Research Article

Over-Expression of Cofilin-1 Suppressed Mobility of Lung Cancer Cells is Associated with Down-Regulation of SNAIL-1 and Induction of Let-7

Wang CY1, Tsai CH2, Chang CY2, Liao MJ2, Liu RS2-4 and Lee YJ2,5*
1Department of Medical Imaging, Cheng Hsin General Hospital, Taiwan
2Departments of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taiwan
3Department of Nuclear Medicine, National PET/Cyclotron Center, Taiwan
4Molecular and Genetic Imaging Core, Medical School, National Yang-Ming University, Taiwan
5Biophotonics & Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taiwan

*Corresponding author: Yi-Jang Lee, Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St. Beitou District, 112, Taipei, Taiwan

Published: 01 Jun, 2016
Cite this article as: Wang CY, Tsai CH, Chang CY, Liao MJ, Liu RS, Lee YJ. Over-Expression of Cofilin-1 Suppressed Mobility of Lung Cancer Cells is Associated with DownRegulation of SNAIL-1 and Induction of Let-7. Clin Oncol. 2016; 1: 1015.


Metastatic lung cancer means the spread of cancer from the primary site to nearby structures or distant organs. Epithelial-mesenchymal transition (EMT) is an important mechanism to be associated with metastasis. Suppression of EMT may prevent the cancer metastasis. We previously found that over-expression of cofilin-1, an actin binding protein belongs to the actin depolymerizing factor (ADF)/cofilin family leads to morphological change and inhibition of invasion of human non-small cells lung cancer (NSCLC). This effect is associated with up-regulation of the tumor suppressive let-7 microRNA through TWIST-1 transcription factor, an important biomarker of EMT. Here we investigated whether other EMT related molecules would be affected by overexpressed cofilin-1. Over-expression of cofilin-1 in human H1299 lung cancer cells also suppressed SNAIL-1 transcription factors, but E-cadherin and N-cadherin were not significantly affected. Importantly, over-expression of cofilin-1 induced let-7 could be suppressed by enforced expression of SNAIL-1, suggesting that EMT related transcription factors can be suppressed by over-expressed cofilin-1 to induce let-7 expression. However, over-expression of cofilin-1 may not suppress EMT. To monitor the effects of cofilin-1 and let-7 on lung cancer migration in vivo, we established a multiple reporter genes transduced lung cancer cell line that can be detected using the reporter gene imaging. The cofilin-1 induced let-7 was suppressed by transfection of locked nucleic acid (LNA) to inhibit let-7. Compared to normal lung cancer cells, over-expression of cofilin-1 suppressed the lung cancer migration, but simultaneously transfection of LNA recovered their migration ability to lungs in small animals. Taken together, over-expression of cofilin-1 can suppress the invasion and migration of lung cancer cells through up-regulation of let-7 in vitro and in vivo. Additionally, cofilin-1 may regulate EMT related transcription factors but not the whole EMT mechanism.

Keywords: Cofilin-1; SNAIL-1; let-7; EMT; Reporter gene imaging; Locked nucleic acid


According to American Cancer Society's estimation, lung cancer is the second most common cancer type independent of sex. However, lung cancer is the leading cause of death in cancer patients worldwide [1]. More than 85% of lung cancer belongs to non-small cells lung cancer (NSCLC) that includes several subtypes, such as adenocarcinoma, squamous cell carcinoma, large carcinoma and less commonly found adenosquamous carcinoma. Metastasis is the primary cause of lung cancer death, including bone and brain metastasis. Therefore, suppression of metastasis is important for tumor control.
Actin cytoskeleton is formed by Rho small GTPase signaling pathway that can form different types of actin architectures for cell morphology, attachment and migration. This signaling mediates the activity of cofilin-1, a non-muscle is form of actin depolymerizing factor (ADF)/cofilin family member to accelerate the actin dynamics [2,3]. Over-expression of cofilin-1 may disrupt the balance of actin dynamics and lead to obstacle of cell motility. However, the underlying mechanisms remain to be addressed.
Metastasis is strongly associated with the epithelial –mesenchymal transition (EMT). EMT is a process of cell morphological change that allows cancer penetrating through the vessel and traveling to distant organs for regrowth [4,5]. Several markers of EMT with the property of transcription factors have been widely reported, including Twist Basic Helix-Loop-Helix Transcription Factor 1 (TWIST-1), Zinc finger protein SNAI1 (SNAIL-1) and SNAI2 (Slug), Zinc finger E-box-binding homeobox 1/2 (ZEB1 and ZEB2) [6,7]. E-cadherin is responsible for cell-cell adhesion and is usually down-regulated by these transcription factors followed by the emergence of mesenchymal shapes [8]. N-cadherin is up-regulated to balance the down-regulated E-cadherin for altered cell adhesion [9]. These transcription factors also promote anti-apoptosis, angiogenesis, chromosomal instability, and are generally regarded oncogenes. Interestingly, recent reports showed that TWIST-1 can suppress the expression of tumor suppressive lethal-7 (let-7) microRNA, which can further regulate Ras and high mobility group A2 (HMGA2) oncogenes [10-12]. Additionally, low let-7 level significantly correlates to the postoperative death [13]. However, it is unclear whether different EMT-related markers will influence the expression of let-7 or not.
Because over-expression of cofilin-1 would disrupt the actin dynamics, it is speculated that cell migration should be affected. Previously, we have found that over-expressed cofilin-1 could suppress the expression of TWIST-1 in NSCLC cells [14]. The let- 7 family members were subsequently up-regulated, and let-7b and let-7e exhibited most significant up-regulation. Because TWIST-1 is involved in EMT, it is interesting to investigate whether other EMT related molecules can regulate the expression of let-7 microRNA. In this study we further examined the expression of SNAIL-1 and E-cadherin after over-expression of cofilin-1 in lung cancer cells. Additionally, we used reporter gene imaging to monitor whether knockdown of let-7 in cofilin-1 over-expressing lung cancer cells would recover the migration ability in small animals. This study would establish a signaling pathway between cofilin-1 and let-7 that regulate lung cancer migration in vitro and in vivo.

Figure 1

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Figure 1
Effects of over-expressed cofilin-1 on EMT related molecules. (a) Morphological change induced by over-expression of cofilin-1. Insets are enlarged photos of normal cells and cofilin-1 over-expressing cells. (b) Western blot analysis for detection of EMT related molecules in response to over-expression of cofilin-1.

Materials and Methods

Cell culture
Human lung cancer H1299 cells with tet-on inducible cofilin-1 over-expression cell line (HCOXP) and reporter genes harboring HCOXP cells (HCOXP-3R) were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, 2mM L-glutamine (Sigma-Aldrich Co, St. Louis, MO, USA), and 0.1mg/ml hygromycine B (Invitrogen, Carlsbad, CA, USA). HCOXP cells were maintained in a humidified incubator with 5% CO2 and 37ºC by passaged every 48 hours.
Western blot analysis and antibodies
The procedure of protein extraction, gel running and electrotransferring has been described previously [15]. The primary antibodies used in this study include: anti-cofilin-1, anti- ser3- phospho-cofilin-1, anti-Twist-1, anti-TWIST-1 (Genetex Inc., Irvine, CA, USA), anti-SNAIL-1, anti-cofilin-1 (Genetex Inc., Irvine, CA, USA), anti-phospho-cofilin-1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-E-cadherin, anti-N-cadherin, anti-HSV1-tk (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and anti- GAPDH (Sigma-Aldrich Co, St. Louis, MO, USA) antibodies.
Quantification of let-7
To measure let-7 microRNA levels before and after cofilin-1 over-expression, quantitative PCR (qPCR) of targeted miRNA was used. In brief, complementary DNA (cDNA) was generated from 5 µg total RNA using SuperScript II reverse transcriptase (LifeTechnologies Co, Carlsbad, CA, USA). The cDNA products were then mixed in the Fast SYBR Green Master Mix (Life-Technologies Co, Carlsbad, CA, USA) and subjected to the Step One Plus RealTime PCR System (Life-Technologies Co, Carlsbad, CA, USA) according to the manufacturer's instructions. The stem loop primers used for let-7b and let-7e were 5'-GTCGTATCCAGTG CAGGGTCCGAGGTATTCGCACTGGATACGACAACcac-3' and 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGC ACTGGAT ACGACAACTAT-3', respectively. The forward primers of qPCR used for let-7b and let-7e were 5'- GCCGCTTGAGGTAGTAGGTTGT-3' and 5'-GCCGCTTGAGGtAGGAGGTTGT-3', respectively. The universal reverse primer was used for both let-7: 5'-CCAGTGC AGGGTCCGAGGT-3'. For internal control, the vertebrate U6 small nuclear RNA was amplified using the primer set: 5'-CGCTTCGGC AGCACATATAC-3' and 5'-TTCACGAATTTGCGTGTCAT-3'. Establishment of HCOXP-3R cells and reporter gene validations – LT-3R plasmid, a multicistronic lentiviral construct was used to establish HCOXP-3R cells for expressing firefly luciferase (fLuc), green fluorescent protein (GFP) and herpes simplex virus type 1-thymidine kinase (HSV1-tk) reporter genes [16]. This plasmid was co-transfected with pCMV-∆R8.91 plasmid and pMD.G plasmid into the 293T packaging cell line to produce virion soup using the calcium phosphate precipitation method. Ultracentrifugation was used to concentrate the virion soup, which was added to HCOXP cell culture for infection. After infection, cells were sorted by the fluorescenceactivated cell sorting (FACS, FACSAria, BD Biosciences, San Jose, CA, USA) based on the GFP emitted fluorescent signals. The obtained stable clone was named HCOXP-3R cells, in which the GFP expression was visualized using the fluorescent microscope. The fLuc activity and expression of HSV1-tk protein were determined by the luciferase assay and Western blot analysis as described before [16].
Knockdown of let-7 – Chemically modified locked nucleic acid (LNATM, Exiqon, Los Angeles, CA, USA) was used to silence the expression of let-7 microRNA. In brief, 30nM LNA was transfected into HCOXP-3R cells using the JetPEI transfection reagent (Polyplustransfection, SA, Illkirch, France). The sequences of LNA for targeting on let-7b and let-7e were 5'-ACCACACAACCTACTACCTC-3' and 5'-ACTATACAACCTCCTACCTC-3', respectively.
Luciferase assay – The expression of luciferase activity in cells with over-expressed cofilin-1 and co-transfected LNA were determined using the in vitro luciferase assay. Cells were cultured in 12-well plates and lyzed by passive lysis buffer and then added with 5-fold diluted reporter assay buffer (50mM glycylglycin, 1M magnesium sulfate, 10mg/ml bovine serum albumin, and 0.5M EDTA) mixed with 100mM adenosine 5'-triphosphate disodium salt (Sigma-Aldrich Co., St. Louis, MO, USA), 1M dithiothreitol and 50mM D-luciferin luciferin (Promega Co., Madison, WI, USA) transferring to a 96-wel black plate. The luminescent signals were detected using a multimode microplate reader (TECAN, Switzerland).
In vitro invasion assay – Cells were trypsinized and five thousand cells were seeded in transwells coated with Matrigel (BD biosciences, San Jose, CA, USA) in serum-free DMEM. Each transwell was placed in a 24-well dish containing DMEM with 10% FBS. After 48 hours of incubation, the transwells were cleaned with a cotton stub, fixed using 4% paraformaldehyde followed by crystal violate (1.25% in ethanol) staining for 30 minutes. The transwells were then rinsed, visualized and counted under a bright-field microscope.
Experimental metastasis animal model and bioluminescent imaging
HCOXP-3R cells (1x106) were injected into the nude mice via tail veins. After injection, the animals were subjected to IVIS-50 imaging system (Caliper Co, Hopkinton, MA, USA) to detect the bioluminescent signals in vivo. Before imaging, the animals were i.p. injected with 150mg/kg D-luciferin (VivoGlo Luciferin, Promega Corp., Madison, WI, USA) and anesthetized with 1% isofluorane for 15 minutes. The images were acquired using the bundled software. The animal studies have been approved by Institutional Animal Care and Use Committee (IUCAC No. 1021208) of National Yang-Ming University.
Statistic analysis
Each datum represented means ± S.D., and the results were analyzed by student's t-test between two samples. For multiple samples, one-way analysis of variance (ANOVA) was used for statistic analysis. In both conditions, p < 0.05 was regarded significance. The analysis and plots were executed using Sigmaplot 10.0 software (Systat Software, Inc, a Jose, CA, USA).


Effects of cofilin-1 over-expression on EMT related molecules
HCOXP cells are derived from H1299 lung cancer cells harboring a tet-on gene expression system for over-expression of cofilin-1. Because induction of cofilin-1 expression in these cells will lead to apparent morphological change likes EMT, we investigated whether over-expression of cofilin-1 would influence the EMT related molecules (Figure 1A). TWIST-1, SNAIL-1, N-cadherin and E-cadherin were examined after over-expression of cofilin-1 using the Western blot analysis. Although TWIST-1 and SNAIL-1 were downregulated by over-expressed cofilin-1, E-cadherin and N-cadherin levels were not changed significantly (Figure 1B). Over-expressed cofilin-1 can be phosphorylated on the serine-3 as described before. Therefore, these data suggest that cofilin-1 would influence the expression of TWIST-1 and SNAIL-1 transcription factors but not the whole EMT related biomarkers.
Over-expression of cofilin-1 induced let-7 up-regulation was suppressed by SNAIL-1
Previously, we have found that over-expression of cofilin-1 can induce let-7 microRNA through suppression of TWIST-1 expression [14]. Over-expression of TWIST-1 can counteract the induced let- 7 microRNA by over-expressed cofilin-1. Here we investigated whether SNAIL-1 transcription factor can also regulate let-7 or not. In HCOXP cells, induction of cofilin-1 expression could induce let- 7b and let-7e that have been reported to be most responsive to overexpressed cofilin-1 (Figure 2A). We next showed that in doxycycline treated HCOXP cells, transfection of pCDH-SNAIL-1 construct could restore the SNAIL-1 level in these cells (Figure 2B). This treatment led to suppression of let-7b and let-7e induced by over-expressed cofilin-1 (Figure 2C). Therefore, down-regulation of SNAIL-1 by over-expressed cofilin-1 is also involved in regulate the expression of let-7 microRNA.
Characterization of HCOXP-3R cells responding to knockdown of let-7 after over-expression of cofilin-1 – We have previously established a lentiviral-based multicistronic reporter construct including green fluorescent protein gene (GFP), firefly luciferase (fLuc), and herpes virus type 1 – thymidine kinase (HSV1-tk) gene, which are used for examination of transfection efficiency, bioluminescent imaging, and radionuclide based imaging in vivo, respectively. Here we transduced this construct into HCOXP cells that have not been examined before. The obtained stable cells were named HCOXP-3R cells. The expression of GFP was examined using the fluorescent microscope (Figure 3A). The activity of fLuc was determined using the luciferase assay with luciferin substrate (Figure 3B). The expression of HSV1-tk was examined using Western blot analysis (Figure 3C). This novel stable lung cancer cell line would be used for in vivo tracking under different conditions. We next compared the cell viability and invasive ability in HCOXP-3R cells before and after over-expression of cofilin-1. The luciferase activity of HCOXP-3R cells were inhibited by induced cofilin-1 expression, but co-treatment of LNA also recovered the luciferase activity (Figure 3D). HCOXP-3R cells invading through the matrigel coated transwells were further compared by above conditions. The results showed that over-expression of cofilin-1 could suppress the invasion, but the effects were compromised after treatment of let-7 targeting LNA (Figure 3E). Thus, the in vivo effect of over-expressed cofilin-1 on lung cancer was subsequently examined using this novel stable clone.
Bioluminescent imaging of lung accumulation by HCOXP-3R cells responding to cofilin-1 over-expression and LNA treatment – We next investigated whether migration of HCOXP-3R cells to lungs will be affected by over-expressed cofilin-1 and co-treated LNA. HCOXP-3R cells were either treated with doxycycline to induce cofilin-1 expression or transfected with LNAs followed by doxycycline treatment. These cells (1x106 each) were then separately i.v. injected into nude mice. After injection, the mice were subjected to IVIS- 50 system and imaged for the bioluminescent signals immediately. Compared to untreated cells, over-expression of cofilin-1 apparently inhibited the migration of HCOXP-3R cells to lungs (Figure 4A and B). However, suppression of let-7b or let-7e by LNAs could recover the lung migration of cofilin-1 over-expressing cells (Figure 4C and D). The photons flux in chest of each group was also semi-quantified according to the analytic software of IVIS system (Figure 4E). Therefore, the effects of cofilin-1 signaling pathways on lung cancer growth and metastasis in vivo would be easily examined using the HCOXP-3R cells in the future.

Figure 2

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Figure 2
Effects of SNAIL-1 on suppression of let-7 induced by over-expression of cofilin-1. (a) Quantification of let-7b and let-7e using qPCR before and after over-expression of cofilin-1. (b) Transfection of pCDH-SNAIL-1 to HCOXP cells followed by Western blot analysis. (c) Over-expression of SNAIL-1 suppressed the expression of let-7b and let-7e induced by cofilin-1.


Previously, over-expression of cofilin-1 was found to induce let-7 microRNA in human H1299 lung cancer cells. This signaling pathway was mediated by TWIST-1 rather than other let-7 regulators, that is, LIN28B and c-Myc [14]. Because TWIST-1 is known to be one of the important markers of EMT, it is of interest to investigate whether over-expression of cofilin-1 induced let-7 is caused by suppression of EMT. Although SNAIL-1 was also down-regulated by over-expressed cofilin-1, another EMT markers E-cadherin and N-cadherin were not significantly affected under this condition. The basal level of E-cadherin in H1299 cells were barely detected as reported previously [17], while suppression of TWIST-1 and SNAIL-1 by over expressed cofilin-1 did not up-regulate E-cadherin. N-cadherin was also not reduced. Therefore, the current data suggest that cofilin-1 may influence certain EMT related molecules rather than the EMT phenomenon.
The expression of let-7 has been reported to be regulated by LIN28A/B, c-Myc and TWIST-1 during cancer development [12,18,19]. As a tumor suppressor, up-regulation of let-7 leads to suppression of tumor metastasis and cancer growth. Interestingly, a recent report showed that SNAIL-1 can temporarily bind to let-7 promoters and reduce its expression for efficient reprogramming of fibroblasts [20]. Here we found that let-7b and let-7e induced by overexpressed cofilin-1 could be suppressed by transfection of SNAIL-1. This result suggests that the SNAIL-1 regulated let-7 expression also plays a role in lung cancer cells. Like TWIST-1, SNAIL-1 also mediates the cofilin-1 regulated let-7 expression. How over-expression of cofilin-1 leads to down-regulation of TWIST-1 and SNAIL-1 but not E-cadherin is unclear and may be important to be studied in the future.
The most critical experiments in this report were the first time to demonstrate the cofilin-1/let-7 signaling pathway would affect the migration of lung cancer cells to lungs in vivo. To this end, we used the reporter gene imaging to track the positions of cancer cells in the nude mice. The multicistronic reporter gene construct, so called LT-3R has been used in tracking the growth of glioblastomas in vivo [16]. Here we transduced this construct to HCOXP cells, which were subsequently i.v. injected into small animals. This experimental metastasis model has been used for investigating the cancer cells migration to lungs in different cancer types [21,22]. The LT-3R plasmid transduced HCOXP cells exhibited similar phenotypes with parental HCOXP cells, including cell growth and growth suppression by over-expressed cofilin-1. Because reporter genes only express in viable cells [23], the luciferase assay should be sufficient to demonstrate the effects of over-expressed cofilin-1 on suppression of cell viability. Transfection of let-7 targeted LNA in cofilin-1 overexpressing HCOXP-3R cells led to recovery of luciferase expression, suggesting that let-7 is important for mediating the effects of cofilin-1 on cell growth and viability. Furthermore, the cellular invasive ability suppressed by over-expressed cofilin-1 was also recovered by LNA. These in vitro studies support that HCOXP-3R cells can used as a surrogate to monitor the behaviors of HCOXP cells in vivo.
To investigate the cancer metastasis in small animals, both "experimental metastasis" (intravascular injection of cells) and "spontaneous metastasis" (orthotopic injection of cells) are usually applied [24]. Because lung cancer model was used here, we first examined whether over-expression of cofilin-1 could affect the migration of these cells to lungs via i.v. injection, and whether LNA would reverse the cofilin-1 effects as found in in vitro studies. The data showed that the in vivo study was consistent with in vitro effects of cofilin-1 over-expression. However, we could not detect the migration of HCOXP-3R cells out of lungs because the bioluminescent signals were disappeared on next day (data not shown). This should not due to the loss of reporter genes because the expression of reporter genes in HCOXP-3R cells remains detectable after one month of continuous culturing. The current data suggest that over-expression of cofilin-1 may inhibit the migration of HCOXP-3R cells to lungs, but suppression of let-7 in these cells will compromise this effect. Whether different levels of bioluminescent signals detected in lungs represents reduced extravasation of HCOXP -3R cells after overexpression of cofilin-1 is of interest to further investigate.
In summary, this is the first study showing that over-expressed cofilin-1 can inhibit the migration of lung cancer in vivo, to the best of our knowledge. We also demonstrate that cofilin-1 induced let-7 is required for inhibition of migration because knockdown of let-7 by LNA can reverse this effect. Although down-regulation of TWIST-1 has been previously reported to be required for cofilin-1 mediated up-regulation of let-7, SNAIL-1 transcription factor is also involved in this signaling pathway. However, it appears that EMT is not the primary role to be ablated by over-expressed cofilin-1. Taken together, the in vivo evidence of cofilin-1/let-7 pathway on controlling the lung cancer migration would be important for clinical consideration of molecular targeting therapy.

Figure 3

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Figure 3
Effects of knockdown of let-7 in HCOXP cells with co-expressed reporter genes. (a) Fluorescent microscope for visualizing GFP. (b) Luciferase assay for detecting the luciferase activity. (c) Detection of HSV1-tk protein by Western blot analysis. (d) Comparison of cell viability by detecting the luciferase activity in cofilin-1 over-expressing cells before and after transfection of LNA. (e) Comparison of in vitro invasion ability according to above conditions. *: p<0.05.

Figure 4

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Figure 4
In vivo imaging of HCOXP-3R cells distribution through intravenous injection into nude mice. (a) Untreated HCOXP-3R cells. (b) Doxycycline treated HCOXP-3R cells. (c) Let-7b and (d) let-7e targeted LNA transfected HCOXP-3R cells that have been treated with doxycycline. (e) The regions of interest (ROI) of each group were marked by red circles and quantified for the BLI signals. *: p < 0.05. **: p < 0.01 (N=4).


This study was supported by a united grant of Cheng-Hsin General Hospital and National Yang-Ming University (103F003C05), and the Ministry of Science and Technology of Taiwan (102-2628-B-010-012-MY3).


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