Expression of microRNA-328 Functions as a Biomarker for Recurrence of Early Gastric Cancer (EGC) After Endoscopic Submucosal Dissection (ESD) by Modulating CD44

Abstract

BACKGROUND This study investigated the molecular mechanism of the effect of CD44 on the recurrence of EGC after ESD, including the potential regulator and signaling pathways of CD44. MATERIAL AND METHODS We searched the miRNA online database (www.mirdb.org) with the "seed sequence" located within the 3'-UTR of the target gene, and performed luciferase assay to test the miRNA/mRNA relationship. We also determined the expression of CD44 in the EGC and control samples. In addition, statistical analysis was used to explore the role of miR-328 as a biomarker to predict the recurrence after ECD. RESULTS We validated CD44 to be the direct gene via luciferase reporter assay system. We also established the negative regulatory relationship between miR-328 and CD44 via studying the relative luciferase activity at different concentrations of miR-328 mimics. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CD44 among different groups (recurrence-positive and recurrence-negative) or cells treated with different concentrations of miR-328 mimics/inhibitors, indicating the negative regulatory relationship between miR-328 and CD44. We also investigated the relative viability of EGC cells when transfected with miR-328 mimics (50 nM and 100 nM) and miR-328 inhibitors (100 nM) to validate miR-328 to be negatively interfering with the viability of EGC cells. miR-328 was also recognized as a potential biomarker to predict recurrence after ESD in EGC patients via analysis of the recurrence-free rate among different groups of EGC patients. CONCLUSIONS The expression level of miR-328 can function as a predictive biomarker of recurrence after ECD in patients with EGC via targeting CD44.

Figure 1

CD44 was identified as the candidate target gene of miR-328 in gastric cancer cells with the ‘seed sequence’ in the 3′UTR of CD44.

Figure 2

Luciferase activity reporter assay was conducted to verify CD44 as the direct target gene of miR-328, as well as to validate the regulatory relationship between miR-328 and CD44. Compared with the scramble control, the relative luciferase activity of cells transfected with wild-type CD44 3′UTR constructs clearly decreased as the concentration of miR-328 mimics increased, exhibiting negative regulation in a stepwise manner. On the contrary, cells carrying mutant CD44 3′UTR constructs exhibited comparable relative luciferase activity index when compared with the scramble controls, indicating CD44 as the direct target gene of miR-328 with the binding site located at the segment that has been mutated.

Figure 3

The recurrence-positive group (N=96) showed evidently lower miR-328 expression level when compared with the recurrence-positive group (N=134).

Figure 4

The mRNA (A) and protein (B) expression level of CD44 of the recurrence-positive group were evidently higher when compared with the recurrence-negative groups.

Figure 5

We investigated the mRNA/protein expression level of CD44 of EGC cells treated with 50-nM miR-328 mimics, 100-nM miR-328 mimics, and 100-nM miR-328 inhibitors to validate the negative regulatory relationship between miR-328 and CD44. The CD44 protein (A) and mRNA expression level (B) of EGC cells treated with 50-nM miR-328 mimics were apparently lower than the scramble control, while those of the sample group treated with 100-nM miR-328 mimics were even lower than the 50-nM treatment group. The miR-328 inhibitors treatment group showed evidently higher expression level of CD44 protein (C) and mRNA (D) when compared with the scramble controls and the miR-328 mimics treatment groups.

Figure 6

Cells transfected with 100-nM miR-328 inhibitors showed evidently upregulated viability when compared with the scramble controls, while cells transfected with 50-nM/100-nM miR-328 mimics showed comparably lower viability.

Figure 7

The recurrence-free period was significantly longer in those with relative high expression of miR-328 than low miR-328, indicating miR-328 might be a potential biomarker to predict recurrence after ESD in EGC patients.