Effects of microRNA-29b on invasion and proliferation of human high peritoneal metastatic gastric cancer cells

Abstract

Objective To investigate the expression of microRNA-29b (miR-29b) in gastric cancer cell line GC9811 and high peritoneal metastatic gastric cancer cell line GC9811-P and its effect on invasion, proliferation and apoptosis. Methods The relative quantitative expression of miR-29b was detected by quantitative realtime polymerase chain reaction(qRT-PCR). GC9811 cells were divided into three groups, miRNA down-regulated and transfected with lentiviruses LV-miR-29b inhibitor group, negative control group with negative transfection, and untransfected blank control group. GC9811-P cells were divided into three groups, miR-29b up-regulated and transfected with lentiviruses LV-miR-29b group, negative control with negative transfection group, and untransfected blank control group. The cell invasion ability was detected with Transwell assay, the cell proliferation ability was measured by methyl-thiazolyl tetrazolium (MTT) test, the colony-forming ability was determined by plate colony formation assay, and the apoptosis was tested by flow cytometry. The expressions of miR-29b and secreted protein, acidic and rich in cystenie (SPARC) in gastric cell line GC9811-P, GC9811, MKN28M, MKN28NM and normal gastric cell line GES were determined by qRT-PCR, and the correlation was analyzed. Two independent samples t test or SNK-q test was performed for mean comparison between two groups, and one-way analysis of variance was used for mean comparison among three groups. Results The relative quantitative expression of miR-29b in GC9811-P (0.21±0.04) was significantly lower than that of GC9811 (1.00±0.03, t=28.140, P<0.01). After GC9811 cells transfected with lentiviruses LV-miR-29b inhibitor, the expression of miR-29b (0.21±0.04) was significantly lower than that of control group (0.89±0.07) and blank control group (1.00±0.04, q=12.76, 14.73, both P<0.01). Compared with negative group, the transmembrane cell number and the clonality of miRNA up-regulated group raised(274.33±9.03 vs 110.67±13.69, t=9.981, P<0.01; 131.33±4.91 vs 69.67±2.33, t=11.340, P<0.01), and the results of MTT test also shows the proliferation was increased. After GC9811-P cells transfected with LV-miR-29b, the expression of miR-29b (4.08±0.20) was significantly higher than that of negative control group (1.15±0.05) and blank control group (1.00±0.10, q=21.73, 22.81, both P<0.01). Compared with negative group, the transmembrane cell number and the clonality of miRNA up-regulated group reduced (51.33±5.55 vs 104.00±6.24, t=6.305, P<0.01; 48.00±5.51 vs 113.33±5.17, t=11.340, P<0.01), and the results of MTT test also shows the proliferation was weakened. Apoptosis assays demonstrated miR-29b promoted apoptosis; however, the difference was not statistically significant. The expression of miR-29b was negatively correlated with SPARC mRNA in gastric cancer cells (r=-0.97, P=0.03). Conclusions The low expression of miR-29b in high peritoneal metastatic gastric cancer cell inhibited the ability of invasion and proliferation. MiR-29b might be a new target of inhibiting peritoneal metastasis in gastric cancer. Key words: Stomach neoplasms; Peritoneal metastasis; Cell invasion; Cell proliferation; miRNA-29b