Cancer Res Treat.  2019 Oct;51(4):1420-1429. 10.4143/crt.2018.638.

MiR-1246 Promotes Metastasis and Invasion of A549 cells by Targeting GSK-3β‒Mediated Wnt/β-Catenin Pathway

• Yang F
• Xiong H
• Duan L
• Li Q
• Li X
• Zhou Y

• KMID: 2460591
• DOI: http://doi.org/10.4143/crt.2018.638

Abstract

PURPOSE
MicroRNAs (miRNAs) are a group of small non-coding RNAs involved in different cancers, including lung cancer. Here, we aim to investigate the expression profiles of circulating miRNAs and their roles contributed to the progress of lung cancer.
MATERIALS AND METHODS
The levels of circulating miRNA in lung cancer patients were investigated by miRNAs assay. Then we predicted the target genes of aberrantly expressing miRNAs by searching genetic databases. Based on the A549 cells transfected with miR-1246 mimics or miR-1246 inhibitor,we further measured the roles of miR-1246 involving in the epithelial-mesenchymal transition (EMT), migration and invasion capacities of lung cancer cells in vitro. Finally, we detected the effects of miR-1246 on glycogen synthase kinase-3β (GSK-3β)/β-catenin pathway by immunofluorescence and Western blot, respectively.
RESULTS
We identified that 14 miRNAs were aberrantly expressed in the serum of lung cancer patients. Among them, miR-1246 was the most up-regulated. The cell assays indicated that miR-1246 significantly increased the migration and invasion capabilities of A549 lung cancer cells. Meanwhile, immunofluorescence analysis revealed that miR-1246 promoted EMT process of A549 cells accompanying with decreasing E-cadherin expression, while increasing vimentin and transforming growth factor β (TGF-β) expression. Furthermore, an online tool predicated that miR-1246 might bind to 3"²-untranslated region of GSK-3β, which was confirmed by overexpression and knockdown of miR-1246 assays.
CONCLUSION
Taken together, the study illustrates that miR-1246 regulates Wnt/β-catenin pathway through targeting GSK-3β/β-catenin, which partly contributing to tumor metastasis. MiR-1246 may play an essential role in the diagnosis and therapeutic of lung cancer.

Keyword

MiR-1246; Epithelial–mesenchymal transition; Metastasis; Glycogen synthase kinase 3β; β-catenin; Lung neoplasms

MeSH Terms

Blotting, Western
Cadherins
Databases, Genetic
Diagnosis
Epithelial-Mesenchymal Transition
Fluorescent Antibody Technique
Glycogen Synthase
Humans
In Vitro Techniques
Lung Neoplasms
MicroRNAs
Neoplasm Metastasis*
RNA, Small Untranslated
Transforming Growth Factors
Vimentin
Cadherins
Glycogen Synthase
MicroRNAs
RNA, Small Untranslated
Transforming Growth Factors
Vimentin

Figure

• 

  Fig. 1. Expression profiling of circulating miRNAs in patients with lung cancer. (A) MiRNA assay analysis of circulating miRNAs from the patients with lung cancer was presented in a heat map (11 lung cancer vs. 5 health control; the 11 lung cancer group includes 4 squamous carcinomas, 4 adenocarcinomas, and 2 small cell lung cancer cases). (B) The 14 miRNAs were differentially expressed in 11 lung cancer vs. 5 health control (p < 0.05, fold-change > 2 or < 0.5). A differentially expressed miRNA was considered if their expression levels showed with fold-changes greater than 2.0 or less than 0.5. Red and blue colors indicate increased and decreased expression, respectively.

• 

  Fig. 2. MiR-1246 enhances the migration and invasion of A549 cells. Migration and invasion assays were performed after A549 transfected with miR-1246 mimics or negative control (NC) miRNA, miR-1246 inhibitor or inhibitor negative control (Inh-NC) for 48 hours. The cells that migrated and invasive were counted, the representative images were shown. Scale bar=100 μm. (A) The migration ability of A549 cells. (B) The invasion capacity of A549 cells. Experiments were performed at least in triplicate and data are represented as mean±standard error of mean (*p < 0.05).

• 

  Fig. 3. MiR-1246 promotes epithelial-mesenchymal transition (EMT) in A549 cells. The mRNA expression of markers (E-cadherin, vimentin, and transforming growth factor β [TGF-β]) related to EMT were measured by real-time quantitative polymerase chain reaction (RT-qPCR) assay. A549 cells were transfected with miR-1246 mimics or negative control (NC), miR-1246 inhibitor, or inhibitor negative control (Inh-NC). After 48 hours, whole cell lysates were prepared and RT-qPCR assay determined the mRNA level of EMT-related markers. (A) The mRNA levels of E-cadherin, vimentin, and TGF-β in A549 cells transfected by miR-1246 mimics or NC. (B) The levels of E-cadherin, vimentin, and TGF-β mRNA in A549 cells transfected with miR-1246 inhibitor or Inh-NC. All experiments were carried out more than three times and results are expressed as mean±standard error of mean (*p < 0.05, **p < 0.01).

• 

  Fig. 4. MiR-1246 regulates Wnt/β-catenin signaling pathway through targeting glycogen synthase kinase-3β (GSK-3β)/β-catenin. (A) GSK-3β was identified as a putative target of miR-1246 through prediction databases for bioinformatics search. Schematics showing the predicted binding site of miR-1246 in the 3' untranslated region of GSK-3β. (B-E) The protein levels of GSK-3β (B, D, E) and β-catenin (C, D, E) from A549 cells transfected with miR-1246 mimics or negative control (NC), miR-1246 inhibitor, or inhibitor negative control (Inh-NC) were determined by immunofluorescence and western blot assay, respectively. All the values are shown as mean±standard error of mean and pooled from three independent experiments (*p < 0.05).

Reference

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