Background
Although substantial effort has been paid to improving patient survival, lung cancer remains a leading cause of cancer-related mortality worldwide [
1,
2]. Non-small cell lung cancer (NSCLC) accounted for approximately 80––85% of all lung cancer cases, with an overall 5-year survival rate of < 20% [
3]. In addition, cisplatin-based traditional chemotherapy and molecular targeting drug therapy have greatly improved the prognosis and quality of life in NSCLC patients [
4,
5]. However, drug resistance is a vital bottleneck, which limits the effect of chemotherapy in most NSCLC patients [
6,
7]. Therefore, elucidating the molecular mechanisms underlying drug resistance in NSCLC is critical.
MicroRNA (miRNA) is an endogenous non-coding RNA between 18 and 25 nucleotides, has been shown to regulate mRNA and protein expression by binding to the 3′-untranslated region (3′-UTR) of their target genes. Several previous studies on miRNA in NSCLC have been exerted and a series of miRNAs have been found to be involved in the development of NSCLC drug resistance [
8,
9]. Moreover, abnormal miR-223 expression has been detected in various cancers [
10‐
15]. In NSCLC, previous studies described a controversial role of miR-223, functioning as either a tumor suppressor or an oncogene. In addition, Huang et al. reported that miR-223 may promote malignant phenotypes of lung cancer in A549 cells via activation of the NF-κB signaling pathway [
16]. Liang et al. reported that platelet-secreted microvesicles (P-MVs) can promote lung cancer cell invasion via targeting the tumor suppressor, EPB41L3 [
17]. In contrast, Zhou et al. reported that miR-223 inhibits tumor development of NSCLC and sensitizes A549 cells to gefitinib via targeting E2F1 [
18]. Moreover, miR-223 was found to enhance the sensitivity of NSCLC cells to erlotinib by targeting the insulin-like growth factor-1 receptor [
19]. However, another study showed that miR-223 could induce doxorubicin resistance through targeting F-box/WD repeat-containing protein 7 (FBXW7)-mediated epithelial mesenchymal transition in NSCLC cells [
20]. Thus, additional studies are essential to further uncover the role of miR-223 in the development and drug resistance in NSCLC.
Autophagy is vital biological process required for the maintenance of cellular biosynthesis, growth, and differentiation. Accumulating evidence has demonstrated that the abnormal activation of autophagy is an important factor involved in the process of chemoresistance [
21,
22]. Moreover, recent studies indicate that miRNAs are frequently dysregulated in chemoresistant lung cancers, in which they have been shown to target autophagy-related genes or modulators. For example, miR-200b was shown to regulate autophagy associated with chemoresistance in human lung adenocarcinoma by targeting ATG12 [
23]. In addition, the downregulation of miR-24-3p could induce etoposide (VP16)-cisplatin resistance in small-cell lung cancer by targeting ATG4A [
24]. However, the relationship between miR-223 and autophagy in cancer remains poorly understood.
In the present study, we aimed to study the role of miR-223 on cisplatin resistance in NSCLC and uncover the potential mechanisms. In the present study, we found that miR-223 could enhance cisplatin resistance in NSCLC by targeting FBXW7 and upregulating autophagy. Thus, we aimed to identify the pro-chemoresistant role of miR-223 in NSCLC cells in vitro.
Materials and methods
Cell lines and reagents
Human NSCLC A549, NCI-H358, and NCI-H1299 cells were purchased from the ATCC (Manassas, VA, USA) and cultured in RPMI 1640 medium containing 10% fetal bovine serum. PC9 was purchased from the Chinese Academy of Science Cell Bank (Shanghai, China) and cultured in DMEM medium containing 10% fetal bovine serum. All cell lines were cultured at 37 °C in a humidified atmosphere (95% air and 5% CO2). Cisplatin, rapamycin, and chloroquine were purchased from Sellerck (Huston, TX, USA).
siRNA and transfection
The miRNA mimics, miRNA inhibitors, and FBXW7 siRNA were synthesized by GenePharma (Shanghai, China). The sequences of primers were placed in Additional file
1: Table S1. Cells were transfected using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol.
Cell viability assay
Cells were seeded into 96-well plates (4 × 103 cells/well) directly or 24 h after transfection. After treatment with different concentrations of cisplatin combinations for 48 h, cell viability was examined using a commercial CCK-8 kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol, and the absorbance was determined at 450 nm using an MRX II microplate reader (Dynex, Chantilly, VA, USA).
Western blot
Cells were washed with PBS, harvested in ice-cold PBS, centrifuged at 2000 rpm at 4 °C, and lysed in RIPA buffer; protein concentrations were determined with a BCA kit (Pierce, Rockford, IL, USA). An equal amount of cell lysate for each condition was subjected to SDS-PAGE, transferred onto nitrocellulose members, and analyzed. The primary antibodies against FBXW7, LC3-I, LC3-II, SQSTM1, and β-actin were purchased from Abcam (Cambridge, USA) and used at a concentration of 1:1000. The corresponding secondary antibody was also obtained from Abcam and used at a concentration of 1:5000.
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted using Trizol (Takara, Japan) reagent. Reverse transcription and qRT-PCR were conducted using a SYBR Prime Script™ miRNA RT-PCR kit (Takara, Japan,) according to the manufacturer’s instructions. The level of miR-223 expression was normalized to
U6 RNA. SYBR Premix Ex Taq (Takara, Japan) was also used to detect the level of FBXW7 mRNA. The sequences of primers were placed in Additional file
1: Table S1. Relative mRNA expression was normalized to β-actin. Data were analyzed using the 2ΔΔCt method.
EdU assay
Proliferation of the NSCLC cell lines was determined using a Click-iTEdU Imaging Kit (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, cells were treated with different conditions for 24 h, and 10 μM EdU was added for 2 h before fixation and permeabilization. Cell nuclei were stained with Hoechst 33342 (Invitrogen) at a concentration of 5 μg/mL for 30 min.
Luciferase assays
The 293T cells were co-transfected with wild-type or mutant FBXW7 3′-UTR plasmid (Promega) as well as miR-223-3p mimics or miR-223-3p inhibitor (Ribo) using Lipofectamine 2000 (Invitrogen). Cell lysates were harvested 48 h after transfection and then firefly and Renilla luciferase activities were measured by a dual luciferase reporter assay kit according to the manufacturer’s protocol. Renilla luciferase activity was used for normalization.
Autophagic flux assay
A549 and NCI-H1299 cells stably transfected with RFP-GFP-LC3 adenovirus were subjected to different treatments. After 48 h, the cells were fixed with 4% paraformaldehyde (Sigma, USA) and photographed using a laser confocal fluorescence microscope. Cells were detected by the expression of green (GFP) or red (RFP) fluorescence. Autophagosomes were characterized by yellow puncta and autolysosomes based on only red puncta in the merged images. Autophagic flux was determined by an increased percentage of only red puncta in the merged images. A total of 300 cells were randomly selected to be counted and the number of autophagosomes and autolysosomes were averaged.
Flow cytometry assay
Cells were treated with cisplatin (IC50) for 48 h. The cells were stained with the Annexin-V and 7AAD according to the manufacturer’s protocol. The rate of apoptosis was determined by flow cytometry.
Immunohistochemistry and terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
The immunohistochemistry assay of Ki-67 was performed on 4 μm of a thickness of paraffin-embedded mouse tissue sections. The sections were then incubated with the anti-Ki-67 antibody (1:500, Abcam) at 4 °C overnight. Then the primary antibodies were detected with an HRP-linked secondary antibody (Abcam) and developed using a 3ʹ-diaminobenzidine substrate kit. The positively stained cells were defined as those with brown nuclei, and the percentage of positive tumor cells was determined. For apoptosis detection, apoptosis of the tumor tissues was determined with TUNEL assay using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s protocol. The apoptotic cells were observed under a light microscope.
Tumor xenograft studies
Animal experiments were conducted in compliance with the Guide for the Care and Use of Animal Ethics Committee of Zhejiang Chinese Medicine University (Hangzhou, China). 5 × 106 prepared A549 cells were injected subcutaneously into the right axillary fossa. Tumor length (L) and width (W) were measured and tumor volumes were calculated using the formula (L × W2)/2. When tumor volumes reached 100 mm3, the mice were randomly divided into four groups (n = 3), as followed: negative control, 5 mg/kg cisplatin, 8 nmol antagomiR-223, or cisplatin combined with antagomiR-223. Cisplatin and antagomiR-223 were administered by intraperitoneal and intratumor injection, respectively, every 2 days for 2 weeks. All the mice were euthanized.
Statistical analysis
Data are presented as the mean ± SD from three independent experiments and analyzed by a two-tailed Student’s t-test using SPSS 18.0. Differences were considered significant at a value of P < 0.05.
Discussion
The cytotoxic-based platinum compound, cisplatin (cisplatin), has been commonly used as a first line treatment in NSCLC for decades. However, drug resistance to cisplatin in cancer cells remains a substantial challenge to a favorable prognosis [
6]. The development of cisplatin resistance is a complex multifactorial process, involving reactive oxygen species (ROS), the aberrant expression of microRNAs, ATP-binding cassette (ABC) transporter effusion, and abnormal signaling pathways [
29,
30]. Accumulating evidence has demonstrated that autophagy is an essential pathway for cellular homeostasis and many studies have demonstrated that autophagy is involved in chemoresistance [
31,
32]. Additionally, autophagy inhibitors have been explored as a means to sensitize chemoresistant cells to chemotherapy in clinical trials [
33]. Recently, the regulation of autophagy by miRNAs has been shown to be a potentially effective strategy to reduce cancer cell chemoresistance [
34]. Regarding NSCLC, while some studies have revealed a potential role of miRNAs and autophagy in cisplatin resistance in NSCLC [
35,
36], no investigations have verified the ability of miR-223 and FBXW7 to directly impact the regulation of autophagy and cisplatin resistance in NSCLC cells. Therefore, additional studies are required to elucidate the relationship between miRNAs, autophagy, and cisplatin resistance in NSCLC. In the present study, we describe a novel mechanism by which miR-223 mediates cisplatin resistance by inhibiting FBXW7 to promote cisplatin-induced autophagy in NSCLC.
Increasing evidence has shown that miR-223 can function as either a cancer inducer or a tumor suppressor and is correlated with chemoresistance or chemosensitivity depending on the tumor type. In human gastric cancer, miR-223 was found to promote cisplatin resistance in human gastric cancer cells by regulating cell cycle through targeting FBXW7 [
37]. In hepatocellular carcinoma, miR-223 was reported to modulate multidrug resistance via the downregulation of ABCB1 [
38]. Moreover, in Glioblastoma, miR-223 was found to promote temozolomide chemoresistance in glioblastoma multiforme cells by targeting paired box 6 signaling [
39]. However, the role of miR-223 on chemoresistance is inconsistent regarding NSCLC. Zhang et al. reported that increasing miR-223 expression could induce cell resistance to erlotinib in HCC827 cells [
40]. In contrast, Han et al. reported that miR-223 could reverse the resistance of EGFR-TKIs through the IGF1R/PI3K/Akt signaling pathway in NSCLC [
41]. In addition, Zhou et al. reported that miR-223 could sensitize cancer cells to gefitinib by targeting E2F1 [
18]. Therefore, additional studies are required to further elucidate the role of miR-223 in NSCLC chemoresistance. In the present study, we showed that miR-223 can regulate cisplatin sensitivity in NSCLC by autophagy, indicating that miR223 may be mediate cisplatin resistance by autophagy. However, of these mechanisms of cisplatin resistance, the altered expression of MDR-related genes is the most common [
42,
43]. Maybe mir223-mediated cisplatin resistance is correlated with altered expression of MDR-related genes.
FBXW7, also known as FBW7, is a F-box-containing protein in the SCF E3 ligase complex, which functions in phosphorylation-dependent ubiquitination [
44]. Moreover, FBXW7 has been identified as a tumor suppressor gene in several cancers and an FBXW7 knockdown was shown to sensitize cancer cells to chemotherapy [
26,
45]. Accumulation studies demonstrated that FBXW7 played a significant role in cancer cells by autophagy. For example, downregulated the expression of FBXW7 induced by high glucose activated mTOR signal, which led to diminished autophagy in renal mesangial cells [
46]. Perifosine induces the degradation of key proteins in the mTOR axis through a GSK3/FBXW7-dependent mechanism in human lung cancer cells [
47]. In addition, the relationship between miR-223 and FBXW7 has been studied in several cancers. In pancreatic ductal adenocarcinoma, miR-223 was reported to promote pancreatic cancer cell growth and invasion by targeting FBXW7 [
48]. Moreover, the down-regulation of miR-223 could reverse the epithelial-mesenchymal transition (EMT) in gemcitabine-resistant pancreatic cancer cells [
49]. In esophageal squamous cell carcinoma, the overexpression of miR-223 was found to promote tumor progression by inhibiting FBXW7-mediated regulation of the cell cycle [
50]. In NSCLC, the miR-223/FBXW7 axis was reported to regulate doxorubicin sensitivity through EMT [
20]. However, there are no existing reports on the miR-223/FBXW7 axis and the regulation of autophagy. In the present study, we have demonstrated for the first time that miR-223 can induce autophagy and enhance cisplatin-induced autophagy by targeting FBXW7.
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