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Introduction

Nasopharyngeal carcinoma (NPC) is a type of cancer arising from the nasopharynx epithelium and has a high prevalence in south Asia and Africa. Its etiological agent includes Epstein-Barr virus (EBV) infection, environmental and diet factors and genetic susceptibility (1). Due to its insidious location and lack of early symptoms, NPC patients tend to present with a late-stage diagnosis. Although NPC is radiosensitive, the patient 5-year survival rate remains low, largely due to regional lymph node and distant metastasis and locoregional recurrence. The outcomes of advanced-stage NPC are markedly worse compared with those in earlier stages (2). Thus, identifying specific biomarkers with diagnostic value and understanding the molecular mechanisms that regulate the invasion and metastasis of NPC is urgently needed.

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs of 19–25 nucleotides in length, which regulate ~60% of gene expression by binding to the 3′ untranslated region (UTR) of the mRNA targets through the seed sequence (3). A growing body of literature indicates that miRNAs play important roles in most biological processes, such as development, proliferation, differentiation, cell cycle control and cell death, tumorigenesis and adaptation to stress (4). Thus, miRNAs have emerged as potential biomarkers or therapeutic targets for human diseases including cancer.

To date, aberrant miRNA expression has been reported in the development and progression of NPC. For example, miR-124 (5), miR-125a-5p (6) and miR-101 (7) acted as tumor suppressors by inhibiting NPC cell growth, or by promoting cell apoptosis. miR-200a and miR-200b suppressed NPC cell migration and invasion by targeting ZEB2, CTNNB1 and Notch1 (8,9). miR-139-5p, miR-3188 and miR-29c were reported to enhance the sensitivity of NPC to chemotherapy and radiotherapy (10–12). Conversely, some miRNAs can act as oncogenes in NPC. Upregulation of miR-19a and miR-222 promoted NPC cell proliferation by directly targeting transforming growth factor β receptor 2 or PTEN (13,14).

In our previous study, whole-genome small RNA deep sequencing was performed on NPC cell lines C666-1, CNE2 and the non-neoplastic cell line NP69, to characterize miRNA expression profiles in NPC cells (unpublished data). The aberrantly expressed miRNAs were subsequently validated by real-time quantitative PCR in NPC and non-cancerous nasopharyngitis biopsies. Of these differentially expressive miRNAs, miR-342-3p was identified to be significantly downregulated in NPC cell lines and tissues. However, thus far, the expression profile of miR-342-3p and its role in NPC have not been characterized.

In the present study, we investigated the tumor-suppressive roles of miR-342-3p in NPC cells. We revealed that overexpression of miR-342-3p inhibited NPC cell proliferation and invasion by directly targeting Cdc42.

Materials and methods

Patients and samples

Twenty-two NPC tissue samples and 15 nasopharyngitis tissues were collected and total RNA was extracted for further RT-qPCR assay. Formalin-fixed, paraffin-embedded tissues (FFPTs) of 10 NPC tissues were acquired from NPC patients who were firstly diagnosed with NPC at the Sun Yat-sen University Cancer Center (SYSUCC; Guangzhou, China) from January 2007 to December 2007. The patients were histologically and clinically diagnosed with NPC and assessed according to the TNM staging of The International Union against Cancer. None of the patients were subjected to radiotherapy or chemotherapy prior to biopsy sampling. The present study was approved by the Research Ethics Committee of SYSUCC, and written informed consent was obtained from all patients.

Immunohistochemical staining

Paraffin sections were prepared from NPC patients and immunohistochemistry (IHC) assays were performed according to the manufacturer's instructions to detect the protein expression. The antibody rabbit anti-Cdc42 (1:200; cat. no. 10155-AP-1) was purchased from Proteintech Group Inc., (Wuhan, China). In brief, slides with paraffin sections underwent deparaffinage and aquation. Then 3% hydrogen peroxide was used to block endogenous peroxidase activity and antigen retrieval was performed by boiling slides in ethylene diamine tetraacetic acid (EDTA) (1 mmol/l, pH 8.0). The slides were incubated with the primary antibody anti-Cdc42 overnight at 4°C. The staining score was assessed by two pathologists independently. The intensities were graded as 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive). The abundance of positive cells was graded from 0 to 100%. The staining score was determined using the following formula: Overall scores = percentage score × intensity score × 100.

NPC cell culture and transfection

The human NPC cell lines C666-1, SUNE1, 6-10B and 5-8F used in the present study, were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.). The human immortalized nasopharyngeal epithelial cell line, NP69, was grown in defined-keratinocyte serum-free medium (KSFM) supplemented with EGF (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.). The aforementioned cells were cultured in a humified atmosphere with 5% CO2 at 37°C. All NPC cell lines and NP69 cell were provided generously by Professor Musheng Zeng (Sun Yat-sen University Cancer Center, Guangzhou, China). All cells were negatively tested for mycoplasma contamination before use, and authenticated based on STR fingerprinting before use at The Medicine Lab of Forensic Medicine Department of Sun Yat-sen University. miR-342-3p mimics were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou, China) as follows: miR-342-3p sense, 5′-UCUCACACAGAAAUCGCACCCGU-3′ and antisense, 5′-GGGUGCGAUUUCUGUGUGAGAUU-3′). miR-342-3p mimics were transfected into 6-10B cells at a final concentration of 15 nM using Lipofectamine 3000 (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.).

Reverse quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from tissues or cells with TRIzol reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. cDNA was obtained using GoScript Reverse Transcription Mix (Promega Corp., Madison, WI, USA) and Bulge-Loop™ miRNA qPCR Primer set for miR-342-3p (Guangzhou RiboBio Co., Ltd., Guangzhou, China). qPCR was performed with GoTaq® qPCR Master Mix (Promega Corp.). The standard cycling program was performed as follows: hot-start activation: 95°C for 2 min, 1 cycle; denaturation: 95°C for 15 sec, 40 cycles; annealing/extension: 60°C for 60 sec; dissociation: 75°C, 1 cycle. The mRNA expression level was examined by the 2−ΔΔCq method (15) and normalized by internal control U6. The sequence of Cdc42 primer for RT-qPCR assay was as follows: Forward, 5′-CCATCGGAATATGTACCGACTG-3′ and reverse, 5′-CTCAGCGGTCGTAATCTGTCA-3′.

Cell proliferation and colony formation assay

Cell growth was determined by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). In brief, 1,000 cells/well were suspended into a 96-well-plate and pre-incubated for 24 h in a humidified incubator (5% CO2 at 37°C). Following the addition of 10 µl CCK-8 solution to each well of the plate and incubation of the plate for 3 h, the absorbance at 450 nm was determined using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were performed in triplicates.

Concerning the colony formation assay, 6-10B miR-342-3p mimics, as well as their control cells were seeded into a 6-well-plate (500 cells/well) with complete medium. The cells were grown for 12 days at 37°C with 5% CO2. To visualize the colony formation, the cells were fixed with methanol and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China) and observed under an inverted fluorescent microscope. The number of colonies >0.1 mm diameter was counted. The colony formation of each cell line was performed in triplicate.

Luciferase reporter assay

The wild-type (wt) or mutant (mt) Cdc42-3′UTR luciferase reporter vector was designed on the basis of miR-342-3p binding sites. In brief, 4×104 293T cells/well were seeded into a 24-well plate. Following incubation for 24 h at 37°C with 5% CO2, using Lipofectamine 3000 reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.), 1,000 ng firefly luciferase reporter plasmid was co-transfected with 10 ng pRL-TK Renilla plasmid into cells, combined with miR-342-3p mimics or negative control. In accordance with the manufacturer's instructions, the luciferase and Renilla signals were determined using the Dual-Luciferase Reporter Assay kit (Promega Corp.). The experiments were repeated independently three times.

Wound healing and invasion assay

A scratch wound healing assay was performed to assess the cell migration rate. Cells transfected with miR-342-3p mimics were cultured in a 6-well plate. Subsequently, as the cells reached sub-confluence, with a sterile micropipette tip, a scratching wound was generated and images were captured under an inverted fluorescent microscope at 0 and 48 h. The culture medium was replaced by serum-free RPMI-1640 medium. The distance between either side of the scratch in images captured by the inverted fluorescent microscope was assessed to evaluate the cell migration rate.

Cell invasion assay was conducted using cell culture inserts with 8-µm PET track-etched membranes (Falcon; BD Biosciences, Franklin Lakes, NJ, USA). Tumor cells were placed into the upper chambers (5×104 cells/well) with 200 µl serum-free medium, whereas the lower chambers in the 24-well-plate contained 650 µl complete medium with 10% FBS. After 16 h, the cells were fixed with methanol and stained with 0.1% crystal violet. Cells in the upper chambers were removed gently and the cells which had invaded into the membranes were imaged under an inverted fluorescent microscope. The number of invasive cells were counted for statistical use. All the aforementioned assays were performed three times.

Western blot analysis

Cells were harvested and lysed in RIPA lysis buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF). Total protein concentration was quantified by BCA assay kit (Beyotime Institute of Biotechnology) and Gen5 Software (CHS 2.06; BioTek Instruments, Winooski, VT, USA). An equal volume of protein samples (30 µg) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were then incubated with the primary antibodies, including Snail, β-catenin, vimentin, cyclin D1, cyclin D3, CDK4, Myc, Cdc42 and GAPDH at 4°C overnight and subsequently with the secondary antibody (anti-rabbit IgG, HRP-linked antibody; 1:5,000; cat. no. 7054; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The signals from protein were detected by an advanced ECL detection kit. The rabbit monoclonal antibodies used in the present study were purchased from Cell Signaling Technology, including those against human Snail (1:1,000; cat. no. 3879), β-catenin (1:1,000; cat. no. 8480), vimentin (1:1000; cat. no. 5741), cyclin D1 (1:1,000; cat. no. 2978), cyclin D3 (1:1,000; cat. no. 2936), CDK4 (1:1,000; cat. no. 12790), c-Myc (1:1,000; cat. no. 13987) and GAPDH (1:1000; cat. no. 5174); Cdc42 (1:800; cat. no. 10155-AP-1) was purchased from Proteintech Group Inc. (Wuhan, China).

MicroRNA sequencing and DataSets

A whole genome microRNA sequencing was carried out by Illumina Hiseq 2500 to detect the dysregulated miRNAs in NPC cell C666-1 and immortalized nasopharyngeal epithelial cell NP69. Data in the GEO DataSets (GSE32960 and GSE36682) was involved in the study (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). starBase v2.0 (http://www.lncrnablog.com/starbase-v2-0-for-decoding-rna-interaction-networks/), miRWalk 2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html) and microRNA.org (http://microrna.org/) were applied to predict the potential targets of miR-342-3p.

Statistical analysis

All statistical analyses were performed using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). To assess the differences among categorical variables, one-way analysis of variance (ANOVA)/Student-Newman-Keuls (SNK) test and independent-sample Student's t-test and Spearman rank correlation were the four major methods used to analyze the results of the present study. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-342-3p is downregulated in NPC cell lines and tissues

To explore the deregulation of miRNAs in human NPC oncogenesis, a whole genome microRNA sequencing using Illumina Hiseq 2500 was performed and its result revealed that the expression level of miR-342-3p was clearly lower in NPC C666-1 cells than in non-tumor NP69 cells (Table I). To ascertain this finding, we determined the expression level of miR-342-3p in four NPC cell lines including C666-1, 6-10B, 5-8F and SUNE2 using RT-qPCR assays, with the immortalized cell line NP69 used as the control. We observed that the expression of miR-342-3p was markedly downregulated in all four NPC cell lines compared with NP69 (Fig. 1B). In addition, the expression of miR-342-3p was also detected in 22 NPC and 15 nasopharyngitis samples. Consistent with results in NPC cell lines, it was observed that miR-342-3p was downregulated in NPC compared with nasopharyngitises (P<0.0001, Fig. 1A). Furthermore, data in the GEO DataSets (GSE32960 and GSE36682) revealed that the expression level of miR-342-3p in NPC was significantly lower than that in normal tissues using microarray, which was consistent with our results (Table II).

Figure 1. miR-342-3p is downregulated in NPC samples and cell lines. Differences in the expression of miR-342-3p were compared between (A) 22 NPC and 15 nasopharyngitis samples and (B) NPC cell lines and the immortalized nasopharyngeal epithelial cell line NP69. ***P<0.001. NPC, nasopharyngeal carcinoma.

Table I. miR-342-3p is downregulated in nasopharyngeal carcinoma C666-1 cells compared with NP69 cells in microRNA-Seq results.

Table I.

miR-342-3p is downregulated in nasopharyngeal carcinoma C666-1 cells compared with NP69 cells in microRNA-Seq results.

	Mean expression value
MicroRNA	NP69	C666-1	log2 (fold change)	P-value
hsa-miR-342-3p	327.16	26.36	−3.64 (C666-1 vs. NP69)	0.006960605

Table II. The expression of miR-342-3p is reduced in nasopharyngeal carcinoma compared with NP in MicroRNA GEO datasets.

Table II.

The expression of miR-342-3p is reduced in nasopharyngeal carcinoma compared with NP in MicroRNA GEO datasets.

		No. of cases
MicroRNA GEO datasets accession no.	miRNA_ID	NP	NPC	log2 (fold change)	P-value	Adjusted P-value
GSE32960	hsa-miR-342-3p	312	18	−1.479853 (NPC vs. NP)	7.95E-32	6.63E-30
GSE36682	hsa-miR-342-3p	62	6	−1.295291 (NPC vs. NP)	0.0004	0.0018

[i] NPC, nasopharyngeal carcinoma; NP, nasopharyngitis.

miR-342-3p inhibits NPC cell proliferation in vitro

To investigate the biological function of miR-342-3p in NPC cells, the miR-342-3p mimics were transfected into 6-10B cells. The overexpression efficiency was validated by RT-qPCR assay (Fig. 2A). miR-342-3p overexpression significantly suppressed NPC cell viability as assessed by CCK-8 assay (Fig. 2B). In addition, compared with the scramble control, colony formation ability of 6-10B cells was also decreased, which was induced by miR-342-3p mimics (Fig. 2C). These findings indicated that miR-342-3p may act as a suppressor in NPC. In addition, cell cycle-related proteins cyclin D1, cyclin D3 and CDK4 were downregulated following miR-342-3p overexpression (Fig. 2D). Our results demonstrated that miR-342-3p suppressed NPC cell proliferation in vitro.

Figure 2. High expression of miR-342-3p inhibits NPC cell growth in vitro. (A) RT-qPCR validation of miR-342-3p overexpression efficiency in 6-10B cells treated with miR-342-3p mimics. (B) A CCK-8 assay revealed that miR-342-3p overexpression in NPC cells delayed cell growth in 6-10B cells. (C) The effects of overexpression of miR-342-3p on the clonogenic ability of 6-10B cells. (D) miR-342-3p-overexpressing 6-10B cells underwent western blot analysis using antibodies specific to cyclin D1, cyclin D3, CDK4, MYC and GAPDH. miR-342-3p mimics markedly inhibited the expression of cyclin D1, cyclin D3, CDK4 and MYC. ***P<0.001 vs. NC. NPC, nasopharyngeal carcinoma.

miR-342-3p inhibits NPC cell migration and invasion in vitro

The effects of miR-342-3p overexpression on NPC cell migration and invasion were determined by wound healing and Transwell assays, respectively. In the wound healing assay, the gap filling was markedly decreased in the miR-342-3p-overexpressing 6-10B cells compared with the control cells (Fig. 3A). In the Matrigel-coated Transwell assay, the number of invading cells was markedly decreased in the miR-342-3p-overexpressing 6-10B cells compared with control cells (Fig. 3B). In addition, the expression of transcription factor Snail and mesenchymal markers β-catenin and vimentin were reduced in the miR-342-3p-overexpressing 6-10B cells (Fig. 3C). Collectively, our results indicated that miR-342-3p inhibited NPC cell migration and invasion.

Figure 3. miR-342-3p regulates the migration and invasiveness of NPC cells in vitro. (A) A wound healing assay demonstrated that miR-342-3p overexpression markedly delayed the migration of 6-10B cells. (B) A Transwell assay demonstrated that miR-342-3p overexpression markedly attenuated the invasion of 6-10B cells. ***P<0.001 vs. NC (C) A western blotting assay revealed reduced levels of β-catenin, vimentin and the EMT regulatory factor Snail in 6-10B cells treated with miR-342-3p mimics compared with NC cells. NPC, nasopharyngeal carcinoma; EMT, epithelial-mesenchymal transition.

miR-342-3p directly targets Cdc42 in NPC

To further explore how miR-342-3p regulated proliferation and migration of NPC cells, we used multiple target algorithms (starBase v2.0, miRWalk 2.0 and microRNA.org) to predict candidate targets of miR-342-3p and observed that the 3′UTR of Cdc42 matched the ‘seed sequence’ of miR-342-3p (Fig. 4A). Luciferase reporter assay revealed that miR-342-3p overexpression inhibited the luciferase activity of wild-type (wt) Cdc42 3′UTR but not the mutant (mt) Cdc42 3′UTR (P<0.01, Fig. 4B). RT-qPCR and western blot assays confirmed that both the mRNA and protein levels of Cdc42 were downregulated when miR-342-3p was overexpressed in 6-10B cells (Fig. 4C and D). Our results determined that Cdc42 was one of the direct targets of miR-342-3p. To further confirm the relationship between miR-342-3p and Cdc42, we analyzed the miR-342-3p expression by RT-qPCR and Cdc42 protein expression by immunohistochemistry assay simultaneously in 10 NPC samples (Fig. 5A). As displayed in Fig. 4E, miR-342-3p expression was inversely correlated with Cdc42 expression (Fig. 4E Spearman's rank correlation coefficient r=−0.749, P=0.013) in the same NPC specimens, which indicated that miR-342-3p was actually involved in the in vivo regulation of Cdc42 in NPC patients.

Figure 4. Cdc42 is a direct target of miR-342-3p in NPC. (A) Schematic of predicted miR-342-3p-binding sites in the Cdc42 3′UTR. (B) Luciferase reporter assays in 6-10B cells co-transfected with either the wild-type Cdc42 3′UTR or mutated 3′UTR construct with miR-342-3p or NC oligonucleotides. (C) RT-qPCR analysis of Cdc42 mRNA levels in the 6-10B cells transfected with miR-342-3p mimics or NC oligos. (D) Representative western blot images of Cdc42 in 6-10B cells transfected with NC or miR-342-3p mimics harvested at 72 h. (E) The expression levels of miR-342-3p and Cdc42 were detected simultaneously in 10 NPC samples. Spearman's rank correlation was calculated on the basis of the relative expression levels of miR-342-3p by RT-qPCR and relative protein expression levels (IHC scores) of Cdc42. **P<0.01, ***P<0.001 vs. NC. NPC, nasopharyngeal carcinoma; IHC, immunohistochemistry.

Figure 5. Cdc42 is upregulated in NPC and promotes cell invasiveness. (A) Representative immunohistochemical staining images of Cdc42 in NPC tissues and nasopharyngitis tissues. The staining density of Cdc42 was higher in tumor cells than in adjacent non-tumor epithelial cells. (B) Western blot analysis revealed that ML141 inhibited Cdc42. (C) A Transwell assay demonstrated that cell invasiveness was markedly reduced in 6-10B cells following treatment with Cdc42 inhibitor ML141 (20 µM). ***P<0.001 vs. DMSO. NPC, nasopharyngeal carcinoma; DMSO, dimethyl sulfoxide.

It has been reported that overexpression of Cdc42 was detected in different types of human cancer and correlated with increased cancer progression and poorer outcome (16). However, the expression profile and exact role of Cdc42 in NPC were not clear. In the present study, we observed that the expression of Cdc42 in NPC cells was higher than adjacent non-tumor epithelium as determined by immunohistochemistry (Fig. 5A). To explore the role of Cdc42 expression in NPC cells, we treated 6-10B cells with Cdc42 inhibitor ML141, and observed that ML141 treatment significantly reduced NPC cell invasion ability (Fig. 5B and C). Our findings indicated that miR-342-3p may act as a tumor suppressor in NPC by inhibiting Cdc42.

Discussion

miR-342 is encoded in an intron of the gene EVL, whose protein product is an actin-associated protein involved in a variety of processes related to cytoskeleton remodeling, cell motility and polarity (17). A previous study reported that both EVL and miR-342 genes were coordinately downregulated in the majority of colorectal cancers due to CpG island methylation upstream of the EVL/hsa-miR-342 locus (18).

Reduced expression of miR-342 has been demonstrated in colorectal cancer, hepatocellular carcinoma, cervical cancer, osteosarcoma and extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTCL) tissues compared with normal tissues (19–22). In addition, research has revealed that miR-342-3p may be used as a potential therapeutic and prognostic biomarker. Breast cancer patients with high miR-342 expression level have significantly better survival compared to patients with low expression (9). Cittelly et al (23) reported that miR-342 was downregulated in tamoxifen resistant breast tumor cell lines and tamoxifen refractory human breast tumors. The expression level of miR-342-3p was reduced in metastatic non-small cell lung cancer samples compared to those in primary tumor samples. Functional studies revealed that miR-342-3p exerted a tumor suppressive role that was operated through regulation of cell proliferation, apoptosis and cell cycle progression, invasion and migration. AGR2 (24), AEG-1 (21), Ikk-g, TAB2 and TAB3 (19), FOXM1 (20), TIAM1 (22) and DNMT1 (25) have been experimentally confirmed as the direct targets for miR-342.

To date, the expression profile and biological function of miR-342-3p in NPC have not been reported. Based on our miRNA sequencing results and RT-qPCR validation, miR-342-3p was identified to be downregulated in NPC tissues and cell lines compared with normal controls. miR-342-3p overexpression inhibited cell proliferation, epithelial-mesenchymal transition (EMT), migration and invasion in NPC cells. These findings were generally consistent with previous studies that suggested a possible tumor suppressor function of miR-342-3p in other cancers.

In the present study, we revealed for the first time that miR-342-3p may exert its function by specifically targeting Cdc42 (cell division control protein) in NPC. Cdc42, a member of the Rho GTPase family, acts as a molecular switch in multicellular pathways. Activated Cdc42 mediates a signaling cascade leading to the activation of more than twenty downstream effectors, and then influences a variety of cellular responses such as cell polarity, cytoskeleton remolding, proliferation, migration, cellular transformation and gene expression. Cdc42 is overexpressed in a number of human cancers, and in some instances, has been correlated with poor prognosis. Despite the prominent association of Cdc42 activation with tumor development and progression, the regulation and oncogenic role of Cdc42 in NPC cells remains to be elucidated. Epstein-Barr Virus-encoded LMP1 was reported to interact with FGD4 to activate Cdc42 and thereby promote migration of NPC cells (26). A traditional Chinese medicine, berberine, may inhibit the anti-migration and anti-invasion properties of NPC cells through effective inactivation of Cdc42 (27). These studies revealed that Cdc42 may be involved in the progression and metastasis in NPC. In the present study, we provided evidence from the luciferase activity assay and western blot analysis that Cdc42 was a direct target of miR-342-3p. In addition, the protein expression levels of Cdc42 in most NPC cell lines were higher than that in non-neoplastic cell line NP69 as determined by using western blot analysis. Cdc42 expression in NPC biopsies was also higher than adjacent non-tumor epithelium as revealed by immunohistochemistry. These findings indicated that Cdc42 expression was activated in NPC and downregulation of miR-342-3p in NPC may be the cause of Cdc42 activation. In addition, the inhibition of Cdc42 activity by ML141 caused marked reduction of NPC cell invasion ability, indicating that Cdc42 may play an important role in NPC progression.

Our results revealed that reduced expression of miR-342-3p in human NPC contributed to the enhanced proliferation and invasion of NPC cells by direct targeting of the Cdc42 pathway. Cdc42-selective small-molecule inhibitors have been used in a variety of cancer models, including colon, breast and skin tumors (28–30). Our study provided evidence that miR-342-3p may be used as a potential biomarker and new therapeutic target for Cdc42 regulation and NPC progression.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the National Nature Science Foundation of China (nos. 81772884, 81572466, 81772991 and 81629004).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

SM, HW and XFSZ conceived and designed the experiments. SM, LS and MW wrote the study. RX and MZ collected and prepared the tissues of NPC patients. LS and RX performed the experiments. LS, NW, XZ and SM analyzed and interpreted the data. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The present study was approved by the Research Ethics Committee of Sun Yat-sen University Cancer Center (SYSUCC; Guangzhou, China), and written informed consent was obtained from all patients involved in this study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References