Abstract
Peritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-β1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-β1. TGF-β1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-β1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. MiR-29b inhibits TGF-β1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.
Highlights
Human peritoneal mesothelial cells (HPMCs) were isolated from omental tissue obtained from patients who underwent sleeve gastrectomy and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (FBS)
Digital PCR analysis revealed that HPMC produced substantial levels of miR-29b, which were more than those from gastric cancer cells, MKN45 and NUGC as well as peripheral blood mononuclear cells (PBMC) and mesenchymal stem cells (MSC) (Fig. 1A)
We found 50 nM miRs are enough for the transfection to more than 90% of the HPMC and expression levels of miR-29b was significantly upregulated with digital PCR system (Supplementary Fig. 1)
Summary
The cells were transfected with miR-29b-3p mimic or negative controls with lipofectamine RNAiMAX in HPMC at a final concentration of 50 nM, and cultured for 48 h at 37 °C, according to the manufacturer’s instructions and used for the following experiments. HPMC (5 × 104) were plated in 24-well collagen coated plates, incubated with 10 ng/ ml of TGF-β1 and transfected miR-29b or negative controls for 48 h. Cells were incubated for 1 h at room temperature with mAbs to E-cadherin (1:200), calretinin (1:500), vimentin (1:1000), and fibronectin (FN) (1:150). Cells were washed 3 times with PBS and incubated for 30 min at room temperature with the appropriate fluorescence conjugated secondary anti-rabbit antibodies conjugated with AlexaFluor 488 or AlexaFluor 595® (1:2000). Glass coverslips were placed on slides and the preparations were visualized under a fluorescence microscope BZ-X710 (Keyense, Osaka, JAPAN) and the figures were generated using BZ-H3A software (Keyense, Osaka, JAPAN) (https://www.keyence.com/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3ae/)
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