MicroRNA-410 suppresses migration and invasion by targeting MDM2 in gastric cancer

Abstract

Gastric cancer is one of the most frequent malignancies in tumors in the East Asian countries. Identifying precise prognostic markers and effective therapeutic targets is important in the treatment of gastric cancer. microRNAs (miRNAs) play important roles in tumorigenesis. However, the mechanisms by which miRNAs regulate gastric cancer metastasis remain poorly understood. In this study, we found that the levels of miR-410 in gastric cancer and cell lines were much lower than that in the normal control, respectively, and the lower level of miR-410 was significantly associated with lymph-node metastasis. Transfection of miR-410 mimics could significantly inhibit the cell proliferation, migration and invasion in the HGC-27 gastric cancer cell lines. In contrast, knockdown of miR-410 had the opposite effect on the cell proliferation, migration and invasion. Moreover, we also found that MDM2 was negatively regulated by miR-410 at the post-transcriptional level, via a specific target site with the 3'UTR by luciferase reporter assay. The expression of MDM2 was inversely correlated with miR-410 expression in gastric cancer tissues, and overexpression of MDM2 in miR-410-transfected gastric cancer cells effectively rescued the inhibition of cell proliferation and invasion caused by miR-410. Thus, our findings suggested that miR-410 acted as a new tumor suppressor by targeting the MDM2 gene and inhibiting gastric cancer cells proliferation, migration and invasion. The findings of this study contributed to the current understanding of these functions of miR-410 in gastric cancer.

Figure 1. miR-410 was downregulated in gastric cancer.

A. qRT-PCR analysis of miR-410 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). (B) qRT-PCR analysis of miR-410 expression in 60 pairs GC tissues and their corresponding adjacent nontumorous tissues. The expression of miR-410 was normalized to U6 snRNA. (C) Relative miR-410 expression levels in GC tissues and adjacent normal regions; (D) Statistical analysis of the association between miRNA level between pM stage (No metastasis and Metastasis, respectively); *p<0.05, and **p<0.01.

Figure 2. Overexpression of miR-410 inhibited the cell proliferation, migration, and invasion in gastric cancer cells (A) Expression levels of miR-410 were examined by qRT-PCR after transfection of 20 nmol/L of miR-410 mimics, inhibitors or sramble or no transfection.

The expression of miR-410 was normalized to U6 snRNA. (B) The cells treated with miR-410 mimics, inhibitors or sramble or no transfection were measured by CCK8 assay at different time periods. (C) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of migrated cells per field is shown below. (D) Transwell analysis of HGC-27 cells after treatment with miRNA mimics, inhibitors or scramble or no transfection; the relative ratio of invasive cells per field is shown below, *p<0.05, ** p<0.01, and ***p<0.001.

Figure 3. miR-410 targets at MDM2 in GC cells.

(A) The sequences of miR-410 binding sites within the human MDM2 3′UTRs and schematic reporter constructs, in this panel, c-MDM2-WT represent the reporter constructs containing the entire 3′UTR sequences of MDM2. C-MDM2-MUT represent the reporter constructs containing mutated nucleotides. (B) The analysis of the relative luciferase activities of MDM2-WT, MDM2-MUT in 293T cells. The error bars are derived from triplicate expriments. (C) qRT-PCR analysis of MDM2 mRNA expression in HGC-27 cells after treatment with miRNA mimics or scramble or no transfection. The expression of MDM2 was normalized to GAPDH. (D) Western blot analysis of MDM2 expression in HGC-27 cells transfected with miR-410 mimics or scramble or no transfection. GAPDH was also detected as a loading control.

Figure 4. Overexpression of MDM2 impaired miR-410-induced inhibition of proliferation and invasion in GC cells (A) Western blot analysis of MDM2 in HGC-27 cells co-transfected with either miR-410 mimic or scramble and 2.0 µg pCDNA-MDM2 or pCDNA empty vector.

(B) Cell growth curves in HGC-27 cells transfected with different combinations. (D) Transwell analysis of HGC-27 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p<0.05, ** p<0.01, ***p<0.001.

Figure 5. miR-410 negatively regulated MDM2 gene expression.

(A) The relative MDM2 mRNA expression levels were determined by qRT-PCR in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). The expression of MDM2 was normalized to GAPDH. (B) Western blot analysis of MDM2 expression in four cell lines derived from gastric cancer and one nonmalignant gastric cell line (GES). GAPDH was also detected as a loading control. (C) qRT-PCR analysis of MDM2 expression in 40 pairs gastric cancer tissues and their corresponding adjacent normal tissues. The expression of MDM2 was normalized to GAPDH. The expression of MDM2 in gastric cancer tissues was significant higher than in the corresponding adjacent normal tissues. (D) Analysis of correlation of miR-410 and MDM2 expression in gastric cancer tissues. *p<0.05, ** p<0.01, ***p<0.001.