MicroRNA-21 promotes cell growth and migration by targeting programmed cell death 4 gene in Kazakh's esophageal squamous cell carcinoma

Abstract

Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of cancer death. There are two main types of EC--squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Although some advances in the exploration of its possible etiological mechanism were made recently including behaviors and environmental risk factors as well as gene alterations, the molecular mechanism underlying ESCC carcinogenesis and progression remains poorly understood. It has been reported that miR-21 was upregulated in most malignant cancers, the proposed mechanism of which was through suppressing expression of programmed cell death 4 (PDCD4). In present study, it is firstly reported that miR-21 was upregulated in Kazakh's ESCC and that miR-21 played a negative role in regulating PDCD4 using in situ hybridization (ISH) and luciferase reporter approach. Morever, in model of ESCC xenografted nude mice, miR-21 maybe used as an effective target in the treatment. The present results demonstrated that miR-21 may be a potential therapeutic target in management of ESCC.

Figure 1

miR-21 directly regulated PDCD4-3′-UTR. (a) Prediction result of targetscan (http://www.targetscan.org/): PDCD4-3′-UTR-containing reporter constructs of miR-21. (b) Reporter assay, the cells were transfected with wild reporter plasmid or mutation reporter plasmid and miR-21 plasmid.

Figure 2

miR-21 promoted tumor cell migration after transfection. (a) Cell migration ability was assessed using wound-healing assay. Eca109 cells were transfected with miR-21 mimics, inhibitor, and scrambled sequence and were assessed for migration by wound-healing assay at 0 h, 24 h, 48 h, and 72 h after transfection. Images of migratory cells from the scratched boundary were observed and acquired with light microscope (10 × 10). Similar results were obtained in three independent experiments, and shown were representative figures. (b) Statistical analysis of wound-healing. There was significant difference between miR-21 mimics and control group (^* P < 0.05).

Figure 3

miR-21 negatively correlated with PDCD4 expression in ESCC tissues. ((a), (b)) miR-21 was detected as a weak positive staining in normal epithelium, strongly positive staining of the tumor. ((c), (d)) PDCD4 was detected as a strongly positive staining in normal epithelium, weak positive staining of the tumor.

Figure 4

Prognostic analysis of miR-21 and PDCD4 expression. 50 ESCC patients with their following information available were classified into negative expression group and positive expression group. Shown are Kaplan-Meier overall survival curves of two group patients; log-rank test was used. (a) Expression of PDCD4 protein is associated with overall survival of ESCC patients. (b) Expression of miR-21 is associated with overall survival of ESCC patients.

Figure 5

miR-21 promotes tumorigenesis of ESCC in xenografted nude mice. Twice per week intratumor injection of miR-21 mimics, inhibitor, and scramble reduces tumor volume followed for 28 d after implantation. (a) Evaluation of tumor size at different therapeutic times; (b) gross morphology of tumors resected from different groups.