = 49). OS, overall survival; PFS, progression‐free survival.

FIGURE 5

Proposed model illustrating the role of CAA‐derived exosomal miR‐199a‐3p in regulating pancreatic cancer. Adipocytes in contact with pancreatic cancer cells are first dedifferentiated into cancer‐associated adipocytes (CAAs) in the microenvironment of pancreatic cancer. Subsequently, exosomal miR‐199a‐3p secreted from CAAs are taken up by surrounding pancreatic cancer cells, then directly bind to the 3′‐untranslated region (UTR) of SOCS7 and suppress its expression, which in turn activates STAT3 and increases SAA1 expression. The secreted SAA1 then promotes pancreatic cancer cell proliferation, metastasis, and chemoresistance by activating NF‐κB, suggesting that miR‐199a‐3p plays a vital role as a mediator of the microenvironment in pancreatic cancer.