Cancer cell‐derived novel periostin isoform promotes invasion in head and neck squamous cell carcinoma

Figures

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  FIGURE 1. POSTN expression in the heterogeneity of cancer cells within the HNSCC tissues. The processed RNA‐seq data obtained from the GEO database (accession number: GSE103322) was used. 31 (A) UMAP plot of cancer cells from 10 patients (indicated by colors) reveals tumor‐specific clusters. Zoomed in UMAP plot of MEEI5/16 with MEEI5 and MEEI16, which can be seen to further divide into two subclusters. Violin plot depicts distributions of the POSTN expression in MEEI5/16 cluster. (B) Cells with POSTN expression are plotted in each cluster. (C) Cells from primary tumors and metastatic lymph nodes are colored black and red. Violin plot depicts distributions of the POSTN expression in the cells.

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  FIGURE 2. The schematic model of screening methods for POSTN isoforms. (A) POSTN expression was examined by RT‐PCR in HNSCC cell lines and normal fibroblast. GAPDH was used as a control. (B) Periostin protein structure and isoforms (Iso1‐11) are shown. Moreover, primers (F1, F2, R1, and R2) and the product size of each primer set F1/R1, F1/R2 and F2/R2 are shown. (C) The screening method for determination of POSTN isoforms using each primer sets.

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  FIGURE 3. Expression of POSTN isoforms in HNSCC cell lines. (A) POSTN isoforms were detected by RT‐PCR using primer set F1/R1 in MSCC‐Inv1, HOC719‐NE, and MRC5 cells. (B) POSTN isoforms were detected by RT‐PCR using primer set F1/R2 in MSCC‐Inv1, HOC719‐NE, and MRC5 cells. To distinguish Iso3 and Iso9, PCR products by using primer set F1/R2 from POSTN Iso3 and Iso9 constructs were used as a marker. (D) Summary of the expression of POSTN isoforms in MSCC‐Inv1, HOC719‐NE, and MRC5 cells.

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  FIGURE 4. The upregulation of POSTN expression and detection of isoforms in TGF‐β1 treated HNSCC cell lines. (A) HOC621 cells were treated with TGF‐β1, FGF2, PDGF, HGF, and EGF for 24 hours. Cells were then collected and analyzed POSTN expression by RT‐PCR. GAPDH was used as a control. (B) POSTN mRNA expression was determined by qRT‐PCR in HOC621 cells after TGF‐β1 treatment. In each group, data were presented as the mean ± SD of triplicates. *P < 0.05 (C) HNSCC cell lines (HOC621, HOC313, HOC719‐NE, HSC2, HSC3, and HSC4) were treated with 10 ng/mL of TGF‐β1 for 24 hours. Cells were then collected and analyzed POSTN expression by RT‐PCR. GAPDH was used as a control. (D) POSTN isoforms were detected by RT‐PCR using primer set F1/R1 or F2/R2 in TGF‐β1 treated HOC621 cells. (E) The invasion ability of HOC621 cells with or without TGF‐β1 treatment was examined by in vitro invasion assay. Representative images were shown. Graph shows the number of invaded cells as mean ± SEM (n = 3). *p < 0.05.

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  FIGURE 5. POSTN Iso5 cooperatively promotes invasion with Iso3 which is conventional isoform. (A) HOC621 cells were engineered to overexpressing GFP, POSTN Iso3 and/or Iso5 by transfection with lentiviral gene transfer. The overexpression of GFP was used as a control. Ectopic expression of POSTN Iso3 and Iso5 was quantified by qRT‐PCR. In each group, data are presented as the mean ± SD of triplicates. ****p < 0.001, ***p < 0.005, **p < 0.01. (B) Ectopic expression of POSTN Iso3 and Iso5 were detected by RT‐PCR using primer set F2/R2. GAPDH was used as a loading control. (C) Ectopic expression of Periostin Iso3 and Iso5 with monensin treatment was examined by immunoblotting with antibodies detecting N‐terminus and exon 21 of POSTN. anti‐Ex21 periostin antibody can detect POSTN Iso5 but not Iso3 lacking Exon 21. β‐Actin was used as a control. (D) The invasion ability of GFP‐, POSTN Iso3‐, Iso5, and Iso3/5‐overexpressing HOC621 cells were examined by in vitro invasion assay. Representative images were shown. Graph shows the number of invaded cells as mean ± SEM (n = 3). ****p < 0.001, ***p < 0.005, **p < 0.01.

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  FIGURE 6. Knockdown of POSTN Iso5 attenuates invasion ability in HNSCC cell line having EMT feature. (A) The structure of POSTN Iso3 and Iso5 are shown. Moreover, siRNA targeting POSTN Iso5 (siEx21) or total POSTN are shown. (B) POSTN mRNA expression in Iso5‐overexpressing HOC621 cells were determined by qRT‐PCR. In each group, data are presented as the mean ± SD of triplicates. ****p < 0.001 (C) POSTN mRNA expression in negative control siRNA (siNC), total POSTN siRNA (siPOSTN), and siRNA targeting POSTN Iso5 (siEx21)‐transfected HOC719‐NE cells were determined by qRT‐PCR. In each group, data are presented as the mean ± SD of triplicates. ****p < 0.001 (D) POSTN isoforms were detected by RT‐PCR using primer set F2/R2 in siNC, siPOSTN, and siEx21‐transfected HOC719‐NE cells. GAPDH was used as a control. (E) The invasion ability of siNC and siEx21‐transfected HOC719‐NE cells were determined by in vitro invasion assay. Representative images were shown. Graph shows the number of invaded cells as mean ± SEM (n = 3). ****p < 0.001.

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  FIGURE 7. Correlation between POSTN and p‐EMT. (A) Heatmap shows top20 genes (columns) that are differentially expressed across POSTN expressing cells (ON) and POSTN nonexpressing cells (OFF) in MEEI16 tumor. (B) Boxplot shows p‐EMT score and TGFBI expression values in ON and OFF cells. (C) Scatter plot of p‐EMT score and POSTN expression value (upper), TGFBI expression value and POSTN expression value (lower) for ON cells in all tumors were shown. Each cell was colored by the reported TCGA molecular subtype.

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  FIGURE 8. Induction of p‐EMT genes by POSTN Iso3 and/or Iso5. (A) HOC621 cells were engineered to overexpressing POSTN Iso3 and/or Iso5 by transfection with lentiviral vector. The overexpression of GFP was used as a control. Expression of p‐EMT genes was evaluated by qPCR in EV‐, POSTN Iso3‐, Iso5, and Iso3/5‐transfected HOC621 cells. The graphs show the results as mean ± S.D. of three independent experiments. *p < 0.05 (compared to EV). (B) Expression of p‐EMT genes was evaluated by qPCR in negative control siRNA (siNC), siRNA targeting POSTN Iso3 and 5 (siPOSTN), and siRNA targeting POSTN Iso5 (siEx21)‐transfected HOC719‐NE cells with both POSTN Iso3 and Iso5 expression. The graphs show the results as mean ± S.D. of three independent experiments. *p < 0.05 (compared to siNC).