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. 2012 Apr;27(4):1019-26.
doi: 10.3892/or.2012.1645. Epub 2012 Jan 19.

microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN

Bao Gui Zhang  1 , Jian Fang Li, Bei Qin Yu, Zheng Gang Zhu, Bing Ya Liu, Min Yan
Affiliations

microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN

Bao Gui Zhang et al. Oncol Rep. 2012 Apr.

Abstract

Gastric cancer is one of the most common carcinomas in China. microRNAs, a type of non-coding RNA, are important specific regulators and are involved in numerous bioprocesses of an organism. microRNA-21 (miR-21) has been identified as the most suitable choice for further investigation because it is overexpressed in nearly all solid tumors; furthermore, it has been demonstrated that miR-21 is involved in the genesis and progression of human cancer. It has been reported that PTEN, an important tumour suppressor, is regulated by multiple miRNAs. Thus, in this study we focused on the expression and significance of miR-21 in gastric cancer tissues, and the role of miR-21 in the biological behaviour and the expression of PTEN in gastric cancer cells. Real-time PCR was used to detect miR-21 expression in gastric cancer tissues, the adjacent normal tissues, and the gastric cell lines. The gastric cancer cell line BGC-823 was transfected with pre-miR-21/miR-21 inhibitor to overexpress/downregulate miR-21. The influence of miR-21 on the biological behaviour of gastric cancer cells was evaluated using the CCK-8 kit, FCMs, the scratch healing assay and the transwell test. Western blotting and the Luciferase Reporter Assay were used to evaluate the change of PTEN expression after lowered expression of miR-21 in gastric cancer cell lines. Real-time PCR analysis indicated that miR-21 exhibited higher expression in gastric cancer tissues compared to the adjacent non-tumor tissues. miR-21 expression was significantly associated with the degree of differentiation of the tumour tissues (P=0.004), as well as local invasion and lymph node metastasis (P<0.01). After transfection, pre-miR21 BGC-823 cells grew faster than the negative and control groups (P<0.01). The reduction in miR-21 expression demonstrated a remarkable effect on the biological behaviour of gastric cancer cells (P<0.05); the pre-miR-21-transfected cells healed more quickly compared to the control cells in the scratch healing assay, whereas the transwell test indicated that cell migration in vitro was notably inhibited with the downregulation of miR-21 (P<0.05). The western blot results and Luciferase Reporter Assay demonstrated that PTEN expression was remarkably increased after miR-21 inhibition (P<0.05). microRNA-21 expression was upregulated in gastric carcinoma tissues and was significantly associated with the degree of differentiation of tumour tissues, local invasion and lymph node metastasis. Overexpression of miR-21 promoted BGC-823 cell growth, invasion and cell migration in vitro, whereas downregulation of miR-21 exhibited a stronger inhibitory effect on the biological behaviour of gastric cancer cells; additionally, miR-21 inhibition may upregulate the PTEN expression level, which indicates that PTEN may be a target gene for gastric cancer initiation and development.

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Figures

Figure 1
Figure 1
Upregulation of miR-21 expression in gastric cancer tissues and gastric cancer cell lines compared with the corresponding controls. (A) qRT-PCR for miR-21 was performed using nine gastric cancer cell lines and one immortalised normal gastric mucosal epithelial cell line (GES-1). The mean and standard deviation of miR-21 expression levels relative to the miR-21 expression level of GES-1 are shown. The data represent triplicate measurements from single RNA samples (P<0.05, compared with GES-1). (B) qRT-PCR for miR-21 was performed using 30 surgical specimens of gastric cancer tissues and matched with non-tumour tissues. The mean and standard deviation of miR-21 expression levels are shown. The data represent triplicate measurements from single RNA samples (P<0.05).
Figure 2
Figure 2
The effect of miR-21 on the proliferation of BGC-823 cells. Cell proliferation was measured by the CCK8 assay. BGC-823 cells were transfected with miR-21 precursor/inhibitor or control at a final concentration of 100 nM and, at 24 h post-transfection, the CCK8 assay was performed every 24 h for 4 days. Results are the mean of three independent experiments ± SD (P<0.05). The Cy3-labeled pre-miR™ negative control #1 was transfected into BGC-823 cells, and the transfection efficiency was assessed by fluorescence microscope (nuclei were stained with DAPI). Nearly all cells exhibited Cy3 staining, indicating that the miR-21 precursor/inhibitor and control were readily transfected into BGC-823 cells.
Figure 3
Figure 3
The effect of miR-21 on cell cycle distribution and apoptosis of BGC-823 cells. (A) Proportion of cells in various phases of the cell cycle. (B) Representative histograms depicting cell cycle profiles of BGC-823 cells transiently transfected with miR-21 inhibitor or control (100 nM). Cells were stained with PI and analysed by flow cytometry at 48 h post-transfection. The results are the mean of three independent experiments ± SD (P<0.05). (C) Cells staining positive for Annexin-V-FITC and negative for PI at 48 h post-transfection were considered to have undergone apoptosis. (D) Representative histograms depicting apoptosis of BGC-823 cells transiently transfected with miR-21 inhibitor or control (100 nM). Average apoptotic rate of three independent experiments ± SD are shown.
Figure 4
Figure 4
microRNA-21 promotes migration of BGC-823 cells in vitro. BGC-823 cells were first transfected with miR-21 precursor/inhibitor and control (100 nM) and then subjected to scratch wound-healing motility assays/transwell migration assays. (A) Representative photographs of Scratch wound-healing motility assays. (B and C) Average migratory cell number of three independent experiments ± SD (P<0.05).
Figure 5
Figure 5
PTEN is a validated target of miR-21. (A) miR-21 inbibitor upregulated luciferase activities controlled by wild-type PTEN-3′-UTR (P<0.05) but did not affect luciferase activity controlled by mutant PTEN-3′-UTR. The results are the mean of three independent experiments ± SD (P<0.05). (B and C) PTEN protein in BGC-823 cells was detected by western blot analysis at 48 h post-transfection with miR-21 inhibitor and control (100 nM). GAPDH was used as an internal loading control. A reproducible result was obtained in three independent experiments. The results are shown as fold-changes relative to the control inhibitor-transfected BGC-823 cells. (D and E) PTEN expression in gastric cancer tissues and non-tumour tissues determined by western blot analysis and immunohistochemistry.
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