Decreased Lung Tumor Development in SwAPP Mice through the Downregulation of CHI3L1 and STAT 3 Activity via the Upregulation of miRNA342-3p.

We previously found that lung tumor development was reduced in a presenilin (PS) Alzheimer's disease (AD) mouse model. Here, we investigated whether this reducing effect could occur in a different AD mouse model. We investigated urethane-induced (1 mg/g) lung tumor development and melanoma growth in Swedish amyloid precursor protein (SwAPP) transgenic mice. The expression of chitinase-3-like-1 (Chi3L1) increased during lung tumor development and melanoma growth, which was accompanied by an increase in the activity of signal transducer and activator of transcription 3 (STAT3) and the downregulation of miRNA342-3p in wild-type mice. Like tumor development, the expression of Chi3L1 and STAT3 activity was reduced in the SwAPP mice, whereas the expression of miRNA342-3p was upregulated. In addition, Chi3L1 knockdown in the lung cancer and melanoma tissues reduced cancer cell growth and STAT3 activity but enhanced miRNA342-3p expression. However, the miRNA342-3p mimic decreased Chi3L1 expression, cancer cell growth, and STAT3 activity. Moreover, a STAT3 inhibitor reduced Chi3L1 expression and cancer cell growth but enhanced miRNA342-3p expression. These data showed that lung tumor development was reduced through the decrease of Chi3L1 expression via the STAT3-dependent upregulation of miRNA342-3p. This study indicates that lung tumor development could be reduced in SwAPP AD mice.

Introduction

Chitinase-3-like-1 (Chi3L1) is a prototypic mammalian chitinase-like protein. It is known to be expressed in a variety of cells, including epithelial cells, smooth muscle cells, macrophages, and neutrophils.2, 3, 4 Elevated levels of Chi3L1 expression have been noted in a wide variety of diseases,5, 6, 7, 8, 9 and many studies have also demonstrated that the levels of Chi3L1 expression are elevated in many cancers.8, 10, 11 Although higher expression of Chi3L1 in cancer cells has been reported, the role of Chi3L1 in tumor development and the related action mechanisms is unknown.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression by binding to a complementary sequence in the 3′ UTR of target mRNAs. Such binding results in either the degradation of the target mRNAs or the inhibition of their translation into proteins. miRNAs have been implicated in biological processes including metabolism, cell proliferation, developmental timing, apoptosis, morphogenesis, and stress response.14, 15 Regulation by miRNAs has been found to correlate with cancer, with roles as both oncogenes and tumor suppressors. Consequently, miRNAs are considered to be potential diagnostic and prognostic targets. Cell proliferation, migration, and the invasion of cervical, lung, and pancreatic cancer cells are regulated by miRNA342-3p.17, 18, 19 It is highly expressed in these cancer cells, inhibiting their growth by repressing target genes such as Ras-related protein Rab-2b and E2F1 in lung cancer, forkhead box protein M1 (FOXM1) in cervical cancer, and Ikβ kinase, TAK1 binding protein 2, and TAK1 binding protein 3 in hepatocellular carcinoma through interactions with their 3′ UTR regions.

Lung cancer is a lethal disease and continues to be the leading cause of cancer-related mortality worldwide. Although smoking is the primary cause of most lung cancer incidences, approximately 10%–15% of lung cancer cases are caused by genetics. Epidemiological data demonstrated that patients with Alzheimer’s disease (AD) have a lower incidence of cancer. We found that an AD suppressed model (presenilin 1 [PS1] and PS2 knockout mouse model) showed higher lung tumor development. Proteomic analysis in AD models showed that the lung tissues of these mutant mice have very low levels of Chi3L1 protein, prompting us to question whether the protein plays a significant role in lung tumor development. In the present study, we investigated whether the expression of Chi3L1 is significant in lung tumor development in Swedish amyloid precursor protein transgenic (SwAPP) mice, an AD mouse model. We assessed the roles of miRNA342-3p in Chi3L1-dependent lung tumor development because we previously found that miRNA342-3p interacts with Chi3L1. We also investigated the signal transducer and activator of transcription 3 (STAT3) pathway because it regulates Chi3L1 and miRNA342-3p expression.

Results

Reduced Lung Tumor Growth and Expression of Chi3L1 in SwAPP Mice

The expression of Chi3L1 was significantly lower in SwAPP mice compared with that in wild-type (WT) mice in most tissues, especially in the lung and brain (Figure 1A). We also identified a change in Chi3L1 expression in urethane-induced lung tumor tissues. Chi3L1 gene (Figure 1B) and protein expression (Figures 1C, western blot, and 1D, immunohistochemistry) were significantly elevated in the lung tumor tissues of WT mice, but the expression was significantly lower in the lung tumor tissues of SwAPP mice. In addition, the lung tumor tissue obtained from human patients showed significantly higher Chi3L1 expression compared with normal lung tissue (Figure 1E). Therefore, we were interested in studying the effects of Chi3L1 on lung cancer development in SwAPP mice. Lung tumorigenesis was induced using urethane injections. Thirty weeks after the initial urethane injections, a significantly higher number of lung tumors were found in WT mice (Figure 2A). Tumor multiplicity was 40.3 ± 7.5 tumors per WT mouse, but only 15.9 ± 5.7 per SwAPP mouse. The histological findings after H&E staining indicated that the tumors in WT mice were well-differentiated lung adenomas. However, tumors from SwAPP mice were significantly smaller than those from WT mice and showed fewer adenocarcinomas (Figure 2B). Western blotting data also showed that the protein levels of proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 (MMP9), cyclin B, cyclin D1, cyclin E, cyclin dependent kinase (CDK2), CDK4, and CDK6 were significantly higher in the tumor tissues of the WT mice than in the SwAPP mice (Figure S1).

Figure 1

Effect of SwAPP Overexpression on the Expression of Chi3L1

Expression of Chi3L1 protein in mouse lung, liver, kidney, heart, and brain tissues (A). The gene expression of Chi3L1 in mouse lung tissues (B). Expression of Chi3L1 protein in mouse lung tissues (C: western blot; D: immunohistochemistry). Cells positive for Chi3L1 in normal and diseased human lung tissues, analyzed by immunohistochemistry (E). *p < 0.01, significant difference from the saline-injected mice; #significant difference between wild-type (WT) mice and SwAPP mice.

Figure 2

Effect of SwAPP Overexpression on the Development of Lung Tumors

At the time of sacrifice, lungs were lavaged, perfused, and fixed in ice-cold Bouin’s fixative solution for 24 h. After fixation, lungs were used for surface tumor number measurements (A). Lung tissues were processed and stained with H&E (B). STAT3, p-STAT3 protein expression (C), as well as the DNA-binding activity of STAT3 were analyzed (D). Expression of mature miRNA342-3p in lung tumors was quantitated using the miScript miRNA qPCR assay (E). *p < 0.01, significant difference between WT mice and SwAPP mice. The results are expressed as mean ± SD.

Decreased STAT3 Activity in the Tumor Tissue of SwAPP Mice

We found that Chi3L1 could be regulated by STAT3, which is an important transcription factor in the development of several tumors. Thus, we attempted to determine whether the activation of transcriptional factors would be lower in the lung tumor tissues of SwAPP mice. Supporting our assumption, the phosphorylation of STAT3 (Figure 2C) was much lower in the lung tumor tissue of SwAPP mice compared with that in the WT mice lung tissue. Lower STAT3 DNA-binding activity (Figure 2D) was also observed in the lung tissue of SwAPP mice compared with that of WT mice. Because STAT3 cooperates with nuclear factor κB (NF-κB) in the control of tumor growth, we investigated the changes of NF-κB activity and the expression of its subunits. We additionally found that the activation of NF-κB and the expression of p50 and p65 were inhibited (Figure S2). We previously found that miRNA342-3p directly binds to Chi3L1 and controls Chi3L1 expression. Furthermore, miRNA342-3p significantly contributes to tumor growth. To further investigate whether the increase in Chi3L1 has an enhancing effect on lung cancer cell growth through decreasing miRNA342-3p expression, we measured the expression of miRNA342-3p in tumor tissues. We found a significant increase in miRNA342-3p expression in the tumor tissue of SwAPP mice compared with that in WT mice (Figure 2E).

Reduced Melanoma Growth in SwAPP Mice

To investigate whether tumor growth was also decreased in SwAPP mice after allograft cancer cell implantation, we injected the mice with melanoma cells instead of lung cancer cells because SwAPP mice are resistant to cancer cell growth due to rejection by the immune system. First, we compared the tumor development between WT mice and SwAPP mice. Similar to the carcinogen-induced lung tumor development, SwAPP mice showed much lower melanoma growth compared with that in WT mice (Figure 3A). In addition, the expression of CDKs, PCNA, and MMP9 were lower in the melanoma of SwAPP mice (Figure 3B). Next, we compared the expression of Chi3L1 and miRNA342-3p, as well as STAT3 activity, between WT mice and SwAPP mice. We found that the expression of Chi3L1 (Figure 3C) was inhibited, whereas the miRNA342-3p expression increased (Figure 3D) in the melanoma-injected SwAPP mice. In addition, the DNA-binding activity of STAT3 and the expression of the STAT3 and phosphorylated STAT3 (p-STAT3) proteins were inhibited (Figure 3E) in the melanoma-injected SwAPP mice.

Figure 3

Effect of SwAPP Overexpression on Melanoma Tumor Growth in Allograft Mouse Models

Four weeks after melanoma injection in WT or SwAPP mice, melanoma tumor growth was measured (A), and the expression of tumor proliferation factors (MMP9, PCNA, CDK2, CDK4, and CDK6) was assessed using a western blot (B). Chi3L1 gene expression (C) and miRNA342-3p gene expression (D) were measured using real-time PCR. The DNA-binding activity of STAT3 and the expression of STAT3 and p-STAT3 proteins were analyzed (E). *p < 0.01, significant difference from the melanoma-injected non-transgenic mice; #significant difference between WT mice and SwAPP mice.

The Upregulation of SwAPP Expression Inhibited Lung Cancer Cell Growth

We investigated the changes in Chi3L1 and miRNA342-3p expression in SwAPP-overexpressed A549 cells. When SwAPP gene expression was overexpressed, Chi3L1 protein (Figure 4A) and gene expression (Figure 4B) were significantly decreased, but the expression of miRNA342-3p was significantly increased (Figure 4C). Next, we investigated the role of SwAPP on cancer cell growth. The cell growth was inhibited by the overexpression of SwAPP (Figure 4D) in A549 cells. To further evaluate the relationship between SwAPP expression and STAT3 activity, we measured STAT3 activity in SwAPP-overexpressed A549 cells. We found that the activity corresponded with the cancer cell growth pattern, because the phosphorylation of STAT3 (Figure 4E) was much lower in SwAPP-overexpressed A549 cells. Lower STAT3 DNA-binding activity (Figure 4F) was also observed in these cells.

Figure 4

Effect of SwAPP Overexpression on Cell Viability, Activation of STAT3, and Their DNA-Binding Activities in A549 Cells

The effect of SwAPP overexpression on Chi3L1 protein expression in A549 was determined with a western blot assay (A). The effects of SwAPP overexpression (50 ng) on Chi3L1 gene expression (B) and miRNA342-3p gene expression were measured with real-time PCR (C). The change in cell viability after SwAPP overexpression was measured with an MTT assay (D). The effects of SwAPP overexpression on the protein expression of STAT3 and p-STAT3 (E) and the DNA-binding activity of STAT3 (F) were measured with a western blot and electrophoretic mobility shift assay (EMSA). Dual-luciferase reporter plasmids containing WT or mutant Chi3L1-3′ UTR were treated with miRNA342-3p or control miRNA. Firefly luciferase activity (normalized to the control Renilla luciferase) indicating Chi3L1 expression was determined using the Luc-Pair miR Luciferase Assay Kit (G). The effect of the miRNA342-3p mimic (10 or 50 nM) on Chi3L1 gene expression (H) and miRNA342-3p gene expression was measured with real-time PCR (I). The change in cell viability after miRNA342-3p mimic treatment was measured with an MTT assay (J). The effects of a miRNA342-3p mimic on the protein expression of STAT3 and p-STAT3 (K) and the DNA-binding activity of STAT3 (L) were measured with a western blot and EMSA. *p < 0.01, significant difference from the control vector; #significant difference between different doses.

Functional Roles of miRNA342-3p on the Expression of Chi3L1 and Lung Cancer Cell Growth

The expression of miRNA342-3p is associated with lung tumors, which suggests its significant role in lung tumor development. We investigated the role of miRNA342-3p on Chi3L1 expression with a luciferase assay using a reporter construct carrying the WT 3′ UTR of Chi3L1 in lung cancer cells. The assay was performed in A549 cells treated with either miRNA342-3p or a scrambled negative control. We observed marked repression of luciferase reporter activity by the miRNA mimic, but the luciferase activity was significantly reversed in the miRNA342-3p mutant (Figure 4G). To study the relationship between the miRNA342-3p and Chi3L1 expression and lung cancer cell growth, we evaluated cultured A549 cells after treatment with a miRNA342-3p mimic. The expression of Chi3L1 decreased with the treatment of the miRNA342-3p mimic (Figure 4H), but the miRNA342-3p level was elevated (Figure 4I). We also found that cell growth was inhibited by the miRNA342-3p mimic treatment (Figure 4J) in A549 cells. To further evaluate the relationship between miRNA342-3p expression and STAT3 activity, we measured STAT3 activity in miRNA342-3p mimic-treated A549 cells. We found that, corresponding with the cancer cell growth pattern, the phosphorylation of STAT3 (Figure 4K) and STAT3 DNA-binding activity (Figure 4L) were much lower in miRNA342-3p mimic-treated A549 cells.

The Upregulation of SwAPP Expression Inhibited Melanoma Cancer Cell Growth

We evaluated the changes in Chi3L1 and miRNA342-3p expression in SwAPP-overexpressed B16F10 cells. When the SwAPP gene expression was overexpressed, the expression of Chi3L1 significantly decreased (Figures 5A and 5B), but the expression of miRNA342-3p significantly increased (Figure 5C). Next, we investigated the role of SwAPP in B16F10 melanoma cancer cell growth and migration. The cell growth (Figure 5D) and migration of B16F10 (Figure 5E) cells were inhibited by the overexpression of SwAPP. To further evaluate the relationship between SwAPP expression and STAT3 activity, we measured STAT3 activity in SwAPP-overexpressed B16F10 cells. Similar to the lung cancer cell results, the phosphorylation of STAT3 (Figure 5F) and STAT3 DNA-binding activity (Figure 5G) were much lower in SwAPP-overexpressed B16F10 cells.

Figure 5

Effect of SwAPP Overexpression on Cell Viability, Activation of STAT3, and Their DNA-Binding Activities in B16F10 Cells

The effect of SwAPP overexpression on Chi3L1 protein expression in B16F10 was determined with a western blot assay (A). The effects of SwAPP overexpression (50 ng) on Chi3L1 gene expression (B) and miRNA342-3p gene expression were measured with real-time PCR (C). The changes in cell viability (D) and migration (E) after SwAPP overexpression were measured. The effects of SwAPP overexpression on the protein expression of STAT3 and p-STAT3 (F) and the DNA-binding activity of STAT3 (G) were measured with a western blot and electrophoretic mobility shift assay (EMSA). Dual-luciferase reporter plasmids containing WT or mutant Chi3L1-3′ UTR were treated with miRNA342-3p or control miRNA. Firefly luciferase activity (normalized to the control Renilla luciferase) indicating Chi3L1 expression was determined using the Luc-Pair miR Luciferase Assay Kit (H). The effect of the miRNA342-3p mimic (10 or 50 nM) on Chi3L1 gene expression (I) and miRNA342-3p gene expression was measured with real-time PCR (J). The change in cell viability after miRNA342-3p mimic treatment was measured with an MTT assay (K). The effects of the miRNA342-3p mimic on the protein expression of STAT3 and p-STAT3 (L) and the DNA-binding activity of STAT3 (M) were measured with a western blot and EMSA. *p < 0.01, significant difference from the control vector; #significant difference between different doses.

Functional Roles of miRNA342-3p on the Expression of Chi3L1 and Melanoma Cell Growth

Like in the lung cancer cells, we also investigated the role of miRNA342-3p on Chi3L1 expression in melanoma cells. The assay was performed in B16F10 cells treated with either miRNA342-3p or a scrambled negative control. The repressed luciferase reporter activity significantly decreased with the treatment of the miRNA342-3p mimic, but its activity was reversed in miRNA342-3p mutant treated cells (Figure 5H). To study the effects of miRNA342-3p on melanoma cancer cell growth, we evaluated cultured B16F10 cells after treatment with a miRNA342-3p mimic. The expression of Chi3L1 decreased with the treatment of the miRNA342-3p mimic (Figure 5I), but the miRNA342-3p level increased (Figure 5J). We also found that cell growth was inhibited by the miRNA342-3p mimic treatment (Figure 5K) in B16F10 cells. To further evaluate the relationship between miRNA342-3p expression and STAT3 activity, we measured STAT3 activity in miRNA342-3p mimic-treated B16F10 cells. We found that the phosphorylation of STAT3 (Figure 5L) and STAT3 DNA-binding activity (Figure 5M) were much lower in miRNA342-3p mimic-treated B16F10 cells.

Discussion

In this study, we found that SwAPP mice developed significantly less carcinogen- and allograft-induced lung tumors. Consistent with the lung tumor development decrease, the expression of Chi3L1 and STAT3 significantly decreased, whereas miRNA342-3p activity significantly increased in the lung tumor tissue of SwAPP mice compared with that in the WT mice. Lower expression of the subunits of STAT3, as well as PCNA, MMPs, cyclins, and CDKs, was also found in the lung tumor tissue of SwAPP mice. Moreover, overexpressed SwAPP decreased lung cancer cell and melanoma cell growth, Chi3L1 expression, and STAT3 activity, but it upregulated miRNA342-3p. We also found that miRNA342-3p significantly reduced Chi3L1 expression and cancer cell growth in accordance with the decrease of STAT3 activity in cultured lung cancer and melanoma cell lines. These data show that decreased Chi3L1 expression is essential for reduced lung tumor growth via the upregulation of miRNA342-3p in SwAPP mice.

Chi3L1 is critical for tumor metastasis and angiogenesis.25, 26, 27 It was also reported that in lung cancer cells, i.e., in the lung tumor tissue and blood of patients, the expression of Chi3L1 is elevated.25, 26, 27 In the present study, we found that lower Chi3L1 expression in SwAPP mice was associated with decreased lung tumor and lung cancer cell growth. Moreover, the overexpression of SwAPP inhibited cancer cell growth and migration. Thus, the lower Chi3L1 expression could be a significant factor in reducing lung tumor development in SwAPP mice. The decreased expression of Chi3L1 in the lung tumor tissues of carcinogen- and allograft-induced SwAPP mice correlated significantly with the reduced expression of PCNA and MMP9. Therefore, the lower expression of Chi3L1 in SwAPP mice may be important in the regulation of genes involved in cell proliferation and metastasis, leading to a decrease in lung tumor development in SwAPP mice. A few studies have demonstrated that Chi3L1 is implicated in colorectal and lung angiogenesis and metastasis.28, 29 It was also reported that the growth of colon and breast cancer was increased by Chi3L1 overexpression, but the inhibition of Chi3L1 blocked colon and breast cancer cell growth.28, 29 In addition to our findings, these data also indicate that reduced Chi3L1 expression could be important for reducing lung tumor development, especially in the SwAPP AD mouse model. However, the exact mechanism(s) of how the lower expression of Chi3L1 could reduce tumor growth in SwAPP mice is not clear.

A few studies have investigated miRNA342-3p alterations in human cancers, including colorectal, breast, and cervical cancer, as well as leukemia, all of which reported its reduced expression.17, 30, 31, 32, 33 DNA (cytosine-5) methyltransferase 1, DNA-binding protein inhibitor 4, and FOXM1 have been identified and verified, using luciferase assays, as direct targets of miRNA342-3p.17, 30, 33 In the present study, we found that miRNA342-3p targets Chi3L1, which was demonstrated by its luciferase activity. Previously, we found that Chi3L1 binds to miRNA342-3p in vascular smooth muscle cells. We also observed that the miRNA342-3p introduction into lung cancer cells resulted in a relatively modest reduction in Chi3L1 expression. Moreover, in lung tissues, there was an inverse relationship between miRNA342-3p expression and tumor development. We also found that miRNA342-3p mimic treatment decreased Chi3L1 expression and lung cancer cell growth. Therefore, these data indicate that miRNA342-3P could downregulate Chi3L1, thus inhibiting lung tumor growth in SwAPP mice.

We measured the STAT3 activity and found that STAT3 was activated by Chi3L1 but was inhibited by miRNA342-3p. These data indicate that STAT3 could conversely regulate miRNA342-3p, but that STAT3 activity is related to Chi3L1 expression. STAT3 is involved in cell proliferation, metastasis, and angiogenesis through the regulation of tumor-promoting genes such as PCNA, MMP, cyclins, and CDKs. In the present study, western blotting revealed that the expression of PCNA, MMPs, cyclins, and CDKs was lower in the tumors of SwAPP mice lung tissue compared with the lung tumor tissue of WT mice. STAT3 activity was also much lower in the lung tumor tissue of SwAPP mice compared with that of WT mice. STAT3 has been known to regulate Chi3L1 and many miRNAs, which results in the development of lung tumors.36, 37, 38 Consistent with the upregulation of miRNA342-3p, the miRNA342-3p mimic reduced STAT3 activity in lung cancer cells, resulting in decreased cancer cell growth and migration. Therefore, a lower level of STAT3 activity in the tumor tissue of SwAPP mice may also play a significant role in carcinogen- and allograft-induced lung tumor growth by the downregulation of genes involved in cancer cell growth through the downregulation of Chi3L1 expression via the upregulation miRNA342-3p.

In our previous study, we demonstrated reduced lung tumor development in a PS1 and PS2 knockin AD mouse model,23, 39 although more epidemiological studies are required to clearly demonstrate the relationship between lung tumor incidence and AD, especially in SwAPP mice. In the present study, we found that lung tumor development in a different AD mouse model (swap mice) was also reduced. The present data thus confirmed the inverse relationship between the incidence of cancer and AD.

Materials and Methods

Animals

In the present study, we used SwAPP mice as a model of AD. These mice overexpress human APP with the Swedish double mutations of K670N and M671L under the control of a hamster prion protein promoter.

Ethics Statement

The 8-month-old SwAPP male mice (n = 10) were purchased from Taconic Farms (Germantown, NY, USA) and were maintained and handled in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Laboratory Animal Research Centre at Chungbuk National University (approval no. CBNUA-1073-17-01).

Carcinogenesis Protocol

Tumors were induced in 8-month-old mice by a single intraperitoneal injection of 1 mg/g urethane (ethyl carbamate; Sigma-Aldrich, St. Louis, MO, USA) once a week for 10 weeks. Ten mice per group were euthanized at time points of up to 6 months after the injection of the carcinogen. After fixation, the lungs were used for surface tumor number and diameter measurements. Tumors on the lung surface were enumerated by at least two experienced readers, who were blinded to sample identifiers under a dissecting microscope; tumor counts were averaged and statistically analyzed. Tumor diameters were measured using Fisherbrand Traceable Digital Calipers (Fisher Scientific, Asheville, NC, USA).

Melanoma Injection Protocol

B16F10 mouse melanomas were subcutaneously injected (1 × 107 tumor cells/200 μL PBS) into 8-month-old mice with a 27G needle. Fourteen days after injection, the tumor volume of the animals was monitored every 3 days. The tumor volumes were measured with Vernier calipers and calculated by the following formula: (A × B2)/2, where A is the larger and B is the smaller of the two dimensions. At the end of the experiment, the animals were sacrificed with inhalants using carbon dioxide. The tumors were separated from the surrounding muscles and dermis, excised, and weighed.

Cell Culture

A549 human lung cancer cells and B16F10 mouse skin melanoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). RPMI 1640, DMEM, penicillin, streptomycin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, USA). A549 cells were grown in RPMI 1640 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 humidified air. The B16F10 cells were grown in DMEM with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2 humidified air.

Plasmids and miRNA Mimics

The miRNA mimics of Pre-miR-342-3p (PM12328) and Pre-miR-NC #2 (AM17111) were purchased from Ambion. A total of 5 nmol/L miRNA mimics was transfected into 1 × 105 cells per well of a six-well plate using RNAiMAX (Invitrogen).

miRNA PCR Array

Total RNA (including miRNA) was isolated from HBEC K-ras and vector cell lines using the miRNeasy Mini Kit (QIAGEN). The expression of 667 miRNAs was evaluated using the TaqMan Human miRNA Array v.2.0 (Life Technologies). In brief, 1 μg of total RNA was reverse transcribed using Megaplex Primer Pools A and B, and the resulting cDNA for each sample was then loaded onto two 96-well microfluidic array cards containing TaqMan primers and probes for miRNA and normalization controls followed by real-time PCR. The PCR data were normalized and analyzed with SDS v.2.3 software (Life Technologies) using the ΔΔCt (cycle threshold) method. To accurately compare miRNA expression across the study samples, the same Ct threshold was utilized for all samples. The wells with Ct values ≤35 were counted as negative.

Luciferase Activity Assay

A549 and B16F10 cells (8 × 103) were seeded in 96-well plates and allowed to settle for 24 h. Cells were co-transfected with 100 ng of WT or mutant 3′ UTR of Chi3L1 and 200 ng of pWPXL-miR-342-3p or pWPXL-miR-NC using Lipofectamine 3000. At 48 h after transfection, both firefly and Renilla luciferase activities were quantified using a Dual-Luciferase Reporter System (Promega Corporation, Madison, WI, USA). All experiments were performed in triplicate.

Immunohistochemistry

All specimens were fixed in formalin and enclosed in paraffin for examination. H&E staining and immunohistochemistry were performed as previously described.

Western Blot Analysis

Western blot analysis was performed as previously described. The membranes were immunoblotted with primary specific antibodies. The blot was then incubated with corresponding conjugated anti-rabbit and anti-mouse immunoglobulin G-horseradish peroxidase (1:2,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive proteins were detected with the enhanced chemiluminescence western blotting detection system. The intensity of the bands was measured using the Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany).

Dual-Luciferase Reporter Assay

A549 and B16F10 cells (1 × 105 per well in a six-well plate) were transfected, together with a pRLTK vector (0.1 μg), with either an empty pGL3 vector or a vector carrying the 3′ UTR of E2F1 with WT or mutant miRNA342-3p target sites (0.3 μg), and sequentially transfected with 50 nmol/L miR-342-3p antisense (AS) or scrambled control (SC) oligonucleotides 24 h after the initial transfection. Luciferase assays were performed 48 h after AS or SC transfection using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Each assay was performed in triplicate.

Electromobility Shift Assay

Electromobility shift assay was performed as previously described. In brief, 1 × 106 cells/mL were washed twice with 1× PBS, followed by the addition of 1 mL of PBS; then cells were scraped into a cold Eppendorf tube. The cells were centrifuged at 15,000 × g for 1 min and the resulting supernatant was removed. Solution A [50 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 10 mm KCl, 1 mm ethylenediaminetetraacetic acid, 1 mm ethylene glycol tetraacetic acid, 1 mm dithiothreitol, 0.1 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL aprotinin, and 0.5% Nonidet P-40] was added to the pellet in a 2:1 ratio (v/v) and incubated on ice for 10 min. A total of 0.5 g of tumor tissue was chopped into 1.5 mL of solution A. The tumor pieces were then homogenized and centrifuged at 12,000 × g for 15 min at 4°C. Solution C (solution A + 10% glycerol and 400 mm KCl) was added to the pellet in a 2:1 ratio (v/v) and vortexed on ice for 20 min. After centrifugation at 15,000 × g for 7 min, the resulting nuclear extract supernatant was collected in a chilled Eppendorf tube. Consensus oligonucleotides, activator protein 1 (AP-1), NF-κB, and STAT3 (Promega), were end-labeled using T4 polynucleotide kinase and (γ-32P) ATP for 10 min at 37°C. Gel shift reactions were assembled and allowed to incubate at room temperature for 10 min followed by the addition of 1 μL (50,000–200,000 cpm) of 32P-labeled oligonucleotides and another 20 min of incubation at room temperature. Subsequently, 1 μL of gel loading buffer was added to each reaction and loaded onto a 4% nondenaturing gel and electrophoresed until the dye was 75% of the way down the gel. The gel was dried at 80°C for 1 h and exposed to film overnight at 70°C. The intensity of the bands was measured using the Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany).

Cell Viability Assay

For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 10% v/v of 5 mg/mL MTT (Sigma) diluted in PBS was added to A549 and NCIH460 cell cultures. After 2 h of incubation, the medium was aspirated, and DMSO was added. Absorbance was measured at 570 nm. The data were normalized to their respective controls and are presented as a bar graph.

Data Analysis

The data were analyzed using GraphPad Prism 4 v.4.03 software (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± SD. The differences in all data were assessed by one-way ANOVAs followed by Dunnett’s tests. A p value < 0.05 was considered statistically significant.

Author Contributions

K.C.K., C.J.H., and J.T.H. conceived and designed the research. K.R.P. and Y.S.J. contributed the development of methodology. S.Y.K., J.Y.K., J.K.S., M.J.S., and M.K.C. supported the experiments and data analysis. K.R.P., Y.S.J., D.Y.H., S.-B.H., and J.T.H. supervised the experiments, technic, materials, and recipes. All authors reviewed the paper.