Background
Lung cancer is the leading cause of cancer related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 70–80% of lung cancer cases [
1,
2]. Recently, advances in clinical and experimental oncology have been made for treating NSCLC [
3‐
6], but its complicated pathology is unclear, and more work is required to identify novel molecules that are involved in the process. Therefore, investigation of the molecular mechanisms underlying NSCLC tumorigenesis may aid in the development of novel therapeutic targets and strategies for the treatment of the malignancy.
MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs of approximately 22 nt that regulate the expression of target mRNA by binding to 3′-untranslated regions (3′-UTRs), resulting in target mRNA degradation or silencing [
7,
8]. Recent studies indicate that microRNAs (miRNAs) are important subtypes of noncoding RNAs in the regulation of diverse biological processes, especially those involved in critical pathways linked to cancer cell proliferation, apoptosis, and metastasis [
9‐
11]. One target gene may be regulated by multiple miRNAs and one miRNA may regulate multiple target genes, which results in the formation of complex regulation networks in tumorigenesis [
12]. Studies show that miRNAs exert oncogenic or tumor suppressor roles in the etiology and pathogenesis of cancer by targeting tumor suppressors or oncogenes [
13,
14].
miR-338-3p is mapped to the seventh intron of the apoptosis-associated tyrosine kinase (
AATK) gene and miR-338-3p regulates gene AATK expression in rat neurons [
15]. miR-338-3p was first reported in prion-induced neurodegeneration: expression of miR-338-3p is reduced in mouse brains infected with mouse-adapted scrape [
16]. In tumorigenesis, miR-338-3p is down-regulated in multiple cancers, including gastric, colorectal, and lung cancers [
17‐
19]. However, little is known about the role of miR-338-3p in NSCLC proliferation and apoptosis so we investigated NSCLC progression and development by identifying miRNA targets.
Sphingolipids are a diverse group of water-insoluble molecules including ceramides, sphingoid bases, ceramide phosphates and sphingoid-based phosphates [
20], all of which contribute to cell proliferation, invasion and apoptosis [
21]. Sphingosine kinases (SphKs) are the rate-limiting enzymes for cellular sphingoid-base phosphates and have two distinct isoforms, SphK1 and SphK2 [
22,
23]. SphK1, which is an oncogenic kinase, is involved in tumor development and progression of various human cancers but biological functions of SphK2 in NSCLC remain unknown. Thus, we studied the regulation of miR-338-3p on SphK2 and the consequent effects on proliferation and apoptosis of human NSCLC cells.
Methods
Ethics statement
This study was approved by the Ethics Committee of ZhengZhou University (ZhengZhou, China) and full informed consent was provided by all of the patients involved prior to sample collection.
Patients and tissue samples
A total of 34 patients diagnosed with primary NSCLC at the Henan Tumor Hospital (Zhengzhou, China) between August of 2015 and June of 2016 were included in this study. No patient received chemotherapy or radiotherapy prior to surgery. Tumor and corresponding non-tumor lung tissue samples were collected and rapidly frozen in liquid nitrogen and stored at −80 °C. Tumors were classified according to World Health Organization classification. Data for patient age, gender, smoking history, differentiation, and TNM stage were obtained from patient records (see Table
1).
Table 1
Expression of SphK2 and miR-338-3p in tissues of 34 lung adenocarcinoma cases
Gender |
Male | 21 | 0.539 ± 0.105 | 0.428 | 0.330 ± 0.201 | 0.485 |
Female | 13 | 0.504 ± 0.148 | 0.385 ± 0.255 |
Age |
<60 | 16 | 0.5.34 ± 0.133 | 0.689 | 0.304 ± 0.160 | 0.243 |
≥60 | 18 | 0.517 ± 0.114 | 0.393 ± 0.260 |
Differentiation |
Well | 14 | 0.551 ± 0.120 | 0.433 | 0.290 ± 0.121 | 0.404 |
Moderate | 13 | 0.491 ± 0.140 | 0.401 ± 0.239 |
Poor | 7 | 0.539 ± 0.104 | 0.380 ± 0.306 |
TNM stage |
I + II | 24 | 0.495 ± 0.108 | 0.017* | 0.406 ± 0.228 | 0.023* |
III | 10 | 0.598 ± 0.127 | 0.220 ± 0.132 |
Smoking history |
Smoker | 18 | 0.523 ± 0.096 | 0.710 | 0.299 ± 0.108 | 0.145 |
No smoker | 16 | 0.512 ± 0.149 | 0.410 ± 0.294 |
Cell lines and cell culture
Normal human bronchial epithelial cell line NHBE and human lung cancer cell lines H460, H1299, A549, SPC-A-1 and Calu-3 were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified cell incubator with 5% CO2.
RNA isolation and qRT-PCR
Total RNA was isolated from tissue samples and cell lines using the Qiagen RNeasy kit (Valencia, CA) according to the manufacturer’s instructions. RNA quality and quantity were assessed by standard electrophoretic and spectrophotometric methods. Mature miR-338-3p expression was measured by qRT-PCR according to the Taqman MicroRNA Assays protocol (Applied Biosystems, Carlsbad, CA) and normalized using U6 small nuclear RNA (RNU6B; Applied Biosystems, Carlsbad, CA) with the 2−ΔΔCt method.
Western blot
Total protein was extracted from tissue samples and cell lines using RIPA buffer containing PMSF. A BCA protein assay kit (Beyotime, Haimen, China) was used to measure total protein. Proteins were electrophoresed via SDS-PAGE and transferred onto PVDF membranes. After blocking, membranes were washed four times with TBST at room temperature and then incubated overnight at 4 °C with diluted primary antibody. Following extensive washing, the membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). Antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) served as an endogenous reference. Protein intensity was scanned on Typhoon PhosphorImager (GE Healthcare, Pittsburgh, PA) for fluorescent signal. Experiments were performed in triplicate.
RNA oligoribonucleotides
The miR-338-3p mimics (GMR-miR MicroRNA-338-3p mimics) used in this study were synthesized by Shanghai GenePharma Co. Ltd. Human NSCLC cells A549 and H1299 were seeded into six-well plates (2 × 105 cells/well) and allowed to settle overnight until they were 50–80% confluent. Cells were transfected with miR-338-3p mimics using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Three groups were generated for the ensuing experiments: non-transfected group (blank control), scrambled miRNA transfected group (negative control, NC) and miR-338-3p mimics transfected group (inhibitor). Then, 24–48 h after the initial transfection, the cells were harvested for further experiments.
Plasmid construction and cell transfection
SphK2 coding sequences lacking the 3′-UTR were cloned into the pcDNA3.1 vector (Invitrogen) to generate the pcDNA3.1-SphK2 expression vector. Cell lines were grown at 37 °C in a humidified atmosphere with 5% CO2. For transfection, cells were cultured to 70% confluence and transfected with plasmids using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations.
Cell proliferation assay
For growth curve experiments, different experimental groups of A549 and H1299 cells were plated in 96-well plates at 1 × 104 cells per well and incubated for 48 h after transfection. Optical density (OD) was measured using water-soluble tetrazolium salt assay and microplate computer software (Bio-Rad Laboratories, Hercules, CA) according to Cell Counting Kit-8 (CCK-8) assay kit instructions (Dojindo Laboratories, Japan). Absorbance at 450 nm was read on a microplate reader (168–1000 Model 680, Bio-Rad), and proliferation curves were plotted. Cell proliferation was measured using the ratio of OD of transfected cells in each group to ODs of blank control cells in each group. Data were expressed as percents of control.
To measure colony-forming activity, three groups of A549 and H1299 cells were counted and seeded into 12-well plates (100 cells/well). Culture medium was replaced every 3 days. Twelve days after seeding, colonies containing more than 50 cells were counted.
Construction of 3′-UTR-luciferase plasmid and reporter assays
The 3′-UTR of the SphK2 fragment was PCR-amplified from human genomic DNA and inserted into the pmiR-GLO control vector (Promega, Madison, WI) at the XhoI and XbaI sites 3′ to the luciferase gene. Primer sequences used for PCR amplification were as follows: forward 5′-AUGGGACCAGACGUGAUGCUGGA-3′, reverse 5′-GUUGUUUUAGUGACUACGACCU-3′. The 3′-UTR of SphK2 was confirmed with sequencing and named pmiR-GLO-WT. Site-directed mutagenesis of the miR-338-3p target site in the SphK2 3′-UTR (pmiR-GLO-mut) was carried out using a Quikchange site-directed mutagenesis kit (Promega, Madison, WI), with pmiR-GLO-WT as the template. For the luciferase reporter assay, A549 and H1299 cells were cultured in 96-well plates. Then, using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), they were each cotransfected with wildtype or mutant reporter plasmid (100 nM) and microRNA (100 nM). At 48 h after transfection, luciferase activity was measured using the dual-luciferase assay system (Promega, Madison, WI).
Apoptosis measurement using flow cytometry
A549 and H1299 cells were harvested 48 h after transfection and cell concentration was adjusted to 1 × 106 cells. Annexin V-FITC/PI Apoptosis Detetion Kit Ι (BestBio, Shanghai, China) was used to measure Annexin V. Results were obtained using FACScan Flow Cytometer (BD Biosciences, San Jose, CA). Tests were repeated in triplicate. Data were analyzed with Cell Quest software.
Animals and subcutaneous tumor growth
Male athymic nude mice (6–8 weeks-of-age) were obtained from the Animal Experimental Center of ZhengZhou University and were acclimated for 2 weeks. This study was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of ZhengZhou University. The protocol was approved by the Committee on the Ethics of Animal Experiments of ZhengZhou University.
For in vivo tumorigenesis assays, all surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. NSCLC A549 and H1299 cell line stably expressing luciferase infected with lentivirus packaged with lentiviral vectors LV6-miR-338-3p or LV6 empty vector (GenePharma, Shanghai, China), and cells were collected and injected into the flanks of nude mice. Thirty minutes after injection, luciferase substrate was added (150 mg/kg) and luciferase activity was measured every 5 days using the same protocol. Live tumor images were measured every 5 days for 3 weeks and monitored with bioluminescent imaging (PerkinElmer, Fremont, CA).
Statistical analysis
Statistical analysis was performed using SPSS software version 16.0. All data are presented as mean ± SD where applicable. Differences were analyzed with the Student’s t test. Differences were considered significant when p < 0.05.
Discussion
miRNAs are small, noncoding RNAs, 21–25 nucleotides in length, which are master gene mediators that form the miRNA-induced silencing complex (miRISC) and lead to mRNA instability or degradation [
24]. Aberrant miRNA expression occurs in many biological processes such as cell proliferation, the cell cycle, apoptosis, invasion, and migration. Depending on the cellular function of certain miRNA targets, miRNAs can behave as oncogenes or tumor suppressor genes.
The apoptosis-associated tyrosine kinase (
AATK) gene is located on chromosome 17 (17q25.3) [
25]. Studies indicate that the role of
AATK in anti-tumorigenesis and aberrant Aatk expression depends on methylation in the CpG island promoter of Aatk [
26,
27]. miR-338-3p suppresses the translation of a select group of cellular mRNA whose protein products are negative regulators of neurite growth. Previously, miR-338-3p was shown to act as a tumor suppressor in some cancers [
28,
29].
Previous studies indicate that miR-338-3p is downregulated in colorectal and hepatocellular carcinomas and gastric cancer [
30‐
32]. However, the expression pattern of miR-338-3p in lung cancer, particularly in NSCLC, is unreported. Data from miRNA arrays indicate that miR-338-3p is downregulated in NSCLC tissues [
33,
34] and miR-338-3p may exert a tumor suppressor role in NSCLC. Using various approaches, we observed that overexpression of miR-338-3p suppressed NSCLC A549 and H1299 cell proliferation and induced apoptosis in vitro and in vivo.
Identification of targets of miR-338-3p in NSCLC is necessary for understanding the underlying regulatory mechanisms so we used bioinformatics for target gene prediction. Considering overlap of the genes identified by TargetScan, miRBase targets and PicTarget, SphK2 was selected to be a potential target for validation. Using luciferase reporter assays, Western blot, and qRT-PCR assays we verified that miR-338-3p directly targets SphK2 by interacting with the first binding site in the 3′-UTR.
Authors’ contributions
GJZ, GWZ, and GQZ: conceived of the study, and participated in its design and coordination and helped to draft the manuscript. GJZ, RRC, and CYL: collected the samples. GWZ, HZ, RRC, CYL and YJG: carried out part of experiments and wrote the manuscript. GWZ, HZ, GJZ, and GQZ: performed the statistical analysis. All authors read and approved the final manuscript.