RON Mediates Tumor-Promoting Effects in Endometrial Adenocarcinoma

Abstract

Endometrial adenocarcinoma is one of the most prevalent female reproductive tract cancers in the world, and the development of effective treatment is still the main goal of its current research. Epithelial-mesenchymal transition (EMT) plays a significant part in the occurrence and development of epithelial carcinoma, including endometrial adenocarcinoma. Recepteur d'origine nantais (RON) induces EMT and promotes proliferation, migration, and invasion in various epithelial-derived cancers, but its role in endometrial adenocarcinoma is still poorly studied. The purpose of this study is to verify the overexpression of RON in endometrial adenocarcinoma and to explore its specific roles. RON expression in tumor lesions was verified by immunohistochemical staining, HEC-1B cells were used to construct stable cell lines with RON overexpression or knockdown to investigate the effects of RON on the function of endometrial adenocarcinoma cells, and xenotransplantation experiment was carried out in nude mice to explore the effect of RON on the growth of endometrial adenocarcinoma in vivo. This study revealed that RON could promote the proliferation, migration, and invasion of HEC-1B cells and induce EMT, and these effects were regulated through the Smad pathway. RON overexpression could promote growth of endometrial adenocarcinoma cells in nude mice, while its inhibitor BMS777607 could restrict this role. RON played an important role in endometrial adenocarcinoma and had a potential to become a new therapeutic target for endometrial adenocarcinoma.

Figure 1

RON was overexpressed in endometrial adenocarcinoma. (a) RON staining on control endometria. (b) RON staining on endometrial adenocarcinoma tissues. Scale bar is 100 μm. (c) Statistical results of immunohistochemical score of RON staining. The values represent the mean ± SD, ^∗∗∗P < 0.001. Adjacent noncancerous tissues (d) (scale bar = 100 μm) and adenocarcinoma endometria (f) (scale bar = 100 μm) from the same patient with endometrial adenocarcinoma ((e) scale bar = 400 μm).

Figure 2

RON promoted proliferation, migration, and invasion of HEC-1B cells. (a) RON expression in HEC-1B cells with RON overexpression (RON-OE). (b) RON expression in HEC-1B cells with RON knockdown (shRON). (c) CCK8 assays of RON-OE and shRON cell lines. #P < 0.05, RON-OE group versus NC group; $P < 0.05, RON-OE+ BMS 0.1 μM group versus RON-OE group; &P < 0.05, RON-OE+ BMS 0.5 μM group versus RON-OE group; §P < 0.05, §§P < 0.01, RON-OE+ BMS 1 μM group versus RON-OE group. (d) The transwell assays of RON overexpressing or knockdown cells. BMS means that the cells were exposed to BMS777607 (1 μM) for 24 h before transwell assay. The scale bars represent 100 μm. All of assays were repeated three times, and values represent the mean ± SD, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 3

RON induced EMT of HEC-1B cells. (a) The effect of RON overexpression or knockdown on morphological changes of HEC-1B cells. The red arrow marked the vacuoles in shRON cells. The scale bars represent 100 μm. (b) The effect of RON overexpression on the expression of EMT markers in HEC-1B cells. (c) The effect of RON knockdown on the expression of EMT markers in HEC-1B cells. Values represent the mean ± SD, ^∗P < 0.05, ^∗∗P < 0.01. All of assays were repeated three times.

Figure 4

RON might play its role in endometrial adenocarcinoma cells through the Smad pathway. (a) Expression of p-smad1/5/9 in RON-OE cells. (b) Expression of p-smad1/5/9 in shRON cells. Values represent the mean ± SD, ^∗P < 0.05. The assays were repeated three times.

Figure 5

RON promoted the growth of endometrial adenocarcinoma in nude mice xenografts. (a) The effect of RON overexpression on the growth of HEC-1B cells in vivo. #P < 0.05, RON-OE group versus NC group; &P < 0.05, RON-OE+ BMS 15 mg/kg group versus RON-OE group; §P < 0.05, RON-OE+ BMS 30 mg/kg group versus RON-OE group; ^P < 0.05, RON-OE+ BMS 15 mg/kg group versus NC group; $P < 0.05, RON-OE+ BMS 30 mg/kg group versus NC group. (b) The effect of RON knockdown on the growth of HEC-1B cells in vivo. n = 4; values represent the mean ± SD, ^∗P < 0.05, ^∗∗P < 0.01.