MiR-1224 Acts as a Prognostic Biomarker and Inhibits the Progression of Gastric Cancer by Targeting SATB1

Abstract

Objective: MiR-1224 has been reported to exhibit abnormal expression in several tumors. However, the expressing pattern and roles of miR-1224 in gastric cancer (GC) remain unclear. Our current research aimed to explore the potential involvement of miR-1224 in the GC progression.

Materials and methods: The expression of miR-1224 was examined in tissue samples of 128 GC patients and cell lines by RT-PCR. Besides, the associations of miR-1224 expressions with clinicopathologic features and prognosis of GC patients were analyzed. Then, the possible influences of miR-1224 on cell proliferation and cell migration were determined. Afterward, the molecular target of miR-1224 was identified using bioinformatics assays and confirmed experimentally. Finally, RT-PCR and Western blot assays were performed to investigate the effect of the abnormal miR-1224 expression on the EMT and Wnt/β-catenin pathway.

Results: miR-1224 was lowly expressed in the GC specimens and cell lines due to T classification and TNM stage. Survival assays demonstrated that GC patients with low expressions of miR-1224 possessed poor overall survivals. Moreover, in vitro and in vivo assays revealed that the overexpression of miR-1224 inhibited cell proliferation, migration, and invasion in GC cells. SATB homeobox 1 (SATB1) was verified as a direct target of miR-1224 in GC. Furthermore, β-catenin and c-myc were significantly inhibited in miR-1224-overexpression cells.

Conclusions: Our findings highlight the potential of miR-1224 as a therapeutic target and novel biomarker for GC patients.

Keywords: SATB1; Wnt/β-catenin; gastric cancer; metastasis; miR-1224; prognosis.

Figure 1

MiR-1224 was down-regulated in GC. (A) The qPCR results of miR-1224 in 128 pairs of GC tissues and adjacent tissues. (B) The qPCR analysis of miR-1224 expressions in GC cell lines. (C) The overall survivals of GC patients who had higher or lower miR-1224 expression were analyzed by Kaplan-Meier assays and log-rank test. (D) Disease-free survivals. **p < 0.01. Experiments were repeated three times with three replicates.

Figure 2

MiR-1224 affected the proliferation of SGC-7901 and BGC-823 cells. (A) The qPCR assays determined the expression of miR-1224 in GC cells transfected with miR-1224 mimics. (B, C) The cellular growth of miR-1224 mimics-transfected GC cells was detected by CCK-8 assays. (D, E) EdU assays evaluated the cell proliferation of GC cells after treatment with miR-1224 mimics. The red color represented proliferative cells. The blue color indicated the nuclei stained by DAPI. (F) The colony formation abilities of GC cells were assessed using the colony formation assays. **p < 0.01. Experiments were repeated three times with three replicates.

Figure 3

MiR-1224 suppressed tumor growth of GC cells in vivo. (A, B) The analysis of tumor growth of miR-1224-overexpressed SGC7901 cells in the nude mice. (C) HE staining and IHC staining of Ki-67 in two groups. (D) RT-PCR assay revealed the expression of miR-1224 and STAT1 in tissues obtained from two groups. **p < 0.01, ***p < 0.001. Experiments were repeated three times with three replicates.

Figure 4

The influence of miR-1224 on the invasion and migration of BGC-823 and SGC-7901 cells. (A) Overexpression of miR-1224 reduced the migration of GC cells. (B) Ectopic expression of miR-1224 depressed the invasion of GC cells. (C) The protein expression of N-cadherin and vimentin was measured with Western blot assays. **p < 0.01. Experiments were repeated three times with three replicates.

Figure 5

SATB1 is a direct target of miR-1224 in GC cells. (A) Venn diagram of the potential target genes of miR-1224. (B) Biological processes of the 55 common genes. (C) The cellular components analyses. (D) The molecular functions analyses for the 55 common genes. (E) The biological pathways analyses for the 55 common genes. (F) The SATB1-genes interaction network. (G) miR-1224 and its binding sequence in the wild-type and mutant 3’UTR of SATB1 mRNA. (H) The levels of SATB1 in GC tissues and matched non-tumor samples were determined using qPCR. (I) Luciferase activities confirmed that miR-1224 directly targeted SATB1. (J) The levels of SATB1 were determined by qPCR after the transfection of miR-1224 mimics or inhibitors. **p < 0.01. Experiments were repeated three times with three replicates. Ns, no significance.

Figure 6

The TFs-miR-1224 interaction network. (A) The potential TFs for miR-1224. (B) The TFs-miR-1224 interaction network. Experiments were repeated three times with three replicates.

Figure 7

The activity of the Wnt/β-catenin signaling pathway was repressed by up-regulation of miR-1224 in BGC-823 and SGC-7901 cells. (A, B) The qPCR assays examined the mRNA levels of β-catenin, cyclin D1, and c-myc in BGC-823 and SGC-7901 cells. (C) The protein expressions of c-myc, cyclin D1, and β-catenin in BGC-823 and SGC-7901 cells were reduced when the cells were transfected with miR-1224 mimics. **p < 0.01. Experiments were repeated three times with three replicates.