Background
Nasopharyngeal carcinoma (NPC) is an endemic malignancy in China [
1]. There are over 60,000 new cases of NPC per year which caused 34,000 deaths every year in China [
2]. Intensity-modulated radiation therapy (IMRT) and its combination with chemotherapy have greatly improved locoregional control of NPC, whereas tumor recurrence and distant metastasis remain as the main failure pattern and disease related death after treatment [
3‐
5]. Thus, there is a great need to disclose molecular mechanisms that underlie the initiation and progression of NPC.
MicroRNAs (miRNAs) are a group of small non-coding RNAs which are dysregulated in many cancer types [
6‐
11]. These newly identified regulators is involved in modulating cancer cell proliferation, differentiation, and migration through regulating genes’ expression by pairing with 3′-UTRs nucleotide sequences of their mRNAs [
12‐
15]. They may function as both oncogenes and tumor suppressors. Up to now, a group of miRNAs have been reported participating in NPC progression and therapeutic response, such as miR-451 [
16], miR-29c [
17] and miR-19b-3p [
18]. These findings indicate that miRNAs have important roles in nasopharyngeal tumorigenesis which worth further exploration.
MiR-203 has been reported down-regulated in NPC tissues through microarray analysis [
19]. Recent studies found that miR-203 participated in NPC radioresistance and chemoresistance through negatively-regulate IL8/AKT pathway and ZEB2 [
20,
21], respectively. However, its role in cancer cell growth and metastasis has rarely been characterized in NPC up to now. In this study, we found that the mature form of miR-203, miR-203a-3p, was downregulated in NPC tissues and could suppress cell proliferation and metastasis both in vitro and in vivo. Additionally, the LIM and SH3 domain protein (LASP1) was identified as a functional target of miR-203a-3p as previously reported in other malignancies such as esophageal squamous cell carcinoma, breast cancer etc. [
22‐
28]. Thus, this study expands our understanding of the mechanisms underlying the development and progression of NPC, and may provide a novel therapeutic target for the treatment of NPC.
Methods
Cell lines and clinical specimens
Eight human NPC cell lines (CNE-1, CNE-2, C666–1, HNE-1, HONE-1, 5-8F, 6-10B and SUNE-1) were maintained in RPMI-1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) [
29]. A human immortalized nasopharyngeal epithelial cell line, NP-69, were cultured in keratinocyte/serum-free medium (Invitrogen) supplemented with bovine pituitary extract (BD Biosciences, San Diego, CA, USA). Sixteen freshly frozen NPC samples and seven normal nasopharyngeal epithelium samples were collected from Jiangsu Cancer Hospital (Nanjing, China). All samples were reviewed by pathologists to confirm the diagnosis. The research protocols were approved by the Institutional Ethical Review Board of Jiangsu Cancer Hospital, and informed consent was obtained from each patient. The expression profiles of miR-203a-3p were also investigated in NPC tissues obtained from Gene Expression Omnibus (GEO) (
http://www.ncbi.nlm.nih.gov/geo).
Total RNA was extracted using TRIzol reagent (Invitrogen) as described previously [
16]. Reverse transcribed using Bulge-Loop miRNA-specific RT primers (RiboBio, Guangzhou, China) for miR-203a-3p or random primers (Promega) for LASP1 with M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative RT-PCR reactions were performed on the ABI 7300 (Applied Bio-systems) using Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen). U6 or GAPDH were used as internal controls for miR-203a-3p and LASP1, respectively. The relative expression levels were calculated as previously described [
16].
Oligonucleotide and plasmid transfection
To explore the effect of miR-203a-3p on NPC cells, CNE-2 and SUNE-1 cells were transfected with miR-203a-3p mimic, miR-203a-3p inhibitor and their respective negative control(RuiboBio) using Lipofectamine 2000 reagent (Invitrogen). To determine that whether LASP1 is a direct target of miR-203a-3p, CNE-2 and SUNE-1 cells were co-transfected with miR-203a-3p mimic or miR-Ctrl (50 nM, RiboBio) and the pSin-EF2-puro-LASP1 (LASP1) or empty pSin-EF2-puro-Vector (Vector) (2 μg; Addgene, Cambridge, MA, USA). The cells were harvested for assays 48hs after transfection.
Generation of stably transfected cell lines
The pre-miR-203a sequence was cloned into the lentiviral plasmid pSin-EF2-puromycin (Addgene, Cambridge, MA, USA); pSin-EF2-miR-203a or negative control pSin-EF2-vector was then co-transfected into 293FT cells with the psPAX2 packaging plasmid (Addgene) and the pMD2.G envelope plasmid (Addgene) using the calcium phosphate method. At 24 h after transfection, lentiviruses expressing miR-203a (Lenti-miR-203a) or negative control empty lenti-vector (Lenti-vector) were harvested and used to infect SUNE-1 cells, and stably transfected cells were selected using puromycin and validated by quantitative RT-PCR.
For the MTT assay, transfected CNE-2 or SUNE-1 cells were seeded in 96-well plates at a density of 1000 cells per well. The absorbance values were measured at 490 nm after 1, 2, 3, 4 and 5 days using an ELX800 spectrophotometric plate reader (Bio-Tek, Winooski, VT, USA). For the colony formation assay, cells were plated at a density of 500 cells per well in six-well plates after transfection, and cultured for 7 to 12 days. The colonies were then stained with 0.2% crystal violet with buffered formalin (Sigma, St. Louis, MO, USA). Colony numbers were manually counted using ImageJ software. Cell numbers >50 were considered as a colony.
Wound healing assay and transwell migration and invasion assays
For the wound healing assay, transfected CNE-2 or SUNE-1 cells were seeded into 6-well plates and then subjected to serum starvation for 24 h in serum-free media. An artificial wound was created using a 200 μl pipette tip and images were taken at 0 and 24 h using an inverted microscope. Migration and invasion assays were performed in transwell chambers (Corning, Corning, NY, USA) coated with or without Matrigel (BD Biosciences) on the upper surface of the 8-μm pore size membrane. Briefly, transfected CNE-2 or SUNE-1 cells were harvested, suspended in serum-free medium and 5 × 104 or 1 × 105 cells were plated into the upper chamber for the migration or invasion assays, respectively. Media supplemented with 10% FBS was placed into the lower chamber. After 12 or 24 h incubation, the membranes were stained and counted using an inverted microscope.
NPC xenograft tumor growth and lung metastasis mode in vivo
Male BALB/c nude mice of 4–6 weeks old were purchased from the Medical Experimental Animal Center of Guangdong Province (Guangzhou, China). For the xenograft tumor growth model, 1 × 10
6 SUNE-1 cells stably overexpressing miR-203a or negative control were suspended in 200 μl PBS, and then subcutaneously injected into the dorsal flank of the nude mice. Tumor size was measured every 3 days, and tumor volumes were calculated as described previously [
30]. Four weeks later, the mice were killed, and the tumors were dissected and weighted. For the metastasis assay, SUNE-1 cells stably overexpressing miR-203a or negative control were suspended in PBS, and 1 × 10
6 cells (200 μl) were injected via the tail vein. Eight weeks later, the mice were killed, the lung tissues were fixed, paraffin embedded and 5 μm tissue sections were stained with hematoxylin and eosin (H&E). The number of macroscopic and microscopic metastatic nodules in the lungs was counted. All animal research protocols were approved by the Institutional Animal Care and Use Ethics Committee.
Luciferase reporter assay
The LASP1 Wt and Mt. 3′-UTR were generated and cloned into the XhoI and NotI restriction sites of the psiCHECK-2 luciferase reporter plasmid (Promega). For the luciferase assay, CNE-2 or SUNE-1 cells were seeded into 6-well plates the day before transfection, and then co-transfected with the LASP1 Wt or Mt. 3′-UTR reporter plasmids (2 μg), and miR-203a-3p mimic (50 nM) or miR-Ctrl (50 nM) using Lipofectamine 2000 reagent (Invitrogen). Renilla and firefly luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega).
Western blot analysis
Cells were washed twice with PBS before being lysed on ice for 30 min with RIPA buffer containing protease inhibitor cocktail (Fdbio Science, Hangzhou, China). Protein concentrations were evaluated using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated by 10% SDS-PAGE gels and transferred onto Westran S membrane (Whatman Inc. Floham Park, NJ). The membranes were then incubated with mouse monoclonal anti-LASP1 antibody (1:2000; Chemicon, Temecula, CA, USA) and incubated with anti-mouse IgG secondary antibody (1: 5000; Epitomics, Burlingame, CA, USA). An anti-α-tubulin antibody (1: 1000; Sigma-Aldrich) was used as the loading control. Bound antibody was detected using the SuperSignal West Pico Chemoluminescence system (Pierce, Inc., Rockford, IL).
Statistical analysis
Data are presented as mean ± S.D. All statistical analysis was performed using SPSS 20.0 software (IBM, Armonk, NY, USA). Two-tailed Student’s t-tests were used for comparisons between two groups. Comparison of means from multiple treatment groups was carried out using one-way ANOVA. A Bonferroni correction was introduced to correct for multiple comparisons. All P values were two-sided and P values less than 0.05 were considered significant.
Discussion
MiR-203, which is now named as miR-203a, have been demonstrated to be an important tumor suppressor participating in the pathogenesis of esophageal [
24,
33], prostate [
23,
34], lung cancers [
27,
35] including nasopharyngeal carcinoma [
19‐
21]. A recent study found that miR-203 reduced radioresistance of NPC cells through inhibiting IL-8/AKT pathway [
21]. In another investigation, miR-203 was reported modulating tumor stemness and chemotherapy resistance in nasopharyngeal carcinoma by interacting with ZEB2 [
20]. However, miR-203 expression in nasopharyngeal carcinoma and its biological function on proliferation and metastasis have barely been investigated in NPC
.
In the present study, we confirmed that the mature form of miR-203, miR-203a-3p, was down-regulated in both NPC cell lines and tissue samples. Ectopic expression of miR-203a-3p significantly suppressed cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in NPC in vivo. Furthermore, LASP1 was identified as a direct target of miR-203a-3p in NPC. LASP1 over-expression partially abrogated the tumor suppression by miR-203a-3p in NPC cell lines. Taken together, our data suggest that miR-203a-3p/LASP1 pathway may play an important role in NPC initiation and progression.
MiRNAs are found dysregulated and lead to cancer initiation and progression in many types of cancers. Up to date, a group of miRNAs including miR-34c [
36], miR-145 [
37] and miR-93 [
38] have been reported dysregulated and play important roles in NPC. A recent study screened for differentially expressed miRNAs in NPC tissues through high-throughput microarray assays and found that miR-203 may down-regulated in NPC tissues [
19]. In the present study, we validated and confirmed this result in both NPC cell lines and tissues. We found that miR-203a-3p level was significantly lower in NPC cancer cells as compared to immortalized nasopharyngeal epithelial cell NP-69. Moreover, miR-203a-3p level was also decreased in NPC tissues in comparison with normal nasopharyngeal tissues. The dysregulation of miR-203a-3p indicated a potential tumor suppressor role in NPC.
We then explored the biological function of miR-203a-3p in NPC. Ectopic expression of miR-203a-3p significantly suppressed the proliferative, migratory and invasive capabilities of NPC cells in vitro. Furthermore, ectopic expression of miR-203a-3p inhibited NPC xenograft tumor growth and lung metastases in vivo. Up to now, this is the first study which confirmed the proliferation, migration and invasion suppressive effect of miR-203a-3p in NPC both in vitro and in vivo. Given our discovery that miR-203a-3p is frequently downregulated in NPC tissues, outlining a potential molecular target for NPC treatment.
LASP1, the LIM and SH3 protein-1, is a scaffold protein which mediates cell migration, proliferation and survival in various cancer entities [
39‐
42]. In the present study, we identified LASP1 as a direct target of miR-203a-3p in nasopharyngeal carcinoma, in agreement with previous findings in head and neck cancer, breast cancer, esophageal carcinoma, prostate and lung cancers [
22‐
28]. Ectopic expression of miR-203a-3p inhibited LASP1 level both on protein and mRNA level. In addition, ectopic expressing of LASP1 partially reversed the miR-203a-3p-mediated inhibition of proliferation, migration and invasion in NPC in vitro. These results indicate that LASP1 is a functional target of miR-203a-3p in regulating NPC cell proliferation and metastasis.
Conclusions
In summary, our study demonstrated that miR-203a-3p is downregulated in NPC, and miR-203a-3p inhibited NPC cell proliferation and metastasis through targeting LASP1. MiR-203a-3p/LASP1 pathway may provide novel insight into the molecular mechanisms regulating progression of NPC, and may provide novel therapeutic targets for the treatment of NPC.
Acknowledgements
The authors would like to greatly thank Dr. Changchun Zheng (Department of otolaryngological, Qianshan County hospital, Anqing 246300, Anhui Province, P.R. China) for his kind contributions to the collection of NPC tissue samples.
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