Resveratrol inhibits the proliferation, invasion, and migration, and induces the apoptosis of human gastric cancer cells through the MALAT1/miR-383-5p/DDIT4 signaling pathway

Abstract

Background: We aimed to study the effects and potential mechanism of resveratrol (RS) in gastric cancer (GC).

Methods: The human GC cell line SGC7901 was treated with different concentrations of RS (0, 1, 5 µM) for 24 hours. The messenger ribonucleic acid or protein expressions levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), micro ribonucleic acid-383-5p (miR-383-5p), and DNA damage-inducible transcript 4 (DDIT4) in GC cells were determined by Western blot and quantitative real-time polymerase chain assays. Cells were then transfected with miR-383-5p inhibitor (inhibitor), inhibitor negative control (NC), MALAT1-interfering RNA (si-MALAT1), si-DDIT4 and negative interference control (si-NC). The Cell Counting Kit-8 method, scratch assay, and transwell assay were performed to evaluate cell proliferation, migration, and invasion. Additionally, flow cytometry was used to examine apoptosis, and the target relationship was examined by a luciferase-reporter gene analysis.

Results: RS treatment downregulated the expression of MALAT1, repressed cell proliferation, inhibited cell migration and invasion (all P<0.05), and induced apoptosis (P<0.05) in GC cells. When the cells were treated with RS and inhibited the expression of MALAT1 meanwhile, the above anti-cancer effects were more significant (all P<0.05). Target prediction and the luciferase-reporter gene analysis showed that MALAT1 targeted miR-383-5p (P<0.05). When suppressing the expression of MALAT1 and miR-383-5p, the anti-cancer effects caused by the silencing of MALAT1 were absent (all P<0.05). We also found that miR-383-5p targeted DDIT4 protein. When the expression of miR-383-5p and DDIT4 in the GC cells was inhibited, the promoting cancer effects caused by the inhibition of miR-383-5p were reversed (all P<0.05).

Conclusions: This study found that RS inhibited the proliferation, migration, and invasion of human GC cells through the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-383-5p/DDIT4 pathway and induced apoptosis.

Keywords: MALAT1/miR-383-5p/DDIT4 pathway; Resveratrol; gastric cancer cell.

Figure 1

Effects of RS treatment on the MALAT1 level and proliferative activity of the GC cells. The human GC cell line SGC7901 was treated with different concentrations of RS [0 (Control), 1, 5 µM] for 24 hours. (A) The level of MALAT1 was measured by qRT-PCR; (B) Cell proliferation activity was measured by the CCK-8 method. Compared to the control group, *P<0.05, **P<0.01, ***P<0.001. RS, Resveratrol; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; GC, gastric cancer; CCK-8, Cell Counting Kit 8; qRT-PCR, quantitative real-time PCR.

Figure 2

Effects of RS treatment and interference with MALAT1 on the proliferation, migration, invasion, and apoptosis of GC cells. (A) The level of MALAT1 was measured by qRT-PCR; (B) cell proliferation activity was measured by the CCK-8 method; (C) cell migration ability was measured by the scratch test. The cell migration width was observed under an inverted microscope with the original magnification of 50 times; (D) the invasion ability of cells was detected by transwell. The number of transmembrane cells stained with crystal violet was observed under an inverted microscope, and the original magnification was 100 times; (E) apoptosis was measured by flow cytometry. Compared to the control or si-NC groups, ***P<0.001; compared to the si-MALAT1 group, ^#P<0.05, ^##P<0.01, ^###P<0.001. RS, resveratrol; NC, normal control; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; GC, gastric cancer; CCK-8, Cell Counting Kit 8; qRT-PCR, quantitative real-time PCR.

Figure 3

MALAT1 targets miR-383-5p and inhibits its expression. (A) Starbase prediction target; (B) the targeting relationship between MALAT1 and miR-383-5p was examined using a luciferase-reporter gene analysis; (C) the level of miR-383-5p was measured by qRT-PCR. Compared to the NC or si-NC groups, ***P<0.001. NC, normal control; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; qRT-PCR, quantitative real-time PCR.

Figure 4

Inhibiting the expression of miR-383-5p reversed the effects of interfering with MALAT1 gene expression on GC cell proliferation, migration, invasion, and apoptosis. (A, B) The level of miR-383-5p was measured by qRT-PCR; (C) cell proliferation activity was measured by the CCK-8 method; (D) cell migration ability was measured by the scratch test. The cell migration width was observed under an inverted microscope with the original magnification of 50 times; (E) the invasion ability of cells was detected by transwell. The number of transmembrane cells stained with crystal violet was observed under an inverted microscope, and the original magnification was 100 times; (F) apoptosis was measured by flow cytometry. Compared to the control, NC inhibitor or si-NC + NC inhibitor group, *P<0.05, **P<0.01, ***P<0.001; Compared to the si-NC+miR-383-5p inhibitor group, ^###P<0.001. RS, resveratrol; NC, normal control; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; GC, gastric cancer; CCK-8, Cell Counting Kit 8; qRT-PCR, quantitative real-time PCR.

Figure 5

MiR-383-5p targets DDIT4 and inhibits its expression. (A) Starbase prediction target; (B) the targeting relationship between miR-383-5p and DDIT4 was examined by a luciferase-reporter gene analysis; (C) the level of DDIT4 was measured by Western blot. Compared to the NC or NC inhibitor group, **P<0.01, ***P<0.001. NC, normal control; DDIT4, DNA damage-inducible transcript 4.

Figure 6

Interfering with the expression of DDIT4 in GC cells reversed the effects of the miR-383-5p inhibitor on cell proliferation, migration, invasion, and apoptosis. (A,B) The level of DDIT4 protein was measured by Western blot; (C) cell proliferation activity was measured by the CCK-8 method; (D) cell migration ability was measured by the scratch test. The cell migration width was observed under an inverted microscope with the original magnification of 50 times; (E) the invasion ability of cells was detected by transwell. The number of transmembrane cells stained with crystal violet was observed under an inverted microscope, and the original magnification was 100 times; (F) apoptosis was measured by flow cytometry. Compared to the control or si-NC or NC inhibitor group, *P<0.05, **P<0.01, ***P<0.001; compared to the miR-383-5p inhibitor group, ^###P<0.001. RS, resveratrol; NC, normal control; GC, gastric cancer; DDIT4, DNA damage-inducible transcript 4; CCK-8, Cell Counting Kit 8.