|Year : 2015 | Volume
| Issue : 7 | Page : 168-171
miR-338-3p suppresses epithelial-mesenchymal transition and metastasis in human nonsmall cell lung cancer
W Hong-yuan, C Xiao-ping
Department of Respiratory, The Sixth Affiliated Hospital of Wenzhou Medical University, People's Hospital of Lishui City, Zhejiang, China
|Date of Web Publication||20-Jul-2016|
Department of Respiratory, The Sixth Affiliated Hospital of Wenzhou Medical University, People's Hospital of Lishui City, Zhejiang
Source of Support: None, Conflict of Interest: None
OBJECTIVES: MicroRNAs are important modulators of the cellular epithelial-mesenchymal transition (EMT) process and are associated with metastasis in human nonsmall cell lung cancer (NSCLC). In this study, we tried to investigate the role of miR-338-3p in NSCLC cells. MATERIALS AND METHODS: Real-time polymerase chain reaction was applied to quantify the expression levels of miR-338-3p, as well as EMT-associated molecules in NSCLC cells. Wound healing and transwell assays were performed to evaluate the migration and invasion capacities, respectively. Dual-luciferase reporter assay was finally performed to determine the targeting of zinc finger E-box-binding protein 2 (ZEB2) by miR-338-3p. RESULTS: We found that miR-338-3p was significantly reduced in NSCLC cell lines. Forced expression of miR-338-3p in A549 cells led to the suppression of migration/invasion capacity and inhibition of epithelial markers. In addition, we proved that miR-338-3p could directly target ZEB2. CONCLUSIONS: In general, we summarized that miR-338-3p could inhibit EMT and metastasis of human NSCLC cells, which probably via directly targeting ZEB2 expression.
Keywords: Epithelial-mesenchymal transition, migration/invasion, miR-338-3p, nonsmall cell lung cancer, zinc finger E-box-binding protein 2
|How to cite this article:|
Hong-yuan W, Xiao-ping C. miR-338-3p suppresses epithelial-mesenchymal transition and metastasis in human nonsmall cell lung cancer. Indian J Cancer 2015;52, Suppl S3:168-71
|How to cite this URL:|
Hong-yuan W, Xiao-ping C. miR-338-3p suppresses epithelial-mesenchymal transition and metastasis in human nonsmall cell lung cancer. Indian J Cancer [serial online] 2015 [cited 2020 Feb 28];52, Suppl S3:168-71. Available from: http://www.indianjcancer.com/text.asp?2015/52/7/168/186569
| » Introduction|| |
Recent studies have found that microRNAs (miRNAs) appear as an important subtype of noncoding RNAs in the regulation of a wide range of biological processes, especially involved in critical pathways linked to cancer cell proliferation, apoptosis, and metastasis. ,,, They are approximately 20-23 nucleotides in length and can regulate target gene expression through degradation of the corresponding messenger RNA (mRNA) or inhibition of translation by base pairing to the 3'-untranslated region (3'-UTR) of the target mRNAs. ,
Previous studies have shown that miR-338-3p is downregulated in some types of human cancers, which including gastric cancer, , colorectal carcinoma,  and neuroblastoma,  in which it functions as a potent tumor suppressor in these cancer cells by regulating cell growth, epithelial-mesenchymal transition (EMT), metastasis, and chemosensitivity. ,, Regarding lung cancer, particularly the most common nonsmall cell lung cancer (NSCLC), miR-338-3p was reported to be downregulated in clinical NSCLC tissues and correlated with NSCLC cancer differentiation, pathological stage, and lymph node metastasis. They also showed that its overexpression significantly suppressed the in vitro proliferation and colony formation of NSCLC cells and enhanced apoptosis, and this regulation might be explained by the targeting of Ras-related protein 14 (RAB14) gene.  However, whether miR-338-3p also regulated EMT or metastasis processes of NSCLC cells remain elusive. To answer this question, we designed experiments to demonstrate it.
According to the statistics, more than 90% of the NSCLC patients die of distant metastasis.  Metastasis is constituted of a series of steps in which cancer cells can leave from the original site and migrate to other parts of the body via the bloodstream and the lymphatic system.  EMT is considered as one of the critical steps in NSCLC cell invasion and metastasis, by which cells lose their epithelial phenotypes and cell-cell adhesion and gain invasive mesenchymal features.  Transcription factors such as zinc finger E-box-binding protein (ZEB) are known to be involved in EMT regulation in cancer cells. , Hence, our efforts on understanding the role of miR-338-3p in EMT and metastasis processes of NSCLC cells might provide crucial evidence and therapeutic targets for its treatment in future.
| » Materials and Methods|| |
Cell culture and transfection
For the culture of cell lines used in this study, the human NSCLC A549, H661, and H1299 cells also human bronchus epithelial BEAS-2B cell were cultured in DMEM (Dulbecco's Minimum Essential Medium) containing 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO 2 . The human NSCLC H460, H520, and H1975 cells were grown in 1640 medium supplemented with 10% FBS at 37°C with 5% CO 2 . miR-338-3p mimics and controls were purchased from Shanghai GenePharma (Shanghai, China). Transfections of miRNAs mimics were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
RNA extraction and quantitative real-time-polymerase chain reaction
RNA was extracted from cells using TRIzol according to the manufacturer's instructions. Reverse transcription was performed using M-MLV with random primers. The relative expression level of miR-338-3p was detected by real-time polymerase chain reaction (PCR) with ABI7500 thermocycler. U6B RNA was used as an endogenous control.
EMT protein expression was measured by Western blot. Total protein was extracted from transfected cells using the radio immunoprecipitation assay lysis buffer. For Western blot analysis, equal amounts of protein for each sample were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E-cadherin and N-cadherin antibodies were purchased from Cell Signaling Technology, whereas vimentin and β-actin antibodies were purchased from (Santa Cruz Biotechnology, Inc.).
Wound healing assay
For the wound healing assay, 5 × 10 5 transfected A549 cells were seeded into 6-well plates and cultured until confluent. A P1000 pipette tip was used to make a straight line simulation "wound." Then, cells were washed several times with phosphate-buffered saline to remove cell debris. The extent of wound closure was monitored at 24 h.
Transwell invasion assays
For the invasion assays, the transfected A549 cells were plated in the top chamber with a BD Matrigel-coated membrane. The cells were plated in a serum-free medium, and a medium supplemented with 10% serum was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h at 37°C and 5% CO 2 in a tissue culture incubator. After 24 h, the noninvading cells were removed from the upper sides of the transwell membrane filter inserts. The invaded cells on the lower sides of the inserts were stained with crystal violet, and the cells were counted.
The pmirGLO-ZEB2-3'UTR-WT or pmirGLO-ZEB2- 3'UTR-MUT vector was cotransfected with miR-338-3p into A549 cells. Cells were harvested and lysed for luciferase assays at 24 h after transfection. The Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used to measure the reporter activity according to the manufacturer's protocol.
The information of human miR-338-3p was obtained in miRBase (http://www.miRBase.org/). The prediction of miR-338-3p targets was acquired from TargetScan program (http://www.targetscan.org/).
SPSS 18.0 software was used for the statistical analysis. The data are presented as the means ± standard deviation. Group comparisons were performed using Student's t-test. Differences were considered statistically significant at P < 0.05.
| » Results|| |
Expression of miR-338-3p was low in nonsmall cell lung cancer cells
We firstly confirmed the expression of miR-338-3p in NSCLC since it was shown to be downregulated in NSCLC tissues previously by Sun et al.  Real-time PCR results shown in [Figure 1] validated its lower expression in these NSCLC cell lines when compared to that of noncancerous BEAS-2B. Among these cell lines, A549 as the high-metastasis NSCLC cell line exhibited the most decreased expression of miR-338-3p [Figure 1], compare lane 2 with lane 1]. However, for the H1299 cell line, the expression level of miR-338-3p was instead a bit increased [Figure 1], compare lane 6 with lane 1], suggesting diverse roles of this miRNA in NSCLC cells.
|Figure 1: Decreased expression of miR-338-3p in nonsmall cell lung cancer cell lines. Relative expression of miR-338-3p was detected by real-time polymerase chain reaction in six nonsmall cell lung cancer cell lines and a normal human bronchial epithelial cell line (BEAS-2B). miR-338-3p expression level was calculated by the 2−ΔCt method and normalized to U6B RNA. **P < 0.01|
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miR-338-3p inhibited migration/invasion of A549 cells
The above observation of miR-338-3p in A549 cells suggested that miR-338-3p could be associated with the migration and invasion of A549 cells. To investigate this hypothesis, we performed wound healing and transwell assays to evaluate the migration and invasion capacities, respectively, after miR-338-3p was enforced expressed in A549 cells. Overexpression of miR-338-3p was done using a mimic of this miRNA, and the effect was proved by real-time PCR shown in [Figure 2]a. The results shown in [Figure 2]b showed that miR-338-3p could inhibit migratory capacity illustrated by wound healing. In addition, the transwell invasion assay demonstrated that overexpressing of miR-338-3p also suppressed invasion of A549 cells [Figure 2]c. These results hence proved that miR-338-3p is a suppressor of NSCLC metastasis, at least in our tested cells. Because EMT is one of the critical steps of cancer cell invasion, we then tested whether miR-338-3p was involved in EMT progression.
|Figure 2: miR-338-3p suppresses the migration and invasion of A549 cells. (a) A549 cells were transfected with miR-338-3p mimics. The expression level of miR-338-3p was detected by real-time polymerase chain reaction. ** P < 0.01. (b) Cell migration activity was measured by conducting the wound healing assay 48 h after transfection (24 h after scratching) by miR-338-3p mimics. (c) For testing cell invasive ability, transwell assay was performed by miR-338-3p mimics|
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miR-338-3p inhibited epithelial-mesenchymal transition progression and negatively regulated zinc finger E-box-binding protein 2
We then analyzed the protein expression levels of the epithelial marker E-cadherin, and two mesenchymal markers including (N-cadherin and vimentin) in our system. Western blot results shown in [Figure 3]a showed altered expression of these markers after miR-338-3p overexpression, which indicated blocking of EMT. These results suggested that miR-338-3p is an important EMT suppressor thus contributing to metastasis inhibition in NSCLC cells. According to Huang's report, which characterized that miR-338-3p could target ZEB2 and led to EMT inhibition, we suspected that in our system, miR-338-3p could also target ZEB2. Bioinformatics analysis predicted that there is a binding site of miR-338-3p in the 3'- UTR region of ZEB2 gene [Figure 3]b. Dual-luciferase reporter assay was then performed to determine the targeting of ZEB2 by miR-338-3p. The results came out that A549 cells cotransfected with mimics of miR-338-3p and ZEB2 3'-UTR showed a significant decrease in luciferase activity, whereas cotransfected with mutated ZEB2 3'-UTR showed no change [Figure 3]c. Therefore, we summarized that miR-338-3p could directly target ZEB2, an important transcription factor involved in EMT transition.
|Figure 3: miR - 338 - 3p inhibits epithelial - mesenchymal transition by target zinc finger E - box - binding protein 2 in A549 cells. (a) Protein levels of epithelial-mesenchymal transition markers, E-cadherin, N-cadherin, and vimentin were detected by Western blot in A549 cells transfected with miR-338-3p mimic. (b) The schematic diagram shows the luciferase reporter of miR - 338 - 3p binding site on the wild - type (blue) and mutant (red) zinc finger E - box - binding protein 2 3′ -untranslated region. (c) Relative Dual-luciferase activity analyses. The pmirGLO-ZEB2-3′ UTR-WT or pmirGLO-ZEB2-3′ UTR-MUT was transfected into A549 cells with miR - 338 - 3p mimic or its control plasmid. Renilla luciferase activity was used as internal controls. **P < 0.01|
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| » Discussion|| |
This study revealed that miR-338-3p was downregulated in NSCLC cell lines. In vitro experiments showed that forced expression of miR-338-3p caused inhibition of EMT progression as well as migration/invasion activities significantly. Regarding the underlined mechanisms, we indicated that directly targeting of ZEB2 might contribute to the functions of miR-338-3p in the above processes. Although substantial work needs to further explore the underlying mechanisms of miR-338-3p in the EMT and metastasis of NSCLC, we still provide vital clues to characterize the key roles of miR-338-3p in NSCLC and facilitate its application for future treatment strategies.
The dysregulation and function of miR-338-3p are widely studied in other malignancies. In particularly, it was proved in NSCLC cancer that miR-338-3p was aberrantly downregulated in clinical NSCLC tissues and suppressed cell survival in vitro.  However, the role of miR-338-3p in NSCLC metastasis is largely unknown. Our studies enrich the knowledge of miR-338-3p in NSCLC cells, at least in high-metastasis A549 cells. However, it was still unclear whether it was the same in other NSCLC cell lines except A549.
Increasing evidence has indicated that EMT can be regulated by miRNAs, in which miR-34 and the miR-200 families are mostly studied.  By affecting EMT and the contrast MET processes, miRNAs are also involved in the regulation of stem cell pluripotency and the control of tumor progression. For miR-338-3p, there is only one report demonstrated that miR-338-3p could inhibit EMT in gastric cancer cells through targeting ZEB2 and MACC1/Met/Akt signaling.  ZEB2 was reported to contribute to the loss of epithelial marker E-cadherin and disruption of cell-to-cell adhesion.  In this study, we also confirmed that miR-338-3p could also target ZEB2 in A549 cells. We know it is better to confirm the correlation of miR-338-3p and ZEB2 expression in clinical NSCLC tissues which facilitate the conclusion that miR-338-3p indeed targets ZEB2.
| » Conclusions|| |
This study provided more evidence that miR-338-3p could inhibit EMT process and migration/invasion of NSCLC cells. Increasing the levels of miR-338-3p in NSCLC cells may be a novel therapeutic strategy for the treatment of patients with advanced NSCLC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
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