Background
According to the World Health Organization (WHO), cancer is the leading cause of death worldwide, and lung cancer is the main cause of cancer-related deaths [
1]. In 2017, there were an estimated 222,500 new diagnoses of lung cancer in the United States, and 155,870 people died of lung cancer [
2]. With advances in medical technology, lung cancer treatment has entered the era of precision medicine [
3‐
5]. Therefore, identifying genes that drive cancer progression and providing effective treatments may significantly prolong the survival of patients with non-small cell lung cancer (NSCLC) [
6,
7].
MicroRNAs (miRNAs) are small, highly conserved, noncoding RNAs of approximately 19–25 nucleotides that regulate gene expression at the post-transcriptional level by base-pairing with the 3′ UTRs of target mRNAs [
8‐
11]. They play key roles in human biological processes, such as migration, cellular metabolism, cell proliferation, apoptosis, and epithelial–mesenchymal transition (EMT) [
12‐
17]. Increasing evidence has demonstrated that miRNAs play an important role in cancer and are closely related to tumourigenesis and prognosis. The miRNA miR-365 gene is located on chromosome 16p13.12, and the mature hsa-miR-365 sequence is cleaved from two precursors: hsa-miR-365-1 and hsa-miR-365-2 [
18]. Abnormal expression of miR-365 is observed in a variety of tumours, with different expression patterns and functions in different human cancer types. In skin squamous cell carcinoma, we first found that miR-365a-3p plays an oncogenic role by downregulating NFIB to promote CDK6 and CDK4 expression, leading to Rb phosphorylation and tumour progression [
18‐
20]. Additionally, high expression levels of miR-365a-3p can be detected in breast and pancreatic cancer, playing a role in promoting tumour development [
21,
22]. In colon cancer, miR-365a-3p inhibits the development of cancer by targeting cyclin D1 and BCL-2 [
23,
24]. This miRNA also serves as a tumour suppressor gene in gastric cancer [
25]. In brief, the functions of miR-365a-3p in cancer are complex. Thus, the roles of miR-365a-3p in lung cancer cells remain unclear and require further study.
Deubiquitinating enzymes are key enzymes that can reverse ubiquitination modifications. Recently, their functions and mechanisms have been of great interest in tumour research [
26,
27].
USP33, also known as VHL-interacting deubiquitinating enzyme 1 (
VDU1), is located on chromosome 1 and encodes two protein subtypes: type I and type II. Type I contains 942 amino acids, and type II contains 911 amino acids, with predicted molecular weights of 107 and 103 kDa, respectively. USP33 has been confirmed to inhibit the development of a variety of tumour cells. USP33 deubiquitinates and thus maintains the stability of ROBO1, thereby regulating the activity of SLIT2 and inhibiting cancer cell metastasis [
28‐
30]. USP33 maintains the stability of CP110 and antagonises the effect of cyclin-F on the S/G2 and G2/M phases of the cell cycle by interacting with CP110, maintaining the steady state of the centriole and ensuring normal functioning of mitosis and genome stability, which reduces tumourigenesis [
31].
Additionally, SLIT2/ROBO1 signalling has been shown to inhibit tumour cell proliferation and migration. Prasad et al. [
32] found that ROBO1 and ROBO2 were expressed in several breast cancer cell lines and that SLIT2 inhibited CXCL12/CXCR4-induced chemotaxis, invasion, adhesion, and secretion of MMP-2 and MMP-9 in breast cancer cells. The loss of SLIT2, SLIT3, or ROBO1 protein in mouse breast cancer models results in an increase in repair processes in tissues, promoting cell proliferation [
33].
Our previous studies showed that in cutaneous squamous cell carcinoma, miR-365a-3p plays an oncogenic role by promoting tumour progression [
18]. Moreover, studies have shown that USP33-mediated SLIT2/ROBO1 signalling participates in the development of cancer [
29,
30]. Thus, in this study, we aimed to elucidate the role of miR-365a-3p in lung cancer cells and the relationship among miR-365a-3p, USP33, SLIT2, and ROBO1 in lung adenocarcinoma.
Methods
Tissue samples
This study was approved by the Ethics Review Board of Nanfang Hospital, Southern Medical University (Guangzhou, China). Twenty pairs of primary lung cancer tissues and their corresponding adjacent non-tumour tissue samples were collected in the Thoracic Surgery Department of Nanfang Hospital from March to June 2017. All experiments were performed according to relevant guidelines; informed consent was obtained from each patient. No patients received prior radiotherapy or chemotherapy. All specimens were removed during surgery and immediately stored at − 80 °C in liquid nitrogen for subsequent extraction of total RNA.
Cell lines and cell culture
A549 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). SPC-A-1 and H1299 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium or DMEM supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA). All cells were maintained in a humidified incubator at 37 °C and 5% CO2.
RNA extraction and real-time quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from cell lines or tissues using a TRIzol Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. cDNA was synthesized using Takara RT reagent (Takara Bio). qRT-PCR was performed on a Light Cycler 480 system (Roche Diagnostics, Basel, Switzerland) using a SYBR Green I Master kit (Roche). We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. Mature miR-365a-3p expression was measured by qRT-PCR according to the Taqman MicroRNA Assay protocol (Takara Bio) and normalised using U6 small nuclear RNA with the 2−ΔΔCt method.
Western blotting
Equal amounts of protein were separated by 10% SDS-PAGE and blotted onto PVDF membranes (Millipore, Bedford, MA, USA) probed with the following primary and secondary antibodies: monoclonal rabbit primary antibodies against SLIT2 and ROBLO1 (1:1000; Cell Signaling Technology, Boston, MA, USA) and β-tubulin (1:10,000; Bioworld Technology Inc., St. Louis Park, MN, USA); polyclonal rabbit primary antibody against USP33 (1:500; Abcam, San Francisco, CA, USA); and secondary fluorescent goat anti-rabbit antibody (LI-COR, Lincoln, NE, USA). Primary antibodies were applied overnight at 4 °C, and secondary antibody treatment was performed for about 1 h at 25 °C. An Odyssey Infrared Imaging System (LI-COR) was used to analyse immunoreactive bands. Western blotting was performed three times.
A549 or SPC-A-1 cells were seeded into a 6-well culture plate (200 cells/well) and incubated for 12 days. Cells were stained with Giemsa solution. Plates were scored by determining the number of colonies containing ≥ 50 cells.
5-Ethynyl-2ʹ-deoxyuridine (EdU) assay
5-Ethynyl-2ʹ-deoxyuridine incorporation assays were conducted using the EdU assay kit (RiboBio Co., Guangzhou, China) according to the manufacturer’s instructions. Cells were incubated with 50 nM EdU for 2 h at 37 °C. Cells were then fixed with 4% formaldehyde for 15 min at 25 °C and treated with 0.5% Triton X-100 for 20 min at 25 °C to permeate cell membranes. After washing with PBS three times, cells were incubated with 1× Apollo reaction cocktail (100 µL/well) for 30 min. DNA was stained with 10 µg/mL of Hoechst 33342 stain (100 µL/well) for 20 min, and staining was visualised with fluorescence microscopy. Five fields of view were randomly selected for each sample. EdU-positive cells were stained with red dye, and the relative proliferation-positive ratios were calculated from the average cell count of the five visualised fields.
Cell migration and invasion assays
The migratory and invasive abilities of cells were assessed using Transwell inserts (Corning, Inc., Corning, NY, USA) in 24-well plates. For invasion assays, each group of cells (5 × 104 cells/100 µL) was resuspended in FBS-free RPMI-1640 medium and seeded into the upper chamber containing a Matrigel-coated membrane. After incubation for 24 h at 37 °C with 5% CO2, the incubation medium and non-invading cells were removed from the upper surface of the membrane with cotton swabs. Invading cells that adhered to the lower surface of the chamber were fixed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 30 min. Invading cells were photographed and manually counted at 200× magnification using a microscope (Olympus, Tokyo, Japan). For the Transwell migration assays, the process was the same, except the Transwell membrane was not precoated with Matrigel. Each assay was performed at least three times independently.
Wound healing assay
When cells had grown to approximately 90% confluency (after 48 h), an artificial wound was created with a 10-µL pipette tip. Cells were then cultured in fresh medium without FBS. Images were taken at 0 and 36 h to visualise wound healing. The relative percentage of the wound healed was calculated using the following formula: (width of wound at 0 h − width of wound at 36 h)/width of wound at 0 h.
Plasmid and oligonucleotide construction
AntagomiR-365, antagomiR-negative control (antagomiR-NC), agomiR-365, and agomiR-negative control (agomiR-NC) were designed and synthesized by GenePharma (Shanghai, China). pcDNA3.1-USP33 and pcDNA3.1 vectors were designed and synthesized by Obio Technology (Shanghai, China).
Transient transfection
A549 and SPC-A-1 cells were seeded in 6-well plates at a density of 30–50%. Transient transfection was performed with Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For all experiments, cells were collected 24–28 h after transfection.
Dual-luciferase reporter assay
The dual-luciferase reporter plasmids psi-CHECK2-USP33 (containing the wild-type USP33 3ʹ UTR binding site) and psiCHECK2-mGPC3 (containing a mutant USP33 3ʹ UTR) were constructed. A549 or SPC-A-1 cells were added to 24-well plates at 70–80% confluence 24 h before transfection. A mixture of 50 nM miR-365a-3p agomiR or antagomiR and 0.5 µg psi-CHECK2 reporter plasmid (psiCHECK2-wUSP33 or psiCHECK2-mUSP33) was co-transfected into cells using Lipofectamine 2000 reagent. At 48 h after transfection, luciferase activity was analysed using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Each experiment was performed in triplicate.
In vivo tumourigenesis assays
All animal experimental protocols were approved by the Animal Research Ethics Committee of Nanfang Hospital and complied with the rules of the specific pathogen-free (SPF) animal laboratory of the Nanfang Medical University. 4- to 6-week-old male mice were purchased from the Animal Laboratory Center of Nanfang Medical University (Guangdong, China). A549/antagomiR-365 cells and A549/antagomiR-NC cells were injected subcutaneously into the left and right axilla of five nude mice (4 × 106 cells on each side) beginning 8 days after the injection of tumour cells. The mice were sacrificed 26 days post-injection to observe changes in tumour volume. Tumour size was measured every 3 days using the same protocol, and tumour volumes were calculated with the following formula: V = (L × W2)/2, where V is the volume (mm3), L is the biggest diameter (mm), and W is the smallest diameter (mm).
The Cancer Genome Atlas (TCGA) data portal provides a platform for researchers to query, download, and analyse data sets generated by TCGA (
http://cancergenome.nih.gov/).
Bioinformatics analysis performed with miRanda/TargetScan/starBase indicated that the 3′ UTR of USP33 is a binding site for miR-365a-3p.
Statistical analysis
Data are expressed as mean ± SD. Each experiment was repeated at least three times unless otherwise indicated. Statistical analyses were performed using SPSS 22.0 software (SPSS, Chicago, IL, USA). Significant differences were analysed using Student’s t tests for continuous variables. Spearman’s correlation was used to analyse the relationship between miR-365a-3p and USP33 mRNA expression; P < 0.05 indicated significance.
Discussion
Identifying the mechanisms of lung cancer metastasis and methods for inhibiting metastasis is an important focus of the international medical community. MiRNAs play a dual role in tumour development and progression and participate in all aspects of tumour development [
35‐
38]. Circulating miR-365a-3p may serve as a molecular marker for the diagnosis and prognostic evaluation of some tumours [
39‐
41]. Thus, the role of miR-365a-3p in cancer should be further explored.
The regulation of miR-365a-3p differs among different tissues and genetic backgrounds, in which it can serve as either a tumour suppressor or oncogene [
42,
43]. The dual nature of miR-365a-3p may be related to its unique topological structure, interactions with various signalling pathways, and induction of different biochemical reactions. It is therefore necessary to study the role of miR-365a-3p in specific tumour contexts. Although lung cancer is the most common cause of cancer-related deaths worldwide, the role of miR-365a-3p in the proliferation, invasion, and metastasis of lung cancer has not been previously reported. As lung adenocarcinoma is the main pathological type of lung cancer, it is important to study the role and regulation of miR-365a-3p in lung adenocarcinoma.
In this study, we found that miR-365a-3p was highly upregulated in cancer tissues and cell lines. Based on these experimental results, we speculated that miR-365a-3p may promote tumourigenesis in lung cancer. Functional assays demonstrated that miR-365a-3p promoted proliferation, migration, and invasion, further confirming our hypothesis. However, the pathway by which miR-365a-3p promotes lung cancer remained unknown. Thus, determining the role and mechanism of miR-365a-3p in lung adenocarcinoma was the first aim of this study.
Ubiquitination is a dynamic and reversible process, and the removal of ubiquitination modifications is mainly mediated by deubiquitinases, which are closely related to tumour occurrence and development [
44‐
46]. In recent years, increasing attention has been paid to the role of deubiquitinases in tumour progression, and the molecular mechanisms underlying their regulation have gradually been clarified [
47]. More than 40 deubiquitinating enzymes have been found to be associated with the occurrence and development of tumours. However, few studies have investigated the relationship between miRNAs and tumourigenesis in the context of deubiquitination. USP33 is a deubiquitinating enzyme that is closely related to the occurrence and development of tumours [
28‐
30,
32]. The list of diseases currently reported to be associated with the dysregulation of USP33 includes breast cancer, acute lymphoblastic leukaemia, and lung cancer [
28,
30,
48]. We sought to identify the downstream target genes of miR-365a-3p and found that
USP33 expression was strongly negatively correlated with that of miR-365a-3p in lung adenocarcinoma tissues. Functional rescue assays demonstrated that miR-365a-3p promoted proliferation, migration, and invasion by downregulating
USP33. This is the first evidence for a lung cancer invasion and metastasis mechanism in which miR-365a-3p directly targets and downregulates
USP33, which further promotes the proliferation, migration, and invasion activities of lung cancer. However, these results did not clarify the signalling pathway through which miR-365a-3p downregulates
USP33 to promote lung carcinogenesis.
Recently, an increasing number of studies have found that the SLIT2/ROBO1 signalling pathway is closely involved in tumourigenesis by inhibiting the proliferation, migration, and invasion of tumour cells [
49‐
51]. USP33 deubiquitinates ROBO1 in lung cancer cells to maintain its stability, thereby regulating SLIT2 activity and inhibiting cancer cell metastasis [
30]. Based on the above experiments, we hypothesised that miR-365a-3p may regulate the SLIT2/ROBO1 signalling pathway by targeting
USP33 to promote lung cancer. Based on western blot assay results, the expression levels of SLIT2 and ROBO1 were both downregulated following the overexpression of miR-365a-3p but restored when
USP33 was overexpressed at the same time. Combined with the above in vitro test results, we concluded that miR-365a-3p promotes lung cancer by downregulating the USP33/SLIT2/ROBO1 pathway.
To further validate this hypothesis, we performed in vivo experiments in a mouse lung cancer tumour model. A549/antagomiR-365 cells were injected into mice to observe subcutaneous tumour formation. The results suggested that the inhibition of miR-365a-3p expression reduced tumourigenicity. Thus, the in vivo experiment further demonstrated that miR-365a-3p plays a role in promoting tumour proliferation in lung adenocarcinoma.
Conclusions
In summary, we not only confirmed that miR-365a-3p targets and downregulates USP33 in lung adenocarcinoma, but we also further confirmed that miR-365a-3p promotes the proliferation, migration, and invasion of lung cancer cells by downregulating the USP33/SLIT2/ROBO1 pathway. To achieve early diagnosis and further improve the clinical outcomes of lung cancer, it is necessary to explore biomarkers for early detection and develop individualised treatment methods for lung cancer. Our elucidation of the mechanism by which miR-365a-3p mediates the USP33/SLIT2/ROBO1 signalling pathway in lung carcinogenesis thus provides a new strategy for the diagnosis and targeted therapy of lung cancer.
Authors’ contributions
SDM guided the entire experiment. YHW performed the experiments and produced the figures. YHW and SHZ wrote the manuscript. HJB and SKM designed the study. BSZ analysed the data. HM was involved in collaborations regarding data acquisition. All authors have contributed significantly to the content of the manuscript. All authors read and approved the final manuscript.