Yonsei Med J.  2017 Nov;58(6):1101-1110. 10.3349/ymj.2017.58.6.1101.

Overexpression of miR-191 Predicts Poor Prognosis and Promotes Proliferation and Invasion in Esophageal Squamous Cell Carcinoma

• Gao X
• Xie Z
• Wang Z
• Cheng K
• Liang K
• Song Z

• KMID: 2418891
• DOI: http://doi.org/10.3349/ymj.2017.58.6.1101

Abstract

PURPOSE
Accumulating evidence has shown that dysregulation of microRNA-191 (miR-191) is closely associated with tumorigenesis and progression in a wide range of cancers. This study aimed to explore the potential role of miR-191 in esophageal squamous cell carcinoma (ESCC).
MATERIALS AND METHODS
miR-191 expression was assessed in 93 ESCC tissue specimens by real-time polymerase chain reaction, and survival analysis was performed via Kaplan-Meier and Cox regression analyses. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, plate colony-forming, BrdU, and Transwell assays were conducted to observe the effect of miR-191 on ESCC proliferation and invasion. Luciferase reporter and western blot assays were taken to identify target genes of miR-191.
RESULTS
miR-191 was overexpressed in 93 cases of ESCC, compared with adjacent normal tissues, and miR-191 expression was significantly related to differentiation, depth of invasion, TNM stage, lymph node metastasis, and distant metastasis of tumor. Kaplan-Meier and Cox regression analyses demonstrated that overexpression of miR-191 was an independent and significant predictor of ESCC prognosis. Both gain-of-function and loss-of-function experiments showed that miR-191 promoted ESCC cell proliferation and invasion activities in vitro. Early growth response 1 (EGR1), a tumor suppressor, was predicted as a direct target of miR-191. Luciferase reporter and western blot assays proved that miR-191 reduced EGR1 expression by directly binding its 3' untranslated region. Moreover, EGR1 knockdown by siRNA enhanced ESCC cell growth and invasion.
CONCLUSION
Our findings provide specific biological roles of miR-191 in ESCC survival and progression. Targeting the novel miR-191/EGR1 axis represents a potential new therapeutic way to block ESCC development.

Keyword

miR-191; esophageal squamous cell carcinoma; early growth response 1; prognosis; proliferation; invasion

MeSH Terms

3' Untranslated Regions
Carcinoma, Squamous Cell/genetics/metabolism/*pathology
Cell Line, Tumor
*Cell Movement
Cell Proliferation
Esophageal Neoplasms/genetics/metabolism/*pathology
*Gene Expression Regulation, Neoplastic
Humans
Lymphatic Metastasis
MicroRNAs/*genetics/metabolism
Middle Aged
Neoplasm Invasiveness/*genetics
Prognosis
RNA, Small Interfering
Real-Time Polymerase Chain Reaction
3' Untranslated Regions
MicroRNAs
RNA, Small Interfering

Figure

• 

  Fig. 1 miR-191 is overexpressed in ESCC tissues. (A) The relative expression levels of miR-191 in adjacent non-cancerous esophageal and ESCC samples. (B) The relative expression levels of miR-191 in Stage I–II and III–IV ESCC tissues. ***p<0.001. miRNA, microRNA; miR-191, microRNA-191; ESCC, esophageal squamous cell carcinoma.

• 

  Fig. 2 Kaplan-Meier survival curves of ESCC patients with different level of miR-191 expression stratified by the TNM stage. (A) Association of miR-191 expression with overall survival (cumulative survival) in all stages. (B) Association of miR-191 expression with overall survival in Stage I–II. (C) Association of miR-191 expression with overall survival in Stage III–IV. ESCC, esophageal squamous cell carcinoma; miR-191, microRNA-191.

• 

  Fig. 3 miR-191 promotes ESCC cell proliferation and invasion in vitro. (A) RT-PCR analysis of miR-191 expression in EC9706 cells transfected with miR-191 mimic or inhibitor and corresponding control. (B) RT-PCR analysis of miR-191 expression in TE-1 cells transfected with miR-191 mimic or inhibitor and corresponding control. (C) MTT assay of EC9706 cells transfected with miR-191 mimic or inhibitor and corresponding control. (D) MTT assay of TE-1 cells transfected with miR-191 mimic or inhibitor and corresponding control. (E) The colony formation assays in EC9706 and TE-1 cells transfected with miR-191 mimic or inhibitor and corresponding control. Colonies were evaluated and values were reported as the ratio. (G) The BrdU assays from EC9706 and TE-1 cells transfected with miR-191 mimic or inhibitor and corresponding control. *p<0.05, **p<0.01, ***p<0.001 miR-191, microRNA-191; ESCC, esophageal squamous cell carcinoma; RT-PCR, real-time polymerase chain reaction; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; OD, optical density. (H) Transwell assays of EC9706 cells transfected with miR-191 mimic or inhibitor and corresponding control. Left panel: representative images. Right panel: quantification of 10 randomly selected fields. (I) Transwell assays of TE-1 cells transfected with miR-191 mimic or inhibitor and corresponding control. *p<0.05, **p<0.01. miR-191, microRNA-191; ESCC, esophageal squamous cell carcinoma.

• 

  Fig. 4 miR-191 down-regulates EGR1 expression by directly targeting its 3′UTR. (A) Diagram of EGR1 3′UTR-containing reporter construct. Mutations were generated at the predicted miR-191 binding site located in the EGR1 3′UTR. (B) Relative Luciferase activity after the wild type or mutant reporter plasmids were co-transfected with miR-191 mimic or mimic control in HEK293T and EC9706 cells. (C) RT-PCR analysis of EGR1 mRNA expression in EC9706 cells transfected with miR-191 mimic or inhibitor and corresponding control. (D) Western blot analysis of EGR1 protein expression in EC9706 cells transfected with miR-191 mimic or inhibitor and corresponding control. *p<0.05, **p<0.01. ns, no significance; EGR1, early growth response 1; miR-191, microRNA-191; mRNA, messenger RNA.

• 

  Fig. 5 Down-regulation of EGR1 promotes ESCC cell proliferation and invasion. (A) Western blot analysis of EGR1 expression in EC9706 cells transfected with EGR1 siRNA or corresponding control. β-actin was used as an internal control. (B) MTT assay of EC9706 cells transfected with EGR1 siRNA or control. (C) The colony formation assays in EC9706 transfected with EGR1 siRNA or control. Colonies were evaluated and values were reported as the ratio. (D) The BrdU assays from EC9706 transfected with EGR1 siRNA or control. (E) Transwell assays of EC9706 cells transfected with EGR1 siRNA or control. Left panel: representative images. Right panel: quantification of 10 randomly selected fields. *p<0.05, **p<0.01, ***p<0.001. EGR1, early growth response 1; ESCC, esophageal squamous cell carcinoma; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; OD, optical density.

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