Introduction
Head and neck squamous cell carcinoma (HNSCC) occurs from the mucosa in the upper aerodigestive tract, including the oral cavity, oropharynx, hypopharynx and larynx, and this disease is the sixth most common cancer worldwide (1). Approximately 550,000 new patients are diagnosed, and 30,000 patients die of this disease annually (2). Due to the local recurrence and distant metastasis of HNSCC, the overall survival of patients with HNSCC has not improved in the last decade (3). Currently developed targeted molecular therapies are not sufficiently efficacious in the management of HNSCC (3). Therefore, improving our understanding of the molecular mechanisms of HNSCC aggressiveness is needed based on current genomic approaches.
MicroRNAs (miRNAs) are small noncoding RNAs (19–22 nucleotides in length) involved in the repression or degradation of target RNA transcripts in a sequence-dependent manner (4). One of the unique features of miRNAs is that a single miRNA regulates a vast number of protein-coding or noncoding RNAs in human cells (5). Thus, aberrant expression of miRNAs disrupts systematically regulated RNA networks in cancer cells. In fact, accumulating evidence has revealed that aberrant expression of miRNAs is deeply involved in the pathogenesis of human cancers (6).
In miRNA biogenesis, precursor miRNA (pre-miRNA) is cleaved in the cytoplasm, generating a miRNA duplex comprised of a guide strand and passenger strand. The guide strand of miRNA is thought to be incorporated into the RNA-induced silencing complex (RISC) to target mRNAs, whereas the passenger strand of miRNA is degraded and is not thought to have regulatory activity in cells (7). However, in contrast to this paradigm, we demonstrated that passenger strands of miRNAs, i.e., miR-144-5p, miR-139-3p, miR-150-3p and miR-145-3p, were downregulated and acted as antitumor miRNAs in several types of cancers (8–13). Moreover, dual strands of pre-miR-145 (miR-145-5p and miR-145-3p) coordinately target oncogenic MTDH and UHRF1 in lung cancer and bladder cancer, respectively (10,11). The involvement of passenger miRNA strands and regulation of cancer networks by passenger miRNAs are novel concepts in cancer research.
Analysis of the miRNA expression signature of HNSCC by RNA sequencing revealed that miR-145-5p and miR-145-3p were significantly downregulated in cancer tissues. The guide strand miR-145-5p has been established as an oncogene in several cancers, including HNSCC (14). However, the functional significance of the passenger strand of miR-145 in HNSCC is still unknown. The aims of this present study were to investigate the antitumor function of miR-145-3p and to identify its target oncogenic genes in HNSCC cells. Elucidation of the antitumor roles of passenger strands of miRNAs and the cancer networks mediated by these miRNAs may provide insights into the molecular pathogenesis of HNSCC.
Materials and methods
Clinical HNSCC specimens, cell lines, and cell culture
A total of 22 clinical tissue specimens were collected from patients with HNSCC who underwent surgical resection at Chiba university Hospital between 2008 and 2014. The clinicopathological features of patients with HNSCC are summarized in Table I. All patients in this study provided informed consent, and the study protocol was approved by the Institutional Review Board of Chiba University. TNM classification and tumor stage were determined by the union for International Cancer Control (UICC) (15).
In this study, we used the following human HNSCC cells: SAS (derived from a primary lesion of tongue squamous cell carcinoma) and HSC3 (derived from human lymph node metastasis of tongue squamous cell carcinoma), as described previously.
Mature miRNA and small interfering RNA (siRNA) transfection into HNSCC cells
The following RNA species were used in this study: mature miRNAs, Pre-miR miRNA Precursors (hsa-miR-145-3p, assay ID: PM 13036; hsa-miR-145-5p, assay ID: PM 11480), negative control miRNA (assay ID: AM 17111) (both from Applied Biosystems, Foster City, CA, USA), siRNA (Stealth Select RNAi siRNA; si-MYO1B P/N: HSS106714 and HSS106716; Invitrogen, Carlsbad, CA, USA). The transfection procedures were described previously (16–20).
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
The procedure for PCR quantification was described previously (16–19). TaqMan probes and primers for MYO1B (P/N: Hs00362654_m1; Applied Biosystems) were assay-on-demand gene expression products. Expression for miR-145-3p (P/N: 002149; Applied Biosystems) and miR-145-5p (P/N: 002278) was used to quantify the expression levels of miRNAs according to the manufacturer's protocol. To normalize the data for quantification of mRNA and miRNAs, we used human GUSB (P/N: Hs99999908_m1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (P/N: Hs02758991_m1) and RNU48 (assay ID: 001006) (all from Applied Biosystems). The relative expression levels were analyzed using the 2−ΔΔCT method.
Cell proliferation, migration, and invasion assays
Cell proliferation, migration and invasion assays were described previously (16–19).
Incorporation of miR-145-3p or miR-145-5p into the RISC by Ago2 immunoprecipitation
SAS cells were transfected with 10 nM miRNA by reverse transfection. After 48 h, immunoprecipitation was performed using a human Ago2 miRNA isolation kit (Wako, Osaka, Japan) according to the manufacturer's protocol. Expression levels of miR-145-3p or miR-145-5p were measured by qRT-PCR. miRNA data were normalized to the expression of miR-150-5p (P/N: PM10070; Applied Biosystems), which was not affected by miR-145-3p and miR-145-5p transfection.
Western blot analysis
Cells were harvested and lysed 48 h after transfection. Each cell lysate (50 µg of protein) was separated using Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed with monoclonal anti-MYO1B antibodies (1:250 dilution; HPA013607; Sigma-Aldrich, St. Louis, MO, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1,000 dilution; ab8245; Abcam, Cambridge, UK) were used as an internal control. The procedures were described in our previous studies (16–19).
Identification of putative genes regulated by miR-145-3p in HNSCC cells
Specific genes regulated by miR-145-3p were identified by a combination of in silico and genome-wide gene expression analyses. Genes regulated by miR-145-3p were listed using the TargetScan database. Oligo microarrays (Human GE 60K; Agilent Technologies) were used for gene expression analyses. The microarray data were deposited into GEO (http://www.ncbi.nlm.nih.gov/geo/), with accession number GSE82108. Upregulated genes in HNSCC were obtained from publicly available data sets in GEO (accession no. GSE9638). To identify signaling pathways regulated in silico, gene expression data were analyzed using the KEGG pathway categories with the GeneCodis program.
Regulation of targets downstream of MYO1B in HNSCC
We investigated pathways regulated by MYO1B in HNSCC cells. We analyzed gene expression using si-MYO1B-transfected SAS cells. Microarray data were used for expression profiling of si-MYO1B transfectants. The microarray data were deposited into GEO (accession no. GSE100746). We analyzed common downregulated genes using the GEO dataset.
Plasmid construction and dual-luciferase reporter assay
The partial wild-type sequence of the MYO1B 3′-untranslated region (3′-UTR) was inserted between the XhoI-PmeI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Alternatively, we used sequences that were missing the miR-145-3p target sites (position 88–94 or position 1117–1123). The synthesized DNA was cloned into the psiCHECK-2 vector. SAS cells were transfected with 20 ng of the vector, 20 nM microRNAs, and 1 µl Lipofectamine 2000 in 100 µl Opti-MEM (both from Invitrogen). The procedure of dual-luciferase reporter assay was described previously (16–19).
Immunohistochemistry
Formalin-fixed, paraffin-embedded (FFPE) tissues were used. Tissue sections were incubated overnight at 4°C with anti-MYO1B antibodies diluted 1:300 (HPA013607; Sigma-Aldrich). The procedure for immunohistochemistry was described previously (21).
The Cancer Genome Atlas (TCGA)-HNSCC data analysis
To explore the clinical significance of MYO1B in HNSCC, we used the RNA sequencing database in TCGA (https://tcga-data.nci.nih.gov/tcga/). The gene expression and clinical data were retrieved from cBioportal (http://www.cbioportal.org/, the provisional data downloaded July 1, 2017).
Statistical analysis
Relationships between two or three variables and numerical values were analyzed using Mann-Whitney U tests or Bonferroni-adjusted Mann-Whitney U tests. Spearman's rank tests were used to evaluate the correlations between the expression of miR-145-3p or miR-145-5p and target genes. Expert StatView software (version 5.0; SAS Institute Inc., Cary, NC, USA) was used for these analyses. Multivariate Cox proportional hazard regression models were used to determine independent factors for survival with JMP Pro 13.
Results
Expression levels of miR-145-5p and miR-145-3p in HNSCC clinical specimens and cell lines
To confirm our miRNA expression signatures in HNSCC by RNA sequencing, we validated the expression levels of miR-145-5p and miR-145-3p in HNSCC clinical specimens and cell lines. In Fig. 1, the expression levels of miR-145-5p and miR-145-3p were significantly reduced in cancer tissues compared with those in corresponding adjacent noncancerous epithelium (p<0.0001) (Fig. 1A). Additionally, the expression levels of miR-145-5p and miR-145-3p in SAS and HSC3 cells were markedly downregulated (Fig. 1A).
Spearman's rank test showed a positive correlation between the expression levels of miR-145-5p and miR-145-3p in clinical specimens (Fig. 1A).
Effects of ectopic expression of miR-145-5p and miR-145-3p on cell proliferation, migration and invasion in HNSCC cell lines
To validate the functional roles of miR-145-3p and miR-145-5p, we carried out gain-of-function assays using miRNA transfection into two HNSCC cell lines (SAS and HSC3). XTT assays revealed that cell proliferation was significantly inhibited in miR-145-3p and miR-145-5p transfectants in comparison with mock or miR-control transfectants (Fig. 1B). Similarly, migration assays showed that cell migration activity was significantly inhibited in miR-145-3p and miR-145-5p transfectants in comparison with mock and miR-control transfectants (Fig. 1C). Matrigel invasion assays also demonstrated that cell invasion activity was significantly inhibited in miR-145-3p and miR-145-5p transfectants in comparison with mock and miR-control transfectants (Fig. 1D).
Incorporation of miR-145-3p into the RISC in HNSCC cells
We hypothesized that the passenger strand miR-145-3p may be incorporated into the RISC and exert important effects in cancer cells. Accordingly, we performed immunoprecipitation with antibodies targeting Ago2, which plays an important role in the RISC. After transfection with miR-145-3p or miR-145-5p, Ago2-bound miRNAs were isolated, and qRT-PCR was carried out to determine whether miR-145-3p and miR-145-5p bound to Ago2. After transfection with miR-145-3p and immunoprecipitation by anti-Ago2 antibodies, miR-145-3p levels were significantly higher than those of mock- or miR-control-transfected cells and those of miR-145-5p-transfected SAS cells (p<0.0001) (Fig. 2A). Similarly, after miR-145-5p transfection, miR-145-5p was detected by Ago2 immunoprecipitation (p<0.0001) (Fig. 2B).
Identification of putative targets of miR-145-3p regulation in HNSCC cells
We performed in silico and gene expression analyses to identify genes targeted by miR-145-3p for regulation (Fig. 3). First, we selected putative miR-145-3p target genes using the TargetScan database and identified 3,164 genes. Next, we performed comprehensive gene expression analysis using miR-145-3p transfectants of SAS, with negative control miRNA transfectants serving as controls (accession no. GSE 82108). A total of 1,187 genes were commonly downregulated (log2 ratio<0). The gene set was then analyzed with a publicly available gene expression data set in GEO (accession no. GSE9638), and genes upregulated in HNSCC were chosen (fold-change >1.5). A total of 14 genes were identified as candidate targets of miR-145-3p regulation (Table II). Next, these genes were validated with TCGA database, and we investigated the correlations between survival rates and target genes with high or low expression. In this study, 3 genes (MYO1B, C16orf74 and RBP1) were selected as genes that affected the patient's overall survival (Table II and Fig. 4). Among them, MYO1B was found to have the greatest effect on the overall survival rate (p=0.00452). In this study, we focused on MYO1B as a candidate target gene of miR-145-3p regulation and investigated the functional roles of HNSCC cells.
Direct regulation of MYO1B by miR-145-3p in HNSCC cells
Next, we investigated whether the expression of MYO1B decreased in miR-145-3p-transfected HNSCC cells. MYO1B mRNA levels were significantly reduced by miR-145-3p transfection compared with the mock or miR-control transfectants (Fig. 5A). Furthermore, MYO1B protein levels were also reduced by miR-145-3p transfection compared with mock or miR-control transfectants (Fig. 5B). In contrast, miR-145-5p transfectants did not show altered expression of MYO1B mRNA or protein (Fig. 5A and B).
We then carried out luciferase reporter assays with a vector that included the 3′-UTR of MYO1B to confirm that miR-145-3p directly regulated MYO1B in a sequence-dependent manner. TargetScan Human database predicted that there were two binding sites for miR-145-3p in the 3′-UTR of MYO1B (positions 88–94 and 1117–1123) (Fig. 5C). Cotransfection with miR-145-3p and vectors significantly reduced luciferase activity in comparison with those in mock and miR-control transfectants in position 1117–1123 of the MYO1B 3′-UTR (Fig. 5D).
Effects of MYO1B knockdown on cell proliferation, migration, and invasion in HNSCC cell lines
A loss-of-function assay using siRNA was performed to examine the function of MYO1B in HNSCC cell lines. The expression levels of MYO1B mRNA and protein were reduced by si-MYO1B in HNSCC cell lines (Fig. 6A and B). Furthermore, we investigated effects of MYO1B knockdown on cell proliferation, migration, and invasion in HNSCC cell lines. Cancer cell proliferation was significantly reduced in si-MYO1B transfectants in comparison with that in mock- or miR control-transfected cell lines (Fig. 6C). Additionally, migration activities were significantly suppressed in si-MYO1B transfectants in comparison with that in mock- or miR control-transfected cell lines (Fig. 6D). Invasion activity was also significantly inhibited in si-MYO1B transfectants in comparison with that in mock- or miR control-transfected cell lines (Fig. 6E).
Expression of MYO1B in HNSCC clinical specimens
Next, we investigated the mRNA expression levels of MYO1B in 22 HNSCC clinical specimens by qRT-PCR. MYO1B was significantly upregulated in HNSCC tumor tissues (Fig. 7A). Spearman's rank test showed a negative correlation between the expression of MYO1B and miR-145-3p (p=0.0025, R=−0.461) (Fig. 7B). Furthermore, we also examined the expression levels of MYO1B in HNSCC clinical specimens by immunostaining. MYO1B was strongly expressed in several cancer tissues (Fig. 7C: 1, patient no. 2; 2, no. 3; 3, no. 7 in Table I).
Correlation between MYO1B expression and clinicopathological characteristics in prognostic prediction in HNSCC specimens
We collected clinical data from TCGA database and analyzed clinicopathological factors and expression of MYO1B as a prognostic predictive factor. The multivariate cox proportional hazards model was used to validate independent predictors for overall survival, including MYO1B expression, clinical T stage, clinical N stage, age, sex and histologic grade. As a result, high expression of MYO1B was an independent predictive factor for survival [hazard ratio (HR), 1.68; 95% confidence interval (CI), 1.13–2.49; p=0.01] (Fig. 8).
Downstream genes affected by silencing of MYO1B in SAS cells
Finally, we performed genome-wide gene expression analysis using si-MYO1B in SAS cells to investigate which genes were mediated by MYO1B signaling. A SurePrint G3 Human GE 60K v3 microarray was used for genome-wide expression analysis. We submitted the raw data to the GEO database (accession no. GSE100746). In this study, we focused on significantly downregulated genes by both si-MYO1B-1 and si-MYO1B-2 transfection (log2 [si-MYO1B/mock] <−1.5). MYO1B was the most significantly downregulated gene, indicating that the array data were worthy of evaluation. Genes significantly downregulated by silencing of MYO1B are listed in Table III. Among MYO1B downstream genes, expression of 5 genes (ANXA10, TRIM9, TCTN3, BTBD16 and CYP19A1) was significantly associated with poor prognosis in patients with HNSCC based on TCGA database (Fig. 9).
Discussion
Accumulating evidence has shown that aberrant expression of miRNAs disrupts the well-ordered RNA networks in cancer cells and is involved in the pathogenesis of human cancers (22). Based on the miRNA expression signatures of human cancers, we have sequentially identified antitumor miRNAs that regulate novel cancer networks (16,23–26). Analyses of our miRNA signature of HNSCC by RNA sequencing showed that several passenger strands of miRNAs were significantly downregulated in cancer tissues (8). Our recent study demonstrated that both strands of pre-miR-150 (miR-150-5p, guide strand; and miR-150-3p, passenger strand) had antitumor functions and that these miRNAs cooperatively regulated oncogenic ITGA3, ITGA6 and TNC in HNSCC cells (8). Our other studies showed that the passenger strand of miR-150 acted as an anti-tumor miRNA in several types of cancers, such as esophageal cancer and prostate cancer (9,27). These findings suggested that miRNA passenger strands also contribute substantially to cancer pathogenesis and that identification of RNA networks mediated by miRNA passenger strands may provide novel insights into the pathogenesis of HNSCC.
Based on our miRNA signature of HNSCC, we focused on the passenger strand miR-145-3p in this study. Similarly, miR-145-5p, the guide strand of miR-145, was significantly reduced in this signature. Downregulation of miR-145-5p is frequently observed in many types of cancer, and prior studies have confirmed the antitumor function of miR-145-5p by demonstration of its effects on several types of oncogenes in cancer cells (10,11). Several studies have shown that downregulation of miR-145-5p is caused by hypermethylation of the promoter region of pre-miR-145 in prostate cancer (28). Importantly, the tumor suppressor p53 has been shown to directly bind p53-response elements in the promoter region of pre-miR-145 and to control the expression of miR-145-5p (29). p53 mutations are found in >50% of patients with HNSCC (30). Thus, downregulation of miR-145-5p and miR-145-3p may be dependent on p53 inactivation in cancer cells.
Expression levels of passenger strand of miR-145-3p was lower than miR-145-5p as a guide strand miRNA in HNSCC clinical specimens and cell lines. Our previous studies of bladder, lung, and prostate cancers showed that expression levels of miR-145-3p was lower than miR-145-5p in each cancer (10,11,31). The results of the present data of HNSCC was similar to our previous data. Explanation is incomplete as to in what kind of molecular mechanisms the expression of the two miRNAs differ. This problem is an important issue in miRNA biosynthesis.
Our functional assays showed that miR-145-3p had antitumor functions similar to miR-145-5p in HNSCC cells. We have also demonstrated miR-145-3p is downregulated in cancer tissues and acts as an antitumor miRNA in bladder, lung, and prostate cancers by targeting several oncogenic genes (10,11,31). Previous studies demonstrated that several oncogenic genes were regulated by miR-145-5p in several types of cancers (32–34). There are few studies for target genes by miR-145-3p regulation in cancer cells, including HNSCC cells. Thus, we evaluated miR-145-3p regulatory oncogenic networks in HNSCC cells; a total of 14 putative targets of miR-145-3p in HNSCC cells were identified in this study. Among these candidates, MYO1B, C16orf74, SP9 and RBP1 were found to be associated with poor prognosis in patients with HNSCC by TCGA data analyses.
In this study, we focused on MYO1B because high expression of MYO1B was strongly associated with poor prognosis in patients with HNSCC. Myosins are actin-associated molecular motor proteins that regulate membrane tension, anchor membrane proteins and organelles, and transport inter-cellular vesicles (35,36). We have demonstrated that antitumor miRNAs inhibited cancer cell migration and invasion through targeting several actin-binding proteins and actin-associate proteins, e.g., FSCN1, LASP1, ARPC5 and ANLN (37–40). Overexpression of these proteins has been detected in cancer tissues and has been shown to contribute to cancer cell aggressiveness.
Our present data of restoration of miR-145-5p or miR-145-3p showed the inhibition of cancer cell proliferation. However, inhibition of cell proliferation was weak by knockdown of MYO1B in HNSCC cells. These data suggest that miR-145-5p or miR-145-3p inhibite cell proliferation genes and pathways which do not rely on MYO1B in HNSCC cells.
MYO1B belongs to a member of the membrane-associated class I myosin family and functions as a linker between membranes and the actin cytoskeleton in several cellular processes (41). Previous studies have demonstrated other functions of MYO1B. For example, MYO1B is localized in the endocytotic compartment and has pivotal roles in endocytosis (42). MYO1B couples with the actin assembly to organelles and controls membrane remodeling at the trans-Golgi network (43). In cancer cells, MYO1B is highly expressed in PC-3 metastatic prostate cancer cells, and knockdown of MYO1B affects the cytoskeleton and cell migration (44). Another study showed that knockdown of MYO1B significantly inhibits migratory and invasive abilities of HNSCC cells in vitro and in vivo (35). Our present data confirmed these findings and suggested that MYO1B may be an effective target for the treatment of HNSCC.
To identify MYO1B-mediated HNSCC pathways, we performed genome-wide gene expression analyses using si-MYO1B transfectants. A total of 54 genes were found to be mediated by MYO1B in HNSCC. Among them, 5 genes (ANXA10, TRIM9, TCTN3, BTBD16 and CYP19A1) were significantly associated with poor prognosis in patients with HNSCC by TCGA database analyses. Annexin family proteins are calcium-dependent phospholipid-binding proteins that regulate cell growth and signal transduction (45). Overexpression of ANXA10 has been reported in oral squamous cell carcinoma, and expression of ANXA10 promotes cancer cell proliferation through regulating mitogen-activated protein kinase signaling pathways (46). Exploration of novel MYO1B-mediated pathways may improve our understanding of the aggressiveness of this disease.
In conclusion, downregulation of miR-145-3p was observed in HNSCC clinical specimens, and this passenger strand acted as an antitumor miRNA through targeting MYO1B in HNSCC cells. MYO1B was highly expressed in HNSCC clinical specimens and was found to promote cancer aggressiveness in functional assays. Elucidation of the pathways mediated by the miR-145-3p/MYO1B axis is expected to contribute to further analyses of oncogenesis mechanisms and treatment strategies in HNSCC.