High miR-718 Suppresses Phosphatase and Tensin Homolog (PTEN) Expression and Correlates to Unfavorable Prognosis in Gastric Cancer

Abstract

BACKGROUND Phosphatase and tensin homolog (PTEN) is a kind of phosphatase which has been demonstrated to suppress progression of gastric cancer. Many micro-RNAs (miRNAs), such as miR-106b, miR-93, and miR-200c, could inhibit expression of PTEN in cell lines; and many miRNAs including miR-21, miR-22, miR-18a, and miR-222 are related to the progression and prognosis of gastric cancer. However, among these miRNAs, the clinical significance of miR-718 has not yet been elucidated. MATERIAL AND METHODS The expression of PTEN and miR-718 in 141 gastric cancer tissues were detected by immunohistochemistry and quantitative real-time PCR respectively. The correlation between PTEN, miR-718, and the clinicopathological factors was analyzed by χ² test. The prognostic significance of PTEN and miR-718 was evaluated by univariate and multivariate analysis. Luciferase reporter assay was performed to evaluate the regulation of PTEN by miR-718. The effect of miR-718 on gastric cancer proliferation and invasion was investigated by MTT assay and Transwell assay. RESULTS Low expression of PTEN and high expression of miR-718 were both significantly associated with unfavorable prognosis, and both were identified as biomarkers predicting poorer prognosis of patients with gastric cancer. Increased miR-718 expression could decrease PTEN expression, thus enhancing phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) signaling. Moreover, the abilities of proliferation and invasion of gastric cells transfected with miR-718 were promoted significantly compared with those transfected with control miRNA. CONCLUSIONS Low expression of PTEN and increased expression of miR-718 in gastric cancer tissues were both independent unfavorable prognostic factors of gastric cancer. Upregulation of miR-718 could increase PI3K/Akt signaling by directly downregulating PTEN, thus promoting the proliferation and invasion of gastric cancer cells.

Figure 1

The representative images of low expression and high expression of PTEN. (A) The representative image and magnified image for PTEN low expression. The score of staining intensity is 1 and the score of positive cells is 1. Total IHC score is 1 and defined as the low-expression of PTEN. The relative ratio of miR-718 in this case was 2.43. (B) The representative image and magnified image for PTEN high expression. The score of staining intensity is 3 and the score of positive cells is 4. Total IHC score is 12 and defined as the low-expression of PTEN. The relative ratio of miR-718 in this case was 0.83. (C, D). The level of PTEN mRNA (C) and miR-718 (D) in the 12 pairs of fresh gastric cancers and their adjacent tissues was detected with qRT-PCR. The PTEN mRNA in adjacent tissues was significantly higher compared gastric cancers, while miR-718 level in adjacent tissues was remarkably lower than gastric cancers.

Figure 2

The survival curves of clinicopathological factors, PTEN and miR-718. (A) The survival curves of group T1+T2 and group T3+T4. (B) The survival curves of group N0 and group N1/2/3. (C) The survival curves of group M0 and group M1. (D) The survival curves of group TNM I+II and group TNM III+IV. (E). The survival curves of poor differentiation and good/moderate differentiation. (F) The survival curves of high-expression and low-expression of PTEN. (G) The survival curves of high level and low level of miR-718.

Figure 3

miR-718 could activate PI3K/Akt signaling by targeting PTEN. (A) The transcription of PTEN in gastric cell line was suppressed by miR-718. The normalized firefly luciferase activity of PTEN was detected 48 hours after the transfection with the PTEN-3′UTR-Luc reporter construct with the absence of miR-718 expression vector or the scrambled control miRNA vector in gastric cancer cell MKN7. Data were from 3 independent experiments and displayed by the mean ±SEM. (B) PI3K/Akt signaling in MKN7 could be activated by miR-718. The effect of miR-718 on Akt signaling of MKN7 cells was evaluated by western blotting. EGF at 100 ng/mL was used to incubate the cells for 10 minutes, 48 hours after transfection of expression vector of miR-718 or scrambled control miRNA. Cells were then lysed, and western blotting was performed to detect the levels of PTEN, phosphor-Ser473-Akt, total Akt, and β-actin. (C) miR-718 could accelerate the proliferation of MKN7. The MTT assay was used to detect the proliferation index of MKN7 48 hours after transfection of expression vector of miR-718 or scrambled control miRNA. Data were from 3 independent experiments and displayed by the mean ±SEM. (D) miR-718 could enhance the invasion of MKN7; 48 hours after transfection of expression vector of miR-718 or scrambled control miRNA, MKN7 was seeded into the Matrigel-precoated chamber and incubated for 24 hours with 10% fetal bovine serum and 100 ng/mL EGF as a chemoattractant. Cells at the bottom compartment were fixed and stained with crystal violet. Invaded cells were counted in at least 8 random visual fields. Data were from 3 independent experiments and displayed by the mean ±SEM. (E) Representative images of invaded cells in D. Image 1, 2, 3, 4 represent the groups with/without EGF stimulation or with/without miR-718 transfection.