Research ArticleInhibiting of Proliferation, Migration, and Invasionin Lung Cancer Induced by Silencing Interferon-InducedTransmembrane Protein 1 (IFITM1)

Jun Yan, Ying Jiang, Jianfeng Lu, JianhuiWu, andMingfang Zhang

Department of Pathology, Tianjin First Center Hospital, Tianjin 300192, China

Correspondence should be addressed to Mingfang Zhang; 3032067811@qq.com

Received 12 December 2018; Revised 27 March 2019; Accepted 18 April 2019; Published 8 May 2019

Academic Editor: Kwang Gi Kim

Copyright © 2019 Jun Yan et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Interferon-induced transmembrane protein 1 (IFITM1), a 17-kDa membrane protein, is generally known as a modulator in manycellular functions. Recent studies showed overexpression of IFITM1 in cancers and relationship between IFITM1 overexpressionand tumor progression. However, the role of IFITM1 in lung cancer remains unclear. Here, we presented the overexpression ofIFITM1 in lung cancer tissues and cell lines A549 and H460 using quantitative Real-Time RT-PCR. In vitro assay indicated IFITM1silencing inhibited lung cancer cell proliferation, migration, and invasion. Further, in vivo assay showed that IFITM1 silencingmarkedly suppressed cell growth and metastasis of lung cancer in tumor-bearing BALB/c nude mice. Mechanistically, we foundthat IFITM1 silencing significantly alleviated the protein levels of 𝛽-catenin, cyclin D1, and c-Mycin lung cancer cells and tumorsamples. Taken together, our study revealed the role of IFITM1 as a tumor promoter during lung cancer development and thepossible molecular mechanism.

1. Introduction

Lung cancer, a common type of cancers in the world, isdivided into two categories: small cell lung cancer (SCLC) andnon-small-cell lung cancer (NSCLC) [1]. The latter accountsfor about 80% of cases of lung cancer and is further classifiedinto adenocarcinoma, squamous cell carcinoma, large cellcarcinoma, and so on [2]. By far, therapeutic approachesfor lung cancer include palliative care, surgery, radiationtherapy, chemotherapy, and target therapy [3–5]. Despite agreat improvement in these therapies, lung cancer still has ahigh mortality owing to late diagnosis and poor response totherapies [6, 7]. On the one hand, we shall explore potentialmarkers to improve the diagnosis of lung cancer. On theother hand, we shall study the mechanisms underlying theprogression of lung cancer to help make the treatment moreeffective.

Interferon-induced transmembrane protein 1 (IFITM1), a17-kDa membrane protein, is part of membrane complexestransducing homotypic adhesion signals in lymphocytes[8–10]. It was originally identified as Leu13, a cell-surface

membrane protein encoded on the short arm of chromosome11 [11–14]. Its expression is induced by IFN-𝛼 and, to a lesserextent, by IFN-𝛾 and has been found in Jurkat cells, U937cells, and K562 cells [15, 16]. IFITM1 is largely known asa player in many cellular functions such as adhesion andproliferation [17]. It is also important for immunity andantiviral activity [18]. In addition, IFITM1 has been reportedto be related to inflammatory bowel disease, osteogenesis,and angiogenesis [19–21]. Recently, there have been studiesshowing overexpression of IFITM1 in some types of can-cers such as gastrointestinal, colorectal, and breast cancers[22, 23]. Furthermore, these studies have demonstrated apositive correlation of IFITM1 overexpression with tumorprogression. However, it is poorly understood what roleIFITM1 plays in lung cancer.

In this study, we presented evidence that IFITM1 washighly expressed in lung cancer tissues and cell lines. IFITM1silencing inhibited lung cancer cell proliferation, migration,and invasion in vitro. Furthermore, the in vivo experimentsshowed that IFITM1 silencing inhibited lung cancer cellgrowth and metastasis. We also found that IFITM1 silencing

HindawiBioMed Research InternationalVolume 2019, Article ID 9085435, 9 pageshttps://doi.org/10.1155/2019/9085435

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decreased the protein levels of 𝛽-catenin, cyclin D1, andc-Myc. Taken together, our study suggested that IFITM1might function as a tumor promoter during lung cancerdevelopment.

2. Materials and Methods

2.1. Patients and Tissue Samples. Lung cancer tissues andmatching normal lung tissues were collected from 30patients hospitalized at Tianjin First CenterHospital (Tianjin,China). No patients had received any adjuvant therapiesbefore surgery. Each patient taking part in the study pro-vided written consent. All tissues were frozen in liquidnitrogen and stored at -80∘C until use. The study wasapproved by the Ethics Committee of Nanjing MedicalUniversity.

2.2. Cell Lines and Cell Culture. Human lung cancer cells(A549 and H460) and bronchial epithelial cells (BEAS-2B)were obtained from FuHeng Cell Center (Shanghai, China).All cell lines were cultured in RPMI 1640 (Sigma, St. Louis,MO, USA) containing 10% fetal bovine serum (FBS; Sigma)and 1% streptomycin/penicillin, followed by incubation at37∘C in a humidified atmosphere with 5% CO

2.

2.3. Quantitative Real-Time RT-PCR. Total RNA was iso-lated from tissues or cells using TRIzol reagent (Invitro-gen, Carlsbad, CA, USA) and then used for cDNA syn-thesis. Quantitative RT-PCR was carried out using Plat-inum SYBR Green qPCR SuperMix-UDG (Invitrogen). Allreactions were performed in triplicate. The primers werelisted as follows: IFITM1, 5’-ATGTCGTCTGGTCCCTGT-TC-3’ (forward) and 5’-GTCATGAGGATGCCCAGAAT-3’ (reverse); GAPDH, 5’-TCCACCACCCTGTTGCTG-TA-3’ (forward) and 5’-ACCACAGTCCATGCCATCAC-3’(reverse). GAPDH was used as internal control. The 2-ΔΔCtmethod was used to calculate the expression changes.

2.4. Western Blot Analysis. Tissues or cells were lysed withice-cold lysis buffer. Proteins were fractionated by 12% SDS-PAGE and then transferred onto PVDF membranes. Afterblocking in 5% skim milk, the membranes were probedwith primary antibodies against IFITM1, 𝛽-catenin, cyclinD1, c-Myc, and GAPDH (Invitrogen). Subsequently, themembranes were washed three times with TBST bufferand then incubated at 4∘C with appropriate secondaryantibodies (Invitrogen). The proteins were detected usingenhanced chemiluminescence (Millipore, Bredford, MA,USA). Immunoreactive bandswere quantifiedwith the ImageJ software.

2.5. Small Interfering RNA (siRNA) andCell Transfection. ThesiRNA targeting human IFITM1mRNA (si-IFITM1: 5’-AAC-TCATGACCATTGGATTCA-3’) and a nonspecific scramblesiRNA (si-Control: 5’-AACGTACGCGGAATACTTCGA-3’)were synthesized and purified by Sigma-Proligo (Woodlands,TX, USA). These siRNAs were transiently transfected into

A549 and H460 cells using Lipofectamine 2000 (Invit-rogen) according to the manufacturer’s instruction. Thetransfection efficiency was determined using western blotanalysis.

2.6. MTT Assay. Cell proliferation was determined usingMTT assay. In brief, cells were seeded into 96-well plates ata density of 2×103 cells/well. MTT solution (5 mg/mL) wasadded to each well at indicated time and cells were incubatedat 37∘C for 4 h. After culture medium was removed, DMSOwas added to each well. The optical density was read at 570nm with a microplate reader.

2.7. Wound-Healing Assay. The migratory capability of lungcancer cellswas examined usingwound-healing assay. Briefly,cells were seeded in 6-well plates and grown to full conflu-ency, followed by overnight incubation in starvationmedium.Cell monolayer was scratched with a 10 𝜇L sterile pipettetip, washed with PBS, and then cultured in serum-freemedium for 24h.Wound gaps were observed under OlympusBX41 microscope and photographed using a Qcolor5 digitalcamera.

2.8. Transwell Assay. Cell invasion was detected using tran-swell chambers with Matrigel-coated inserts (8𝜇m pore).5×104 cells in 200 𝜇L of serum-free medium were added tothe upper chamber. 500 𝜇L of DMEM containing 10% FBSwas added to the low chamber. After 24 h, cells remainingon the upper side of the insert were removed while thoseinvading to the lower side were stained with 0.2% crystalviolet. The number of invading cells was counted using amicroscope.

2.9. Xenograft Tumor Assay. Male BALB/c nude mice (5weeks old) were purchased from Shanghai Laboratory Ani-mals Center (Shanghai, China) and used for xenograftexperiments. All animal experiments were performed withthe approval of the Ethics Committee of Nanjing MedicalUniversity. For tumorigenesis experiments, 2×106A549 cellstransfectedwith si-IFITM1 or si-Control were resuspended in0.1 mL PBS and subcutaneously injected into the right flankof nude mice (n = 6) and the mice were daily intratumoralinjection of si-IFITM1 and si-Control for 35 days. The tumorsize was measured every 7 days. After 35 days, mice wereeuthanized and tumors were resected and weighed. Formetastasis experiments, 2×106 transfected A549 cells in 0.1mL PBSwere intravenously injected into the tail of nudemice(n = 6). 35 days later, mice were euthanized and metastasisnodules in lungs were counted.

2.10. Statistical Analysis. Data from three independent exper-iments were shown as means ± standard deviation (SD).Statistical analysis was performed via SPSS 16.0 software.Comparisons between different groups were made usingthe Student’s t-test. P< 0.05 was considered statisticallysignificant.

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Figure 1: IFITM1 is highly expressed in lung cancer tissues and cell lines. (a, b) RT-PCR and western blot analysis of IFITM1 expression in lungcancer tissues and matching normal lung tissues. (n=30). (c, d) RT-PCR and western blot analysis of IFITM1 expression in lung cancer celllines (A549 and H460) and a bronchial epithelial cell line (BEAS-2B). ∗P< 0.05.

3. Results

3.1. IFITM1Is Highly Expressed in Lung Cancer Tissues andCell Lines. Before exploration of the role of IFITM1 in lungcancer, we examined its expression in lung cancer tissuesand corresponding normal lung tissues using RT-PCR andwestern blot analysis. As shown in Figures 1(a) and 1(b),the mRNA and protein levels of IFITM1 were higher inlung cancer tissues than in the normal lung tissues. We alsodetected the expression of IFITM1 in two lung cancer celllines (A549 and H460) and a bronchial epithelial cell line(BEAS-2B). The results showed that all lung cancer cell linesexpressed higher mRNA and protein levels of IFITM1 incomparison with the BEAS-2B cell line (Figures 1(c) and1(d)).

3.2. IFITM1 Silencing Inhibits Lung Cancer Cell Proliferation.In order to silence IFITM1, we transfected si-IFITM1 intoA549 and H460 cells. The efficiency of IFITM1 silencing wasexamined by western blot. As shown in Figures 2(a) and 2(b),the protein levels of IFITM1weremarkedly decreased inA549and H460 cells after transfection with si-IFITM1.

Next, we determined whether IFITM1 silencing affectedthe lung cancer cell proliferation. For this purpose, we per-formedMTT assay.The results showed that IFITM1 silencing

remarkably inhibited the proliferation ofA549 andH460 cells(Figures 2(c) and 2(d)). In addition, cell cycle progression ofA549 and H460 cells was slowed down in IFITM1 silencedcells (Figures 2(e) and 2(f)).

3.3. IFITM1 Silencing Inhibits Lung Cancer Cell Migrationand Invasion. To investigate the effect of IFITM1 silencingon lung cancer cell migration, the wound-healing assay wasperformed. In both A549 and H460 cells, IFITM1 silencingsignificantly inhibited the wound-healing process, indicatinga decreased cell migration induced by IFITM1 silencing(Figures 3(a) and 3(b)). We further examined the functionof IFITM1 silencing in the invasive capability of lung cancercells. As shown by the transwell assay, the number of invadingA549 and H460 cells was greatly reduced after IFITM1silencing in comparison with the control cells (Figures 3(c)and 3(d)).

3.4. IFITM1 Silencing Inhibits Lung Cancer Cell Growth andMetastasis In Vivo. We examined the in vivo tumorigenesiseffect of IFITM1 silencing via subcutaneous injection of A549cells into nude mice. Every 7 days, the tumor size wasmeasured. 35 days later, mice were euthanized and tumorswere weighed. The results showed that tumor volume and

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Figure 2: IFITM1 silencing inhibits lung cancer cell proliferation. (a, b) IFITM1 silencing by siRNA notably reduced the protein expressionlevels of IFITM1 in A549 and H460 cells. (c, d) Cell proliferation was measured in si-IFITM1 or si-Control transfected A549 and H460 cellsby MTT assay. (e, f) Cell cycle phase patterns of A549 and H460 cells. ∗P< 0.05.

tumor mass were significantly reduced by IFITM1 silencingin comparison with the control group (Figures 4(a) and4(b)). In metastasis experiments, we intravenously injectedsi-IFITM1-transfected A549 cells or control cells into nudemice. 35 days later, mice were euthanized to check for lungmetastasis. The results indicated a remarkable decrease inthe number of metastasis nodules in lungs (Figure 4(c)). Thewestern blot assay revealed that the protein levels of𝛽-cateninand its downstream targets cyclin D1 and c-Myc in tumorsamples were significantly lower than that in control group(Figure 4(d)).

3.5. IFITM1 Silencing Inhibits the Activity of Wnt/𝛽-CateninSignaling Pathway. To investigate the underlying mecha-nisms by which IFITM1 played its role in lung cancerdevelopment, we examined the effect of IFITM1 silencing onthe activity of Wnt/𝛽-catenin signaling pathway which hasbeen reported to be closely related to IFITM1 [23, 24]. As

shown in Figure 5, IFITM1 silencing significantly decreasedthe protein levels of 𝛽-catenin and its downstream targetscyclin D1 and c-Myc in A549 cells compared with the controlcells, suggesting an inhibitory effect of IFITM1 silencing onthe Wnt/𝛽-catenin signaling pathway.

4. Discussion

With a steadily rising incidence in the world, lung cancer is aleading cause of cancer-related death. According to statistics,more than 1 million people die of lung cancer every yearand this number continues to rise [25]. Such situation hasattracted worldwide attention. With medical development,many potential markers for prognosis of lung cancer havebeen found such as serum carcinoembryonic antigen, neuronspecific enolase, cytokeratin fragment, tissue polypeptidespecific antigen, and progastrin-releasing peptide [26–28].But their clinical values are limited due to their low specificity

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Figure 3: IFITM1 silencing inhibits lung cancer cell migration and invasion. (a, b) The wound-healing assay was performed to measure theeffect of IFITM1 silencing on the migratory ability of A549 and H460 cells. (c, d) The transwell assay was performed to detect the effect ofIFITM1 silencing on the invasive ability of A549 and H460 cells. ∗P< 0.05.

and sensitivity. So it is necessary to explore new specialmarkers for improvement in the diagnosis of lung cancer.

IFITM1, an encoder of secretary or cell-surface proteinsinvolved in cell adhesion, angiogenesis, and growth factor,was recently reported to be a novel candidate for oncogenes

[29]. For example, IFITM1 has been found overexpressed inesophageal cancer, colon cancer, ovarian cancer, and cervicalcancer [22, 30–32]. In agreement with data obtained fromthe other cancer studies mentioned above, we measuredthe expression levels of IFITM1 in lung cancer tissues and

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Figure 4: IFITM1 silencing inhibits lung cancer cell growth and metastasis in vivo. (a, b) The growth curve and weight of tumors from si-IFITM1-transfected A549 cells or control cells. (c) Quantification of metastasis nodules in lungs 35 days after intravenous injection of thetransfected A549 cells or control cells. (d) Levels of 𝛽-catenin, cyclin D1, and c-Myc in tumor samples were detected by western blot. (n=6)∗P< 0.05.

cell lines. Our study results showed that IFITM1 was highlyexpressed in lung cancer tissues and cell lines. In the followingexperiments, we found that IFITM1 silencing inhibited lungcancer cell proliferation, migration, and invasion. These invitro results were verified by our xenograft tumor assayswhich showed that IFITM1 silencing suppressed lung cancercell growth and metastasis in vivo. More importantly, Yanget al. showed that IFITM1 was closely associated with apoor prognosis of non-small-cell cancer in patients; thiswas evidenced by a significantly low overall survive rateof IFITM1-positive patients versus IFITM1-negative patients[33]. However, it is still not clear how IFITM1 is induced inthe lung cancer. Kim et al. reported that CD147 stimulation,which was implicated in tumor invasion, metastasis, andangiogenesis, could induce the expression of IFITM1 using aleukemic cell line [34].Thus, we expect that unknown factors,

which need to be explored further, may be enriched in lungcancer cell, and induce IFITM1 expression, could enhancetumor proliferation, invasion, and metastasis.

Consistently, Yu et al. reported that knockdown ofIFITM1 had an inhibitory effect on the proliferation, migra-tion, and invasion of glioma cells [35]. Other studies onIFITM1 provided further evidence for the oncogenic role ofIFITM1. For instance, He et al. demonstrated that IFITM1enhanced the invasive and metastatic capacity of colorectalcancer cells [36]. Hatano et al. found that IFITM1 promotedinvasion during the progression of head and neck cancer [37].These observations supported the idea that IFITM1 exertedan oncogenic function in cancer development.

It was documented that the cytoplasmic protein 𝛽-catenin served as the key switch in the Wnt pathway [38],and the perturbation of Wnt signaling could dramatically

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Figure 5: IFITM1 silencing inhibits the activity of Wnt/𝛽-catenin signaling pathway. (a) The western blot showed a significant decrease in theprotein expression levels of 𝛽-catenin, cyclin D1, and c-Myc in si-IFITM1-transfected A549 cells compared with the control cells. (b) Therelative protein expression levels of 𝛽-catenin, cyclin D1, and c-Mycin A549 cells were quantified by Image J software. ∗P< 0.05.

suppress lung tumor progression [39]. The Wnt/𝛽-cateninsignaling pathway takes part in modulation of some crucialcellular processes including proliferation, invasion, apop-tosis, and differentiation [40–42]. More importantly, theWnt/𝛽-catenin signaling pathway has been found playing asignificant role during lung cancer development. Previousstudies on NSCLC demonstrated that the proliferation andmigration of lung cancer could be inhibited by targeting theWnt/𝛽-catenin signaling pathway [43, 44]. Earlier studieshave reported that theWnt/𝛽-catenin signaling is a controllerof the expression levels of IFITM1 and that activation ofthe Wnt/𝛽-catenin signaling may induce IFITM1 duringintestinal tumorigenesis [23, 24]. To know the mechanismunderlying the tumor-promotion role of IFITM1 in lungcancer cells, we investigated the effect of IFITM1 silencingon the Wnt/𝛽-catenin signaling pathway. In this study, weobserved that IFITM1 silencing significantly decreased theprotein levels of 𝛽-catenin, cyclin D1, and c-Myc in lungcancer cells and tumor tissues, indicating an inhibitory effectof IFITM1 silencing on theWnt/𝛽-catenin signaling pathway.Based on all the results in our study, we suggested that IFITM1silencing inhibited lung cancer progression, at least in part,via regulating the Wnt/𝛽-catenin signaling pathway.

In conclusion, our data demonstrated for the first timethat IFITM1 was overexpressed in lung cancer tissues and celllines. Silencing of IFITM1 inhibited proliferation, migration,and invasion of lung cancer cells. Furthermore, we foundthat IFITM1 silencing inhibited lung cancer cell growth andmetastasis in vivo. We further showed that IFITM1 silencinghad a suppressive effect on the Wnt/𝛽-catenin signalingpathway. Taken together, our study suggested IFITM1 as anovel molecular target for treatment of lung cancer.

Data Availability

The data used to support the findings of this study areincluded within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The article is supported by grant NSFC81300346 from theNational Natural Science Foundation of China and BureauFund 2015KZ029.

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