MicroRNAs Associated with Epithelial-Mesenchymal Transition Can Be Targeted to Inhibit Peritoneal Dissemination of Human Scirrhous Gastric Cancers

Abstract

Objectives: Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. Methods: We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. Results: miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. Conclusions: EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers.

Long Noncoding RNA HOTAIR Promotes Epithelial-Mesenchymal Transition and Is a Suitable Target to Inhibit Peritoneal Dissemination in Human Scirrhous Gastric Cancers

“…We used these 2 cell lines for our experiments in the present study: HSC-60 is a parental cell established from a patient, and 60As6 is a metastatic cell isolated from the parental cells through repeated steps of orthotopic inoculation in nude mice, gathering ascites fluids from the mouse, and then re-inoculation on the stomach wall into the other new nude mice [19]. To maintain the cells, we used RPMI 1640 medium with 10% fetal bovine serum as described previously [19][20][21][22][23][24]. For control and confirming experiments, we also used our original cell lines (a parental line, HSC-44PE, and a metastatic line, 44As3) [20,21,24] established from the other patient with human scirrhous gastric cancer.…”

“…We isolated total RNA from the cells and tumor tissues of patients using TRIzol ® (Invitrogen) as reported previously [22][23][24][25][26][27]. The total RNA quality was analyzed by using an Agilent 2100 Bioanalyzer (Agilent Technologies) as reported previously [20][21][22][23][24][25][26][27]. The concentration of each total RNA was determined by using a NanoDrop ND-1000 spectrophotometer (Invitrogen).…”

“…The expression of the micro-RNAs (miRNAs) was measured by TaqMan micro-RNA assays as reported previously [22][23][24]. Each total RNA (5-10 ng) was reverse-transcribed with a TaqMan micro-RNA RT kit and TaqMan micro-RNA assay (Applied Biosystems), and the cDNA was analyzed using a specific TaqMan probe (miR-217) and TaqMan Universal PCR Master Mix as described previously [22][23][24]. The expression level of miRNAs was normalized with small nuclear RNA (RNU6B), and the expression level of HOTAIR was also normalized with glyceraldehyde-3-phosphate dehydrogenase.…”

“…Cells of the metastatic line 60As6 were transfected with miR-217 expression vector (1 µg; Takara Bio, Japan) using a Lipofectamine ® 2000 reagent (Invitrogen) as reported previously [22,24]. The transfected cells were selected with geneticin to obtain a stable overexpressing clone (60As6-miR-217).…”

“…The various cell lines were subjected to invasion assay. For this purpose, we used a BD BioCoat Fluoroblok Tumor Invasion System (BD Biosciences) as reported previously [22][23][24].…”