Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer

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  Figure 1. Periostin expression level and its prognostic value in lung cancer specimens (A) Periostin gene expression profiles in lung cancer specimens were obtained from the Oncomine database. Datasets in a single panel were from the same study. (B) Expression levels of periostin in paired adjacent normal and lung tumor tissues retrieved from TCGA dataset. (C and D) Correlation between periostin, Snail and Twist expression levels in lung cancer specimens retrieved from the TCGA dataset. (E) Kaplan-Meier analysis of overall survival, subgrouped by expression levels of periostin. (F) Lung cancer and normal tissue specimens were analyzed by IHC staining using periostin, Snail and Twist antibodies. The stained specimens were photographed by optical microscope and scored from 1-5 for expression levels. (G) Correlation between periostin, Snail and Twist expression levels in lung cancer specimens. *p < 0.05 compared with normal tissue specimens.

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  Figure 2. Periostin induces EMT in lung cancer cells (A) A549 cells were incubated with periostin (0-100 ng/ml) for 24 h. Western Blot analysis examined E-cadherin, N-cadherin, vimentin, Snail and Twist expression. Actin was used as an internal control. (B and C) A549 and CL1-0 cells were treated as described in (A), and E-cadherin, N-cadherin, vimentin, Snail and Twist expression was examined by qRT-PCR. (D) A549 and CL1-0 cells were incubated with periostin (0-100 ng/ml) for 24 h, and the scattering morphology was photographed. (E and F) A549 and CL1-0 cells were incubated with periostin (0-100 ng/ml) for 24 h, and in vitro migration was measured using the wound healing assay. (G and H) A549 and CL1-0 cells were treated with periostin (0-100 ng/ml) for 24 h, after which the Transwell assay was used to measure in vitro migration and the Matrigel invasion assay measured cell invasion. (I) A549 cells were transfected with Twist or Snail siRNA for 24 h, then incubated with periostin (100 ng/ml) for 24 h. In vitro migration was measured using the Transwell assay; cellular invasion was measured by the Matrigel invasion assay. Results are expressed as the mean ± S.E.M. *p < 0.05 compared with…

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  Figure 3. ERK and p38 signaling pathways are involved in periostin-promoted EMT in lung cancer cells (A) A549 cells were incubated with periostin (100 ng/ml) for the indicated times, and phosphorylation of EKR, p38 and JNK was determined by Western Blot analysis. (B and C) A549 and CL1-0 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min and then incubated with periostin (100 ng/ml) for 24 h, and expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (D) A549 cells were treated as described in (B), and scattering morphology was photographed. (E-H) A549 and CL1-0 cells were treated as described in (B), and the wound healing assay, migration and invasion assays were assessed. (I and J) A549 and CL1-0 cells were transfected with ERK, p38 and JNK siRNA for 24 h, and incubated with periostin (100 ng/ml) for 24 h, while expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (K-O) A549 and CL1-0 cells were treated as described in (I), and scattering morphology, the wound healing assay, migration and invasion assays were assessed. Results are expressed as the mean ± S.E.M. *p…

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  Figure 4. Periostin induces Twist and Snail expression by miR-381 repression in lung cancer cells (A) Schematic selection of candidate miRNAs. miR-381, which targets Twist and Snail 3’ UTR regions, was selected by using 3 online computational algorithms TargetScan, miRWalk and miRanda. (B) A549 cells were incubated with periostin (0-100 ng/ml) for 24 h. Total RNA was extracted and miR-381 expression was assessed by qRT-PCR. (C-D) A549 cells were transfected with control microRNA or miR-381 mimic for 24 h, then incubated with periostin for 24 h. Total RNA or protein was extracted, and Twist and Snail expression was assessed by qRT-PCR and Western Blot analysis. (E-G) A549 cells were transfected with miR-381 mimic or control mimic for 24 h, and incubated with periostin (100 ng/ml) for 24 h, and assessed by the wound healing assay, migration and invasion assays. (H and I) A549 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min or transfected with ERK, p38 and JNK siRNA for 24 h, then incubated with periostin (100 ng/ml) for 24 h. miR-381 expression was assessed by qRT-PCR. Results are expressed as the mean ± S.E.M. *p < 0.05 compared with control. #p <…

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  Figure 5. Knockdown of periostin expression represses EMT in vitro (A) Total protein was extracted from A549 cells stably expressing Neg shRNA and periostin shRNA. Expression levels of periostin were determined. (B) Cell proliferation was assessed by the MTT assay. (C and D) Protein and mRNA expression levels of E-cadherin, N-cadherin, vimentin, Twist and Snail were assessed by Western Blot and qRT-PCR. Actin was used as an internal control. (E-G) The wound healing and cell migration assays were performed in Neg shRNA and periostin shRNA cells. (H) miR-381 expression was assessed in Neg shRNA and periostin shRNA cells by qRT-PCR. Results are expressed as the mean ± S.E.M. *p < 0.05 compared with the Neg shRNA group.

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  Figure 6. Knockdown of periostin expression represses tumor growth in vivo (A) Male nu/nu mice (6 weeks of age; N = 5 in each group) were intravenously injected with 5 × 105 cells suspended in 50 μL PBS. Tumor growth was monitored using the IVIS protocol. The panels depict quantification of fluorescence imaging data obtained on days 1 and 42. (B) The mice were sacrificed on day 42. Lung sections were stained with H&E to monitor tumor growth. (C) The number of lung tumor nodules was counted under a dissecting microscope. (D) Lung specimens from sacrificed mice stained with periostin, Snail and Twist antibody. Stained specimens were photographed using an optical microscope (left panel). The staining intensity of periostin, Snail and Twist were scored from 1-5 to quantify the expression levels in lung specimens (right panel). Results are expressed as the mean ± S.E.M. *p < 0.05 compared with the Neg shRNA group.