MiR-185-3p regulates epithelial mesenchymal transition via PI3K/Akt signaling pathway by targeting cathepsin D in gastric cancer cells

Abstract

Background: Recently research reported that miR-185-3p could serve as an independent prognosis factor in gastric cancer (GC). However, the functional role and underlying mechanism of miR-185-3p in GC and epithelial-mesenchymal transition (EMT) progression remains largely elusive.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to analyze the expression of miR-185-3p and cathepsin D in patient-derived GC samples and various GC cell lines. Scratch assay and Transwell assay were used to evaluate the migration ability. The influence of miR-185-3p on the cell cycle distribution and cell apoptosis was evaluated using flow cytometry. Western blotting assay was performed to detect the expression of EMT associated proteins and the activity of PI3K/Akt signaling pathway. Furthermore, the interaction between miR-185-3p and cathepsin D was explored by dual-luciferase reporter assay.

Results: Our data revealed that miR-185-3p was down-regulated, while cathepsin D was up-regulated in both patient-derived GC samples and GC cells. Apart from inducing apoptosis, overexpression of miR-185-3p also inhibited EMT process and migration of GC cells. Mechanically, we firstly verified that miR-185-3p directly targeted the cathepsin D. Furthermore, miR-185-3p exerted its function on EMT process and migration via inhibiting cathepsin D to mediated PI3K/Akt signaling pathway.

Conclusions: Our findings suggested that miR-185-3p targeted cathepsin D inhibiting EMT process via PI3K/Akt signaling, which may serve as a potential prognosis factor and therapeutic target to reduce the malignancy of GCs.

Keywords: MiR-185-3p; PI3K/Akt signaling pathway; epithelial-mesenchymal transition (EMT); gastric cancer (GC).

Figure 1

Expression level of miR-185-3p and cathepsin D in GC cells. (A) qRT-PCR analysis of miR-185-3p and cathepsin D in patient-derived tumor tissues and paired adjacent normal tissues. Expression level of miR-185-3p was decreased, while the expression of cathepsin D was elevated in tumor tissue compared with normal tissue. (B) qRT-PCR analysis of miR-185-3p in various GC cell lines. Expression level of miR-185-3p in GC cells was noticeably lower than that in gastric epithelial cells, where AGS cells showed the lowest level of miR-185-3p among all tested cells. (C) qRT-PCR analysis of cathepsin D in various GC cell lines. Expression level of cathepsin D in GC cells was higher than that in gastric epithelial cells, where AGS and SGC7910 cells showed the two highest level of cathepsin D among all tested cells. (D) Western blotting analysis of cathepsin D in GC cells. The quantification of cathepsin D expression level was normalized against GAPDH. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 2

MiR-185-3p could inhibit EMT in GC cells. (A) qRT-PCR analysis of miR-185-3p in AGS and SGC7901 cells after transfection with miR-185-3p mimics, NC mimics, miR-185-3p inhibitor, or NC inhibitor. Transfection with miR-185-3p mimics successfully boosted the expression of miR-185-3p, while transfection with miR-185-3p inhibitor reduced the expression of miR-185-3p in the investigated cells. (B) qRT-PCR analysis of EMT markers in AGS and SGC7901 cells after transfection. In both cell lines, transfection with miR-185-3p mimics could increase the expression level of epithelial marker E-cadherin while reducing the expression level of mesenchymal markers vimentin and N-cadherin. (C) Western blotting analysis of EMT markers in AGS and SGC7901 cells after transfection. The results showed the same trend as the results of qRT-PCR analysis. The quantification of EMT markers was normalized against GAPDH. (D) Cell scratch assay of AGS cells after transfection. Left, images of the migration of cells after incubation for 24 h; right, quantification of the cell migration. Cells transfected with miR-185-3p mimics showed the least migration. (E) Transwell assay of AGS cells after transfection. Left, images of migration cells in the lower chamber; right, cell counts of the migration cells in the lower chamber. Cells transfected with miR-185-3p mimics was least migration. (F) Cell cycle distribution of AGS cells after transfection. Overexpressed miR-185-3p blocked more cells in G0/G1 phase. (G) Cell apoptosis study of AGS cell after transfection. Overexpression of miR-185-5-3p contributed an augment in the cell apoptosis. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3

MiR-185-3p could targeting inhibit cathepsin D in GC cells. (A) Prediction of the targeting relationship between miR-185-3p and cathepsin D. (B) Dual-luciferase reporter analysis in AGS cells after transfection. The signal intensity of luciferase was lowest in WT cells transfected with miR-185-3p mimics. (C) qRT-PCR analysis of cathepsin D in AGS cells after transfection. Cells transfected with miR-185-3p mimics showed the lowest level of cathepsin D. (D) Western blotting analysis of cathepsin D in AGS after transfection. Cells transfected with miR-185-3p mimics showed the lowest level of cathepsin D. The quantification of cathepsin D was normalized against GAPDH. *, P<0.05; **, P<0.01.

Figure 4

MiR-185-3p inhibited the activity of PI3K/Akt signaling pathway by inhibiting cathepsin D. (A) qRT-PCR analysis of cathepsin D level after transfection with p-cathepsin D or p-NC. (B) Western blotting analysis of PI3K/Akt related proteins. Transfection with p-cathepsin D could increase the level of p-PI3K and p-Akt (Ser 473), which was compromised by either transfection with miR-185-3p mimics or the administration of LY294002. The quantification of investigated proteins was normalized against GAPDH. *, P<0.05; **, P<0.01.

Figure 5

MiR-185-3p regulated EMT via cathepsin D mediated PI3K/Akt signaling pathway. (A) qRT-PCR analysis of EMT markers in AGS and SGC7901 cells after transfection with miR-185-3p mimics, p-cathepsin D, miR-185-3p mimics and p-cathepsin D, as well as p-cathepsin D followed by administration of LY294002. (B) Western blotting of EMT markers in AGS and SGC7901 cells after transfection. The quantification of EMT markers was normalized against GAPDH. (C) Cell scratch assay of AGS cells after transfection. Left, images of the migration of cells after incubation for 24 h; right, quantification of the cell migration. (D) Transwell assay of AGS cells after transfection. Left, images of migration cells in the lower chamber; right, cell counts of the migration cells in the lower chamber. *, P<0.05; **, P<0.01.