Snail1-expressing cancer-associated fibroblasts induce lung cancer cell epithelial-mesenchymal transition through miR-33b

Abstract

Lung cancer has a high propensity for metastasis. Cancer-associated fibroblasts (CAFs) are the main type of stromal cells in cancer tissue, are activated by tumor cells, and play a significant role in tumor development. However, whether CAFs induce lung cancer cell metastasis, as well as pathway involved in CAF-induced lung cancer cell metastasis, is uncertain. Snail1 is a transcriptional factor whose expression in the stroma is associated with lower survival rates in patients with cancer. However, how Snail1 regulates the crosstalk between stromal cells and tumor cells when it is expressed in the stroma has not been determined. Altered microRNA (miRNA) expression is correlated with lung cancer metastasis. Our previous study of microRNAs showed that miR-33b levels were clearly reduced in lung cancer cell lines and lung cancer tissues, and miR-33b suppressed tumor cell epithelial-mesenchymal transition (EMT) when its expression was elevated. In this study, we found that co-culturing CAFs with lung cancer cells induced miR-33b downregulation and promoted epithelial cells EMT. Moreover, we found that miR-33b overexpression in lung cancer cells counteracted CAF-induced EMT. Interestingly, Snail1 expression in fibroblasts activate the inductive effects of CAFs on lung cancer cell EMT. Hence, understanding the molecular mechanism underlying the communication between stromal cells and tumor cells mediated by miR-33b may lead to the identification of novel targets for the treatment of lung cancer. Additionally, understanding the role of Snail1 driving CAFs to induce lung cancer cell EMT may provide with a new perspective on the treatment of lung cancer.

Keywords: Snail1; cancer-associated fibroblasts; epithelial-mesenchymal transition; lung cancer; microRNA.

Figure 1. Characterization of primary CAFs and NFs

(A) The mRNA expression levels of FAP, FSP1, ACTA2, which are CAF specific genes, in A549 cells (an epithelial cell control), NFs and CAFs were detected by qRT-PCR, GAPDH gene was used as the normalization control. We also found that SNAI was overexpressed in CAFs only. (B) The protein expression levels of E-cadherin, Vimentin and α -SMA in A549 cells, NFs and CAFs by immunoblotting, using GAPDH protein as the loading control. We also found that Snail1 was overexpressed in CAFs only. (C) Immunofluorescence staining revealed the subcellular location and the expression of E-cadherin, Vimentin and α -SMA in A549 cells, NFs and CAFs. (D) The expression levels of E-cadherin and a-SMA, which are CAF specific biomarkers, in CAFs isolated from different primary lung cancer tissues were detected by immunobotting. All the primary CAFs showed positive staining for a-SMA and negative staining for E-cadherin, presenting characteristics of CAFs.

Figure 2. Co-culturing lung cancer cells with CAFs induced cell migration and invasion

(A, B) Cell motility ability was measured by wound-healing assay. The percent of wound closure was determined at 36 h in A549 cells, 24 h in H1299 cells, 48h in SPC-a-1, and 36 h in LTEP-a-2 cells, respectively, before the complete would closure. (C–E) Cell migration (C, E) and cell invasion (D, E) were measured by the Transwell cell migration/invasion assay. ^*P < 0.05, ^**P < 0.01.

Figure 3. CAFs induced miR-33b downregulation and promoted the EMT phenotype in lung cancer cells

(A) The protein expression levels of E-cadherin, Vimentin, ZEB1, Twist1 and Snail1 in the lung cancer cells co-cultured with CAFs were deteced by immunoblotting, using GAPDH protein as the loading control. (B) The relative level of miR-33b in different NSCLC cell lines co-cultured with CAFs, was detected by qRT-PCR, GAPDH gene was used as the normalization control. (C–I) The mRNA expression levels of CDH1 (epithelial marker, encoding E-Cadherin) (C), VIM (mesenchymal markers, encoding vimentin) (D), MMP2 and MMP9 (invasion markers) (E, F), and ZEB1, TWIST1, SNAI1 (EMT-associated transcription factors) (G–I) in different NSCLC cell lines co-cultured with CAFs were detected by qRT-PCR, GAPDH gene was used as the normalization control. ^*P < 0.05, ^**P < 0.01.

Figure 4. miR-33b prevented CAF-induced lung cancer cell EMT

(A) A549 and H1299 cells were transfected with miR-33b or miR-NC and then the relative level of miR-33b was measured by qRT-PCR analysis. (B, C) A549 and H1299 cells were transfected with miR-33b or with miR-NC and, after 48 h, treated or not with CAFs for an additional 48 h. Cell motility was measured by wound-healing assay. (D–F) Migration and invasion of A549 and H1299 cells, treated as in (B), was analyzed. (G) The levels of E-cadherin and vimentin in A549 and H1299 cells, treated as in (B), were assessed by immunoblotting. (H, I) The mRNA expression levels of CDH1 and VIM in A549 and H1299 cells, treated as in (B), were assessed by qRT-PCR.

Figure 5. Knockdown of miR-33b promotes CAF-induced EMT of lung cancer cells

(A) A549 cells and H1299 cells were transfected with anti-miR-33b or anti-miR-NC and then and then the relative level of miR-33b was measured by qRT-PCR analysis. (B, C) A549 and H1299 cells were transfected with anti-miR-33b or with anti-miR-NC and, after 48 h, treated or not with CAFs for an additional 48 h. Cell motility was measured by wound-healing assay. (D–F) Migration and invasion of A549 and H1299 cells, treated as in (B), was analyzed. (G) The levels of E-cadherin and vimentin in A549 and H1299 cells, treated as in (B), were assessed by immunoblotting. (H, I) The mRNA expression levels of CDH1 and VIM in A549 and H1299 cells, treated as in (B), were assessed by qRT-PCR.

Figure 6. miR-33b inhibited CAF-induced lung cancer cell growth and EMT in vivo

(A–C) Tumor xenograft growth curves in mice (n = 6 per group). A549 cells transfected with miR-33b or negative control were inoculated with or without CAFs into nude mice for indicated days. At the experimental end point, tumor xenografts were dissected and photographed. (D) Tumor xenografts were processed and stained with HE and immunohistochemically for E-cadherin, vimentin. (E, F) Levels of vimentin, E-cadherin mRNA and protein in tumor xenografts were assessed by qRT-PCR and western blot analysis. (^*P < 0.05, ^**P < 0.01).

Figure 7. Snail1 promoted CAF regulation of lung cancer cells

(A) CAFs were transfected with SNAI1 or with negative control and then the expression of Snail1 was detected by immunoblotting analysis. (B, C) CAFs were transfected with SNAI1 or with negative control and, after 48 h, co-cultured with A549 and H1299 cells for an additional 48 h. Lung cancer cells motility was measured by wound-healing assay. (D–F) Migration and invasion of A549 and H1299 cells, treated as in (B), was analyzed. (G) The levels of E-cadherin and vimentin in A549 and H1299 cells, treated as in (B), were assessed by immunoblotting. (H, I) The mRNA expression levels of CDH1 and VIM in A549 and H1299 cells, treated as in (B), were assessed by qRT-PCR.

Figure 8. Knockdown of SNAI1 make CAF failed to induce EMT of lung cancer cells

(A) CAFs were transfected with si-SNAI1 or negative control and then the expression of Snail1 was assessed by immunoblotting analysis. (B, C) CAFs were transfected with si-SNAI1 or with negative control and, after 48 h, co-cultured with A549 and H1299 cells for an additional 48 h. Lung cancer cells motility was measured by wound-healing assay. (D–F) Migration and invasion of A549 and H1299 cells, treated as in (B), was analyzed. (G) The levels of E-cadherin and vimentin in A549 and H1299 cells, treated as in (B), were assessed by immunoblotting. (H, I) The mRNA expression levels of CDH1 and VIM in A549 and H1299 cells, treated as in (B), were assessed by qRT-PCR.

Figure 9. Snail1-expressing CAFs induced tumor cell growth and EMT in vivo

(A–C) Tumor xenograft growth curves in mice (n = 6 per group). A549 cells were inoculated with negative control transfected CAFs (NC-CAFs) or Snail1- overexpessed CAFs into nude mice for indicated days. At the experimental end point, tumor xenografts were dissected and photographed. (D) Tumor xenografts were processed and stained with HE and immunohistochemically for vimentin, E-cadherin. (E, F) Levels of E-cadherin, vimentin mRNA and protein in tumor xenografts were detected by qRT-PCR and western blot analysis. (^*P < 0.05, ^**P < 0.01).