microRNA-342-3p targets FOXQ1 to suppress the aggressive phenotype of nasopharyngeal carcinoma cells
- 198 Downloads
microRNA (miR)-342–3p is frequently dysregulated in human cancers. In the present study, we aimed to explore the expression, prognostic significance, and biological relevance of miR-342-3p in nasopharyngeal carcinoma (NPC).
We examined miR-342-3p expression in 79 paired NPC specimens and corresponding normal tissues and analyzed its prognostic impact on overall survival of NPC patients. Gain- and loss-of-function experiments were conducted to determine the biological roles of miR-342-3p.
Compared with matched normal nasopharyngeal tissues, miR-342-3p was significantly downregulated in NPC (P = 0.0038). Low miR-342-3p expression was significantly correlated with reduced overall survival (P = 0.0084). Ectopic expression of miR-342-3p significantly suppressed proliferation, colony formation, and invasion of NPC cells. In contrast, depletion of miR-342-3p facilitated NPC cell proliferation and invasion. In vivo xenograft studies confirmed that overexpression of miR-342-3p restrained the growth of NPC xenograft tumors. Mechanistically, FOXQ1 served as a functional target of miR-342-3p. There was a significantly negative correlation between miR-342-3p and FOXQ1 expression (r = − 0.487, P = 0.004) in NPC specimens. Overexpression of FOXQ1 rescued the inhibitory effects of miR-342-3p on NPC cell growth and invasion.
miR-342-3p downregulation predicts poor prognosis in NPC patients and shows suppressive activity against NPC growth and invasion through repression of FOXQ1. Restoration of miR-342-3p may represent a potential therapeutic strategy for NPC.
KeywordsInvasion FOXQ1 Growth miR-342-3p Nasopharyngeal carcinoma
Nasopharyngeal carcinoma (NPC) is a common tumor of the head and neck in Southeast Asia and China . Radiotherapy in combination with chemotherapy is the primary treatment for locally advanced NPC. Due to high recurrence and metastasis rates, the prognosis of advanced NPC is still unsatisfactory, with a 5-year survival rate of approximately 50% in Chinese population [2, 3]. However, the exact mechanism for NPC development and progression has not yet been clarified.
Forkhead box (FOX) proteins are a superfamily of transcription factors that are evolutionarily conserved in eukaryotic organisms . Deregulation of FOX proteins has been linked to tumorigenesis, metastasis, and drug resistance . For example, FOXO1 phosphorylation overcomes chemoresistance in cancer cells by blocking IQGAP1-MAPK interactions . FOXR2 is required for the growth and spread of prostate cancer cells . Targeted reduction of FOXM1 was found to exert suppressive effects on NPC growth . FOXC2 overexpression induces chemoresistance in NPC cells . FOXQ1 acts as an oncogene in NPC . These studies suggest FOX proteins as promising targets for anticancer treatments.
microRNAs (miRs) are a class of short non-coding RNAs that post-transcriptionally regulate gene expression through base pairing to partially complementary sites in the 3′-untranslated region (3′-UTR) of target mRNAs . miRs play a critical role in the growth and progression of cancers . For instance, it was reported that miR-519 inhibits the proliferation of NPC cells by repressing URG4/URGCP . miR-342-3p is frequently dysregulated in human cancers, such as colon cancer  and pancreatic cancer . This miR shows growth-suppressive activity in hepatocellular carcinoma , lung cancer , and gallbladder cancer . miR expression profiling of NPC has revealed the downregulation of miR-342-3p in NPC . However, the biological function of miR-342-3p in NPC is still unknown.
In the present study, we collected 79 paired NPC tissue specimens and adjacent normal tissues and measured the expression of miR-342-3p. The prognostic impact of miR-342-3p was evaluated. To dissect the biological roles of miR-342-3p, gain- and loss-of-function experiments were carried out.
Clinicopathological parameters of NPC patients (n = 79)
No. of cases
Lymph node status
Four human NPC cell lines (C666, SUNE1, HNE1 and CNE2) and NP69 immortalized nasopharyngeal epithelial cells were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). The identity of the cell lines have been validated by short-tandem repeat analyses. They are free of mycoplasma contamination.
RNA extraction and real-time PCR analysis
Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA). Reverse transcription was performed using the TaqMan Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) and miR-342-3p specific stem-loop primer. Real-time PCR was conducted using the TaqMan MicroRNA Assay Kit (Applied Biosystems) according to the manufacturer’s instructions. U6 was used an endogenous control. The expression of miR-342- 3p was normalized against U6.
Stable expression of miR-342-3p in NPC cells
To generate miR-342-3p stable clones, C666 and CNE2 cells were transfected with a miR-342-3p-expressing plasmid (Cell Biolabs, San Diego, CA, USA) using Lipofectamine 2000 Reagent (Invitrogen). Control cells were transfected with the pEP-miR Null vector (Cell Biolabs). Forty-eight hours post-transfection, cells were selected in the presence of 2 μg/mL puromycin (Sigma-Aldrich) for 10 days.
C666 and CNE2 cells were transfected with anti-miR-342-3p inhibitor or negative control inhibitor (50 nM; Exiqon, Vedbaek, Denmark) using Lipofectamine 2000 Reagent. In some experiments, a FOXQ1-expressing plasmid or empty vector was transfected to C666 cells with stable expression of miR-342-3p. The FOXQ1-expressing plasmid was generated by inserting a full-length open reading frame of FOXQ1 (lacking the 3′-UTR) into pcDNA3.1(+) expression vector. Twenty-four hours after transfection, cells were tested for gene expression, proliferation, and invasion.
Cell proliferation and colony formation assays
Cells were seeded onto 96-well plates (4 × 103 cells/well) and cultured for 3 and 5 days. Each well was added with 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (0.5 mg/ml, Sigma-Aldrich) and incubated for 4 h at 37 °C. Dimethyl sulfoxide was used to dissolve the resultant crystals. Absorbance was recorded at 570 nm. For evaluation of colony formation capacity, cells were plated onto 6-well plates (500 cells/well) and cultured for 10 days. After staining with 1% crystal violet (Sigma-Aldrich), the number of colonies was determined using a microscope.
Transwell invasion assay
We used the methodology previously described by Zhao and colleagues . Cells in serum-free medium containing 10 μg/ml mitomycin C (Sigma-Aldrich) were seeded in the upper chamber of 24-well Transwell plates (2 × 104 cells/well). The inserts (8 μm in pore size) were precoated with Matrigel (BD Biosciences, San Jose, CA, USA). The lower chamber was filled with RPMI 1640 medium containing 10% FBS. The cells were allowed to invade through the Matrigel-coated inserts for 24 h. Afterwards, invaded cells were stained with 1% crystal violet and counted under a microscope.
Dual-luciferase reporter assay
Luciferase reporter assay was performed as previously described . The 3’-UTR of FOXQ1 mRNA was inserted into the pMIR-REPORT Luciferase miRNA Expression Reporter Vector (ThermoFisher Scientific, Waltham, MA, USA). A mutant form with disruption of the miR-342-3p binding site was constructed using the QuikChange site-directed mutagenesis kit (Stratagen, Santa Clara, CA, USA). C666 cells were co-transfected with the reporter constructs together with the miR-342-3p-expressing plasmid or empty vector. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) 48 h after transfection. The relative luciferase activity was determined after normalization against Renilla luciferase activity.
Tumorigenic studies in nude mice
For tumorigenic studies, C666 and CNE2 cells with stable overexpression of miR-342-3p or empty vector (2 × 106 cells/mouse) were subcutaneously inoculated into the flanks of male BALB/c nude mice (5 weeks of age; Shanghai Laboratory Animals Center of Chinese Academy of Sciences, Shanghai, China). Tumor growth was monitored for 5 weeks. After the last measurement of tumor volume, animals were are euthanized by carbon dioxide inhalation followed by cervical dislocation. The xenograft tumors were resected and weighed. Afterwards, tumor samples were fixed, embedded in paraffin, and subjected to immunostaining for Ki-67 using an anti-Ki-67 monoclonal antibody (Thermo Fisher Scientific; 1:400 dilution). The experiment protocol was approved by the Animal Care and Use Committee of Zhengzhou University.
Western blot analysis
Protein samples from NPC tissues and cells were prepared in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with the Protease and Phosphatase Inhibitor Cocktail (Abcam, Cambridge, UK). Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were probed with rabbit anti-FOXM1 polyclonal antibody (Abcam; 1:500 dilution), anti-FOXQ1 polyclonal antibody (Abcam; 1:500 dilution), or anti-β-actin polyclonal antibody (Thermo Fisher Scientific; 1:5000 dilution), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich). Signals were visualized by enhanced chemiluminescence (Thermo Fisher Scientific). Protein bands were quantified by densitometric analysis using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
Data are expressed as the mean ± standard deviation (SD). Differences in the means were determined using the Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The correlation between miR-342-3p and FOXQ1 protein levels in NPC specimens was determined by Pearson’s correlation coefficient analysis. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. P < 0.05 was considered statistically significant.
miR-342-3p is downregulated in NPC and predicts poor prognosis
miR-342-3p suppresses NPC cell growth and invasion in vitro
miR-342-3p restrains the growth of NPC xenograft tumors in vivo
miR-342-3p targets FOXQ1 in NPC cells
FOXQ1 rescues NPC cells from miR-342-3p-induced growth and invasion suppression
In this study, we showed that miR-342-3p was significantly underexpressed in NPC tissues compared to adjacent normal nasopharyngeal tissues. In line with our findings, the dysregulation of miR-342-3p is also described in colon cancer , pancreatic cancer , and hepatocellular carcinoma . We further analyzed the relationship between miR-342-3p expression and survival of NPC patients. It was found that downregulation of miR-342-3p had a poor prognostic impact on the survival of patients with NPC. Similarly, low miR-342-3p expression is significant associated with reduced overall survival in patients with hepatocellular carcinoma . These observations favor the idea that miR-342-3p may govern the progression of NPC.
To clarify the biological relevance of miR-342-3p downregulation in NPC, we performed gain- and loss-of-function experiments. The results demonstrated that ectopic expression of miR-342-3p significantly suppressed proliferation, colony formation, and invasion of NPC cells. In contrast, depletion of miR-342-3p led to a significant enhancement of NPC cell proliferation and invasion. In vivo tumorigenic studies confirmed that miR-342-3p overexpression hampered the growth of NPC xenograft tumors. These data indicate that miR-342-3p acts as a tumor suppressor in NPC. In support of our point, several previous studies have documented that miR-342-3p plays a tumor-suppressive role in multiple malignancies including hepatocellular carcinoma , colorectal cancer , osteosarcoma , and small cell lung cancer . However, a recent study has provided evidence that miR-342-3p has no significant impact on the progression of pancreatic acinar carcinoma in a mouse model . Therefore, the functional consequence of miR-342-3p depends on cellular contexts.
It is well accepted that a single miR has the ability to modulate a large number of target genes . Different targets have been identified to mediate distinct biological activities of miR-342-3p [23, 26, 27]. For instance, miR-342-3p regulates lumen formation in mammary gland morphogenesis through repression of ID4 and DNMT1 . This miR shows the ability to induce adipogenesis of mesenchymal stem cells by suppressing CtBP2 . Both FOXM1 and FOXQ1 mediate the tumor-suppressive activity of miR-342-3p in colorectal cancer cells . Similarly, we found that miR-342-3p overexpression caused a significant downregulation of FOXQ1 in both C666 and CNE2 cells. However, the level of FOXM1 remained unchanged in response to miR-342-3p overexpression. The selective repression of FOXQ1 implies its critical role in mediating the activity of miR-342-3p in NPC. Clinical evidence indicates a negative correlation between miR-342-3p and FOXQ1 expression in NPC specimens. Rescue experiments using a miR-resistant variant of FOXQ1 demonstrated that enforced expression of FOXQ1 significantly reversed the suppressive effect of miR-342-3p on NPC cell proliferation and invasion. FOXQ1 functions as an oncogene in colorectal carcinoma , gastric cancer , and NPC . It has been documented that FOXQ1 can facilitate the metastasis of gastric cancer cells through upregulation of Snail . FOXQ1 is upregulated in NPC and can be regulated by a number of miRs including miR-124  and miR-506 . Our data expand the list of miR regulators of FOXQ1 to include miR-342-3p and confirm the upregulation of FOXQ1 as a consequence of dysregulation of miRs in NPC.
However, it should be mentioned that apart from FOXQ1, other target genes may also be involved in the activity of miR-342-3p in NPC. Genome-wide identification of target genes would be necessary to uncover the mechanism by which miR-342-3p governs the aggressive phenotype of NPC cells. The mechanism underlying the downregulation of miR-342-3p in NPC is still elusive. A previous study has indicated that long non-coding RNAs such as FTX  show the capacity to repress the expression of miR-342-3p. The reduced expression of miR-342-3p can also be explained as a result of abberrant methylation of the promoter region of its host gene, EVL . Ongoing studies are conducted to address this issue. Another limitation of this study was the enrollment of patients from a single center.
In summary, our data demonstrate that miR-342-3p has a poor prognostic impact on the survival of NPC patients. Restoration of miR-342-3p suppresses NPC cell growth and invasion via targeting of FOXQ1 and may have therapeutic benefits in the treatment of this malignancy.
No funding was obtained for this study.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
ZC and YZ made substantial contributions to conception design, acquisition of data, analysis and interpretation of data, and write/edit manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Written informed consent for research was obtained from each patient. This retrospective study was approved by the Institutional Human Experiment and Ethics Committee of the First Affiliated Hospital of Zhengzhou University.
Consent for publication
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 23.Zhang S, Liu L, Lv Z, Li Q, Gong W, Wu H. MicroRNA-342-3p inhibits the proliferation, migration, and invasion of osteosarcoma cells by targeting astrocyte-elevated Gene-1 (AEG-1). Oncol Res. 2017. https://doi.org/10.3727/096504017X14886485417426 [Epub ahead of print].
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.