Effects of TRPC1 on epithelial mesenchymal transition in human airway in chronic obstructive pulmonary disease

Abstract

Background: We investigated the effects of TRPC1 on epithelial mesenchymal transition (EMT) in human airway in chronic obstructive pulmonary disease (COPD).

Methods: A total of 94 patients who underwent lobectomy were selected and divided into COPD (49 cases) and control (45 cases) groups. Immunohistochemistry was applied to detect expression of E-cadherin and vimentin and TRPC1. Correlation of TRPC1 expression with E-cadherin and vimentin expression, and correlations of lung function indicators in COPD patients with expression of TRPC1, E-cadherin, and vimentin were analyzed. Human airway epithelial cells (16HBE) were used for cell experiments; and cigarette smoking extract (CSE) was adopted to establish the COPD model using TRPC1 recombinant plasmids and siRNA. Cells were assigned into the control, CSE, CSE + vector, CSE + TRPC1, CSE + si-NC, and CSE + si-TRPC1 groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were implemented to detect expression of TRPC1, E-cadherin, and vimentin.

Results: Compared with the control group, expression of TRPC1 and vimentin significantly increased while expression of E-cadherin decreased in the COPD group, and protein expression of TRPC1 was positively correlated with the protein expression of vimentin but negatively correlated with the protein expression of E-cadherin. Patients exhibiting positive expression of TRPC1 had lower FEV1, FEV1%Pred, and FEV1/FVC, compared with the patients exhibiting negative expression of TRPC1. Compared with the control group, expression of TRPC1 and vimentin increased, whereas expression of E-cadherin decreased in the CSE, CSE + vector, CSE + TRPC1, and CSE + si-NC groups. Compared with the CSE and CSE + vector groups, the expression of TRPC1 and vimentin increased but the expression of E-cadherin decreased in the CSE + TRPC1 group. Compared with the CSE and CSE + si-NC groups, the expression of TRPC1 and vimentin decreased but the expression of E-cadherin increased in the CSE + si-TRPC1 group. No significant differences were observed among the CSE, CSE + vector and CSE + si-NC groups.

Conclusion: Overexpression of TRPC1 in COPD promoted EMT process and TRPC1 may be a new and interesting focus for COPD new treatment in the future.

Figure 1

Correlation as well as mRNA and protein expression of TRPC1, vimentin, and E-cadherin. Note: (A) immunohistochemistry images of expression of TRPC1, vimentin and E-cadherin (×400); (B) mRNA expression of TRPC1, vimentin, and E-cadherin detected using RT-PCR. COPD = chronic obstructive pulmonary disease, EMT = epithelial mesenchymal transition, RT-PCR = reverse transcription-polymerase chain reaction, TRPC1 = transient receptor potential canonical 1.

Figure 2

Effect of CSE on 16HBE cell morphology, E-cadherin and vimentin. Note: (A) effect of CSE on 16HBE cell morphology observed under an inverted microscope at different time periods (observed at a magnification of 200×); (B) effect of CSE on E-cadherin and vimentin in 16HBE cells detected by Western blot assay at different time periods, compared with the 0 h, P < .05, compared with the 24 h time period, P < 0.05, compared with the 48 h time period, P < .05. CSE = cigarette smoking extract, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, TRPC1 = transient receptor potential canonical 1.

Figure 3

Cell morphology after transfection and expression of TRPC1. Note: (A) cell morphology after transfection observed under an inverted microscope (observed at a magnification of 200×); (B) protein expression in cells determined by Western blot assay; (C) histogram of protein expression levels for each group after transfection; (D) histogram of mRNA expression levels for each group after transfection. ∗, compared with the control group, P < .05; ^#, compared with the CSE, CSE + vector, and CSE + si-NC groups, P < .05. CSE = cigarette smoking extract, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, TRPC1 = transient receptor potential canonical 1.

Figure 4

Protein expression of E-cadherin and vimentin in each group detected by Western blot assay and RT-PCR posttransfection. Note: (A) protein expression of E-cadherin and vimentin after transfection; (B) histogram of protein expression of E-cadherin and vimentin after transfection; (C) histogram of mRNA expression of E-cadherin and vimentin determined by RT-PCR after transfection. ^∗, compared with the control group, P < .05; ^#, compared with the CSE + vector and CSE + si-NC groups, P < .05. CSE = cigarette smoking extract, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, RT-PCR = reverse transcription-polymerase chain reaction, TRPC1 = transient receptor potential canonical 1.