Micro-RNA-21 Regulates Cancer-Associated Fibroblast-Mediated Drug Resistance in Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths due to its highly aggressive biological nature and resistance to chemotherapy. Previous studies indicate that miR-21 is an important regulator in the activation of cancer-associated fibroblasts (CAFs). However, whether miR-21 in CAFs would regulate PDAC's tumor microenvironment and lead to drug resistance remain unknown. In this study, we evaluated the relationship between CAF activation, miR-21 expression, and drug resistance using tumor samples from PDAC patients. We changed the miR-21 expression level in CAFs and tested its roles in regulating the function of CAFs. In addition, we explored the roles of miR-21 in CAFs in the development of PDAC using an animal model. We found that PDAC patients who were resistant to gemcitabine treatment tended to have higher miR-21 expression and more activated CAFs. An in vitro study showed that CAFs with high miR-21 expression had elevated MMP-3, MMP-9, PDGF, and CCL-7 expression and promoted the invasion of PDAC cell lines. miR-21 overexpression also contributed to the activation of CAFs by regulating the PDCD4 gene. The in vivo study showed that upregulating miR-21 in CAFs promoted PDAC desmoplasia and increased its drug resistance to gemcitabine treatment, but downregulating miR-21 in CAFs suppressed desmoplasia and enhanced the effect of gemcitabine. We concluded that miR-21 promoted the activation of CAFs and contributed to the drug resistance of PDAC.

Figure 1

The relationship between the number of cancer-associated fibroblasts (CAFs), miR-21 expression, and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC). (A, B) Representative pictures of high α-SMA expression and low α-SMA expression in human PDAC tissues, respectively [stained by immunofluorescence (IF)]. (C) Each case of human PDAC tissue sample was evaluated by α-SMA, miR-21, and drug resistance to gemcitabine and plotted. Each star represents a PDAC patient. The scatterplot shows that patients who were resistant to gemcitabine treatment tended to have a higher miR-21 expression and a lower α-SMA expression compared to patients who were sensitive to gemcitabine treatment (higher ΔCT value indicates lower miR-21 expression).

Figure 2

miR-21 in CAFs was increased by gemcitabine treatment. (A) Two PDAC cell lines, MIA Paca-2 and Panc02, were treated with gemcitabine, and the miR-21 within these cells was measured. There was no apparent difference in the amount of miR-21 between the groups, whether they accepted gemcitabine or not. (B) Two kinds of CAFs, human CAFs (hCAFs) and murine CAFs (mCAFs), were treated with gemcitabine. The CAFs treated with gemcitabine had over three times the amount of miR-21 as the untreated CAFs. *p < 0.05; **p < 0.01.

Figure 3

miR-21 regulates the function of CAFs and promotes PDAC invasion. In the different experiment groups, hCAFs and mCAFs were transfected with various micro-RNAs (miR-21 mimic, miR-21miR inhibitor, scramble, or miR-21 mimic scramble). Expression of matrix metalloproteinase-3 (MMP-3), MMP-9, chemokine (C-C motif) ligand 7 (CCL-7), and platelet-derived growth factor (PDGF) was evaluated by enzyme-linked immunosorbent assay (ELISA). All data were normalized to the wide-type CAFs. (A–D) MMP-3, MMP-9, CCL-7, and PDGF were significantly increased in the CAFs with increased miR-21, while their expression was decreased in the CAFs with suppressed miR-21. (E) Representative pictures of the Transwell assay using the MIA Paca-2 and Panc02 cell lines and CAFs with different levels of miR-21. (F) Quantified data of invaded tumor cells in the Transwell assay. The groups with high miR-21 expression CAFs showed a highly increased number of invaded tumor cells, while the groups with low miR-21 expression CAFs had a decreased number of invaded tumor cells. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

miR-21 promoted the activation of CAFs by targeting PDCD4. PCDC4 and α-SMA expressions were measured in the murine CAFs transfected with different micro-RNAs and treated with TGF-β. (A, B) Sequences of miR-21 used to transfect human CAFs and murine CAFs, respectively. (C) PDCD4 expression was decreased in the CAFs with increased miR-21, but it was increased in the CAFs with miR-21 inhibitors. (D) In contrast to PDCD4, α-SMA expression was decreased in the murine CAFs with low miR-21 but was enhanced in the CAFs with high miR-21. (E) Murine CAFs transfected with PDCD4 siRNAs were treated with TGF-β to detect their activation by measuring α-SMA expression. The CAFs of the control groups were transfected with scrambled siRNA and treated with complete medium. The CAFs with PDCD4 deficiency had an increased α-SMA expression. (F) CAFs with the miR-21 inhibitor and PDCD4 deficiency showed an increased α-SMA compared with the CAFs with the miR-21 inhibitor alone. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

miR-21 in CAFs regulates drug resistance to gemcitabine in a PDAC mouse model. A PDAC mouse model was established with Panc02 cell lines and CAFs with various levels of miR-21 and accepted gemcitabine treatment after 1 week of implantation. (A) Increase in tumor volume over time after implantation. The mice with CAFs having the miR-21 mimic grew the fastest, while the mice with CAFs having the miR-21 inhibitor grew the slowest. (B) Survival curves of the mice with CAFS having different levels of miR-21. Consistent with tumor volume growth, the mice with high miR-21 expression CAFs had the shortest survival time, and the mice with low miR-21 expression CAFs had the longest survival time.

Figure 6

Desmoplasia in the PDAC mouse model. Collagen amount and α-SMA expression of the tumor tissues from the PDAC mouse model were detected by Sirius red staining and IF staining, respectively. (A–C) Representative pictures showing high, medium, and low levels of collagen within the tumor tissues, respectively. (D) Quantified data showing the amount of collagen in the tumor tissues. Tumor tissues derived from mice with high miR-21 expression CAFs showed the highest amount of collagen, and the tumor tissue of mice with low miR-21 expression CAFs had the lowest amount of collagen. (E–G) Representative pictures showing high, medium, and low levels of α-SMA within the tumor tissues, respectively. (H) Tumor tissues from the mice with high miR-21 expression CAFs had the highest α-SMA expression, and tumor tissues from the mice with low miR-21 expression CAFs had the lowest α-SMA expression. *p < 0.05; **p < 0.01; ***p < 0.001; ****p <0.0001.