Effect of the miR-96-5p inhibitor and mimic on the migration and invasion of the SW480-7 colorectal cancer cell line

Abstract

The objectives of the present study were to identify the aberrant expression of microRNA (miRNA) in colorectal carcinoma (CRC) tissues from published miRNA profiling studies and to investigate the effects of the identified miRNA inhibitor and mimic miR-96-5p on CRC cell migration and invasion. The altered expression of the regulators of cytoskeleton mRNA in miR-96-5p inhibitor-transfected cells was determined. The miR-96-5p expression level in five CRC cell lines, HCT11, CaCo2, HT29, SW480 and SW620, and 26 archived paraffin-embedded CRC tissues were also investigated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). Cell viability in response to the miR-96-5p inhibitor and mimic transfections was determined by an MTT assay. A Matrigel invasion assay was conducted to select the invasive subpopulation designated SW480-7, by using the parental cell line SW480. The effects of miR-96-5p mimic- or inhibitor-transfected SW480-7 cells on cell migration and invasion were evaluated using the Transwell and Matrigel assays, and the change in expression of the regulators of cytoskeleton mRNAs was identified by Cytoskeleton Regulators RT²-Profiler PCR array followed by validation with RT-qPCR. CRC tissues exhibited a significant increase in miR-96-5p expression, compared with their matched normal adjacent tissues, indicating an oncogenic role for miR-96-5p. The results demonstrated that the miR-96-5p inhibitor decreased the migration of SW480-7 cells, but had no effect on invasion. This may be due to the promotion of cell invasion by Matrigel, which counteracts the blockade of cell invasion by the miR-96-5p inhibitor. The miR-96-5p mimic enhanced SW480-7 cell migration and invasion, as expected. It was determined that there was a >2.5 fold increase in the expression of genes involved in cytoskeleton regulation, myosin light chain kinase 2, pleckstrin homology like domain family B member 2, cyclin A1, IQ motif containing GTPase activating protein 2, Brain-specific angiogenesisinhibitor 1-associated protein 2 and microtubule-actin crosslinking factor 1, in miR-96-5p inhibitor-transfected cells, indicating that they are negative regulators of cell migration. In conclusion, the miR-96-5p inhibitor blocked cell migration but not invasion, and the latter may be due to the counteraction of Matrigel, which has been demonstrated to stimulate cell invasion.

Keywords: Matrigel; colorectal cancer; cytoskeleton; invasion; microRNA-96-5p; migration.

Figure 1.

(A) The relative expression level of miR-96-5p in five CRC cell lines. Data represent the level of miRNA expression relative to that of reference gene, U6. (B) Quantitative analysis of miR-96-5p by reverse transcription-quantitative polymerase chain reaction in CRC tumor tissues and the corresponding normal adjacent tissues. The miRNA level was expressed as log(tumor/relative adjacent tissue). Each bar represents an individual patient. CRC, colorectal cancer; miR, microRNA.

Figure 2.

Effects of miRNA-96-5p inhibitor on cell viability of (A) SW480, (B) SW620 and (C) HCT116. The values of cell viability were normalized with that of the corresponding negative control group. Untreated, only medium treated cells; LF, only Lipofectamine 2000 with medium treated cells; HiPerFect, only HiPerFect with medium treated cells; negative control, transfection with miR inhibitor control and cultured with medium; miR-96-5p, transfection with miR-96 inhibitor and cultured with medium. Data represent the mean ± SD of three independent experiments, each performed in triplicate.

Figure 3.

The invasive capabilities of the three colorectal cancer cell lines and in vitro selection of invasive subpopulation from SW480 cells by a Matrigel assay. The two images in each row are the photos of upper (blue) and lower (red) membrane surfaces captured from the same field which was observed with Olympus BX51 fluorescence microscope (Olympus Corp.) in a zig-zag pattern under ×200 magnification. (A) The invasive capabilities of HCT116, SW480 and SW620. (B) An invasive subpopulation, SW480-7, was selected from SW480 cells. (C) The expression of miR-96-5p in SW480 and SW480-7 cells. Data represent the level of miRNA expression relative to that of reference gene, U6. The means ± standard deviations were calculated from the data of three independent experiments. PI, propidium iodide; miRNA, microRNA.

Figure 4.

Transwell assay of SW480-7 colorectal cancer cells transfected with miR negative control, miR-96-5p mimic and inhibitor. The blue cells in upper membrane were stained by DAPI, and cells in the lower membrane were stained by PI. The cells were counted under a light microscope (×200 magnification) in five randomly selected fields. Bars represent the number of invaded cells. (A) The effect of miR-96-5p inhibitor on cell migration. (B) The effect of miR-96-5p inhibitor on cell invasion. (C) The effect of miR-96-5p mimic on cell migration. (D) The effect of miR-96-5p mimic on cell invasion. ^##P<0.01, ^#P<0.05 vs. negative control. PI, propidium iodide; miR, microRNA.

Figure 5.

The mRNA expression of selected mRNAs (MYLK2, PHLDB2, CCNA1, IQGAP2, BAIAP2 and MACF1) in miR-96-5p inhibitor/mimic transfected SW480-7 cells by reverse transcription-polymerase chain reaction. The relative mRNA expression in SW480-7 cells transfected with (A) miR-96-5p inhibitor and (B) miR-96-5p mimic. The ratio of mRNA expression in the inhibitor/mimic-transfected groups relative to the corresponding negative control was calculated. Data are presented as the mean ± standard deviation from three independent experiments. Statistical analysis was conducted using one-sample Student's t-test. P-values are as shown in the bar chart. MYLK2, myosin light chain kinase 2; PHLDB2, pleckstrin homology like domain family B member 2; CCNA1, cyclin A1; IQGAP2, IQ motif containing GTPase activating protein 2; BAIAP2, Brain-specific angiogenesis inhibitor 1-associated protein 2; MACF1, microtubule-actin crosslinking factor 1; miR, microRNA.

Figure 6.

Hypothetical model showing downregulation of the cytoskeleton genes and promotion of migration by miR 96 5p in CRC.