MicroRNA‑144 inhibits migration and proliferation in rectal cancer by downregulating ROCK‑1

Abstract

Cancer of the colon and rectum are two distinct entities, which require different treatment strategies and separate treatment. MicroRNAs (miRNAs) act as critical regulators of genes involved in several biological processes. Aberrant alterations of miRNAs have been found in several types of cancer, including colon cancer and rectal cancer. Extensive catalogues of downregulated miRNAs have been identified for colon cancer, whereas only limited data are available for rectal cancer. An example of miRNA profiling in a previous study found that miRNA (miR)‑144 showed aberrant expression and appeared to be rectal cancer‑specific, its expression not being reported in colon cancer. In the present study, the role of miR‑144 in rectal cancer was investigated. SW837 and SW1463 cell lines were selected as rectal cell carcinoma cells. Using reverse transcription-quantitative polymerase chain reaction, western blot, BrdU, cell migration and cell viability assays, it was found that the expression levels of miR‑144 were significantly reduced in the SW837 and SW1463 cell lines, and the overexpression of miR‑144 suppressed rectal cancer cell viability, migration and proliferation. In addition, Rho‑associated coiled‑coil containing protein kinase 1 (ROCK1) was identified as a target of miR‑144 in the rectal cancer cells. The supplementation of ROCK1 markedly restored the cell migration and proliferation, which was inhibited by miR‑144. Together, the data of the present study demonstrated that miR‑144 acts as a tumor suppressor by targeting ROCK1, and indicates the potential of miR‑144 as a novel biomarker and target in the treatment of rectal cancer.

Figure 1

Expression of miR-144 in rectal carcinoma cell lines. (A) NC and rectal carcinoma cell lines (SW837 and SW1463). The expression levels of miR-144 were normalized to RNU48. Data are presented as the mean ± standard deviation of three independent experiments, and compared with the level of miR-144 in NC cells (normalized as 1). (B) Expression levels of miR-144 were determined using reverse transcription-quantitative polymerase chain reaction 72 h following transfection. Data are presented as the mean ± standard deviation of three independent experiments, and compared with the expression level of miR-144 in the miR control-transfected cells (normalized as 1). miR, microRNA; NC, normal control.

Figure 2

Cell viability, migration and proliferation in miR-144 overexpressed rectal carcinoma cells (SW837 and SW1463). (A) Cell viability was analyzed using an MTT assay following transient transfection for different durations. ^*P<0.05, compared with the corresponding miR-control (Student's t-test). (B) Numbers of SW837 (blue lines) and SW1463 (red lines) cells, which migrated into the lower wells. Data were obtained 24, 48 and 72 h following transfection. ^*P<0.05, compared with the corresponding miR-control group (Student's t-test). (C) Relative percentage proliferation of the SW837 cells percent. (D) Relative proliferation of SW1463 cells. Data were obtained 0, 24, 48 and 72 h following transfection,. ^*P<0.05, compared with the miR-control group at the corresponding time point. The data are presented as the mean ± standard deviation of three independent experiments. miR, microRNA.

Figure 3

Expression of levels of ROCK1 and ROCK2 in miR-144-overexpressing rectal carcinoma cells (SW837 and SW1463). mRNA levels of ROCK1 and ROCK2 mRNA were detected in the (A) SW837 and (B) SW1463 cells using reverse transcription-quantitative polymerase chain reaction following miR-144 transfection for 72 h. Data were normalized based on the mRNA levels of β-actin. (C and D) Protein expression levels of ROCK1 and ROCK2 were detected using western blot analysis following miR-144 transfection for 72 h. The bands on the western blot were analyzed using Gel-Pro analyzer 4.0 software. Data were normalized based on the levels of β-actin. Each data point was obtained from three repeated experiments and expressed as the mean ± standard deviation. ^#P<0.01 and ^*P<0.05 (Student's t-test). miR, microRNA; ROCK, Rho-associated coiled-coil containing protein kinase.

Figure 4

Cell migration and proliferation in miR-144 and ROCK1-co-transfected rectal carcinoma cells (SW837 and SW1463). Numbers of (A) SW837 and (B) SW1463 cells migrating into the lower wells. Data were obtained 24, 48 and 72 h following transfection. ^*P<0.05, compared with the corresponding miR-144 and mock ROCK1-co-transfected group. Relative percentages of proliferation of the (C) SW837 and (D) SW1463 cells. Data were obtained 0, 24, 48 and 72 h following transfection. ^*P<0.05, compared with the miR-144 and mock ROCK1-co-transfected group at the corresponding time point. All data are presented as the mean ± standard deviation of three independent experiments. miR, microRNA; ROCK, Rho-associated coiled-coil containing protein kinase.