MiR-96-5p inhibition induces cell apoptosis in gastric adenocarcinoma

Abstract

Background: Gastric adenocarcinoma (GAC) mortality rates have remained relatively changed over the past 30 years, and it continues to be one of the leading causes of cancer-related death.

Aim: To search for novel miRNAs related to GAC prognosis and further investigate the effect of miR-96-5p on MGC-803 cells.

Methods: The miRNA expression profile data of GAC based on The Cancer Genome Atlas were obtained and used to screen differently expressed miRNAs (DEMs) and DEMs related to GAC prognosis. Then, the expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by qRT-PCR. The target gene, ZDHHC5, of miR-96-5p was predicted using TargetScan, miRTarBase, and miRDB databases and confirmed by luciferase reporter assay. Furthermore, MGC-803 cells were transfected with inhibitor NC, miR-96-5p inhibitor, si-ZDHHC5, or miR-96-5p inhibitor + si-ZDHHC5, and then cell apoptosis was detected by flow cytometry. The expression of ZDHHC5, Bcl-2, and COX-2 was detected using western blotting.

Results: A total of 299 DEMs and 35 DEMs related to GAC prognosis were screened based on The Cancer Genome Atlas. Then compared with adjacent normal samples, the levels of miR-96-5p, miR-222-5p, and miR-652-5p were remarkably increased, while miR-125-5p, miR-145-3p, and miR-379-3p levels were reduced in GAC tumor samples (P < 0.01), which were consistent with bioinformatics analysis. Furthermore, ZDHHC5 was defined as a direct target gene of miR-96-5p. miR-96-5p inhibition increased the number of apoptotic cells as well as promoted the expression of ZDHHC5, Bcl-2, and COX-2 in MGC-803 cells (P < 0.01). After ZDHHC5 inhibition, the number of apoptotic cells and the expression of ZDHHC5, Bcl-2, and COX-2 were reduced. The addition of an miR-96-5p inhibitor partly reversed these effects (P < 0.01).

Conclusion: Our findings identified six miRNAs related to GAC prognosis and suggested that downregulated miR-96-5p might induce cell apoptosis via upregulating ZDHHC5 expression in MGC-803 cells.

Keywords: Cell apoptosis; Differently expressed miRNAs; Gastric adenocarcinoma; MicroRNA-96-5p; Prognosis; The Cancer Genome Atlas.

Figure 1

Differentially expressed miRNAs between gastric adenocarcinoma sample and normal sample. A: Volcano plot for differentially expressed miRNA (DEM) expression. Dark dots represent upregulated miRNAs, whereas lighter dots represent downregulated miRNAs; B: Hierarchical gene clustering analysis of DEMs represented by a heat map. Dark represents upregulated miRNAs, whereas lighter represents downregulated miRNAs.

Figure 2

Association of differentially expressed miRNAs with overall survival of gastric adenocarcinoma. Dark lines represent high expression, and lighter lines represent low expression.

Figure 3

The identification of differentially expressed miRNA levels in gastric adenocarcinoma patients and healthy subjects. A: The levels of miR-96-5p, miR-222-5p, miR-652-5p, miR-125-5p, miR-145-3p, and miR-379-3p in tumor samples and adjacent normal samples by qRT-PCR; B: The miR-96-5p level in the blood of gastric adenocarcinoma patients and healthy subjects by qRT-PCR (^bP < 0.01). Values are mean ± SD.

Figure 4

Target gene prediction and identification of miR-96-5p. A: The intersection of the predicting target genes of miR-96-5p among the TargetScan, miRTarBase and miRDB databases using a Venn diagram; B: Prediction of the binding site of miR-96-5p to zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) by bioinformatics; C: Target regulation of miR-96-5p to ZDHHC5 observed by the luciferase reporter system. ZDHHC5: Zinc finger DHHC-type palmitoyltransferase 5.

Figure 5

miR-96-5p inhibition might induce cell apoptosis in MGC-803 cells. A: The number of apoptotic cells after transfection with inhibitor NC, miR-96-5p inhibitor, si-zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5), or miR-96-5p inhibitor + si-ZDHHC5 in MGC-803 cells by flow cytometry analysis; B: The expression of ZDHHC5, Bcl-2, and COX2 after cells were treated with inhibitor NC, miR-96-5p inhibitor, si-ZDHHC5, or miR-96-5p inhibitor + si-ZDHHC5 in MGC-803 cells by western blotting (^aP < 0.01). Values are mean ± SD. ZDHHC5: Zinc finger DHHC-type palmitoyltransferase 5.