Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor.
Methods: MiR-29b mimics and negative control miRNA were transfected by lipofection method. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC-4, were examined with MTT assay and transwell assay, respectively. MMT of human peritoneal mesothelial cells (HPMCs) was induced 10 ng/ml of TGF-β. Proliferation and migration of HPMCs were examined with the same methods, and MMT was evaluated with immunofluorescence staining for epithelial, mesothelial and mesenchymal markers. To evaluate the effect of miR-29b in vivo, we developed a highly metastatic subclone YTN16P2 from a murine gastric cancer cell line, YTN16, with in vivo selection, and inoculated the YTN16P2 in peritoneal cavity of syngenic C57BL/6 mice. MiR-29b mimics or negative control miRNA with atelocollagen were intraperitoneally (ip) injected every 3 days from tumor implantation. The mice were sacrificed at 14 days after tumor implantation, and formation of peritoneal metastasis were evaluated.
Results: Transfection of miR-29b mimics significantly decreased the proliferation of MKN45 and NUGC-4 by 20-60% (p<0.01) as compared with negative control miRNA. Migration was inhibited more strongly by 90% (p<0.001). After 48 hr-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b mimics significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMCs. MiR-29b also decreased the proliferation and migration of HPMCs by 20% (p<0.05) and 90% (p<0.001) as compared with negative control miRNA. Finally, mice treated with miR-29b developed less PM nodules than those treaded with negative control miRNA and only atelocollagen.
Conclusion: MiR-29b efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell.
Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Yuko Kumagai, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MiR29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2380.