Periostin regulates autophagy through integrin α5β1 or α6β4 and an AKT‐dependent pathway in colorectal cancer cell migration

Figures

• 

  Figure 1. Immunohistochemical staining of PN in CRC tissues. A, Representative pictures of high and low levels of PN at different CRC stages: (i and ii) stage IV, (iii and iv) stage III, (v and vi) stage II and (vii and viii) stage I; and (ix) shows normal colorectal tissue. Original magnification of 200x. Kaplan‐Meier log rank for survival analysis using (B) 3‐years and (C) 5‐years as a cut‐off value to be evaluated

• 

  Figure 2. PN induces CRC cell growth and invasion in 3D‐spheroid assay. A‐D, Light microscopy images of CRC spheroid growth in treatment of rPN (100 ng/mL) at day 4, 7, 10, 13 and 15 compared with CRC cells in 10% FBS or 1% FBS media. Spheroid sizes at each time point were calculated and are plotted in volumes (µm3). Statistical significance is based on the results of Student's t test. * P‐value < .05; ** P‐value < .01. E, HCT‐116 and (F) HT‐29 spheroid cells moved into Matrigel was observed in rPN treatment. Scale bar = 200 µM; Original magnification of 200x

• 

  Figure 3. PN promotes CRC cell migration through ITGα5 and ITGβ4. A‐B, Migration ability of HCT‐116 cells transfected with or without siITGα5 and HT‐29 cells transfected with or without siITGβ4 under rPN treatment were identified by wound‐healing migration assays. C‐D, Percentages of migrated cells are at each time point after PN treatment in CRC cells treated with or without siITGα5 or siITGβ4. Bar graph represents mean ± SD; *P‐value < .05; **P‐value < .01 compared between conditioning of treatments. E‐F, Representatives of the expression of LC3 and PN at the migration front in the wound area. Scale bar = 20 µM; Original magnification of 63x

• 

  Figure 4. PN promotes CRC cell migration through reduction of autophagy. Expressions of proteins that involved autophagy and EMT pathways were demonstrated by Western blotting and immunofluorescence. A and C, Western blot analyses show the levels of expressions of ITGα5 (A), ITGβ4 (C), pAKT, pERK, LC3, E‐cad, α‐SMA and VIM in HCT‐116 (A) and HT‐29 (C) cells upon incubation for 24 h in rPN. Densitometry of the bands is reported as the ratios of each protein versus β‐actin internal control. Bar graphs represent mean ± SD of three independent experiments. B and D, the marker of autophagy (LC3, green colour) and ITGα5 or ITGβ4 (red colour) were assessed by immunofluorescence in HCT‐116 or HT‐29. The nuclei were counterstained with DAPI. Scale bar = 20 µM; Original magnification of 63x. *P‐value < .05

• 

  Figure 5. PN and autophagy in CRC cell lines. (A‐C) CRC cells, HCT‐116 and HT‐29 were treated with 100 ng/ml of PN in 1% FBS medium, 30 µM of CLQ was added at 8 h before harvest, and then subjected for IF staining of PN (orange colour) and LC3 (green colour). Bar graph represents mean ± SD; *P‐value < .05; **P‐value < .01. D, E, Co‐localization of PN and LC3 in tumour clinical sections of CRC patients. Scale bar = 20 µM; magnification = 63×. F Schematic diagram illustrates the potential of PN secreted from cancer‐associated fibroblasts (CAFs) to reduce autophagy in CRC‐mediated migration through ITGα5β1 or ITGα6β4 and AKT‐dependent pathways