Impact of miR-1/miR-133 Clustered miRNAs: PFN2 Facilitates Malignant Phenotypes in Head and Neck Squamous Cell Carcinoma

Based on our original RNA sequence-based microRNA (miRNA) signatures of head and neck squamous cell carcinoma (HNSCC), it was revealed that the expression levels of miR-1-3p, miR-206, miR-133a-3p, and miR-133b were significantly suppressed in cancer specimens. Seed sequences of miR-1-3p/miR-206 and miR-133a-3p/miR-133b are identical. Interestingly, miR-1-3p/miR-133a-3p and miR-206/miR-133b are clustered in the human genome. We hypothesized that the genes coordinately controlled by these miRNAs are closely involved in the malignant transformation of HNSCC. Our in silico analysis identified a total of 28 genes that had putative miR-1-3p/miR-133a-3p and miR-206/miR-133b binding sites. Moreover, their expression levels were upregulated in HNSCC tissues. Multivariate Cox regression analyses showed that expression of PFN2 and PSEN1 were independent prognostic factors for patients with HNSCC (p < 0.05). Notably, four miRNAs (i.e., miR-1-3p, miR-206, miR-133a-3p, and miR-133b) directly bound the 3′untranslated region of PFN2 and controlled expression of the gene in HNSCC cells. Overexpression of PFN2 was confirmed in clinical specimens, and its aberrant expression facilitated cancer cell migration and invasion abilities. Our miRNA-based strategy continues to uncover novel genes closely involved in the oncogenesis of HNSCC.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is a malignant neoplasm that arises mainly from the mucosa of the oral cavity, pharynx, and larynx [1]. HNSCC is the sixth most common cancer worldwide, with 890,000 new cases and 450,000 deaths in 2018 [2]. Epidemiological studies have shown several risk factors for HNSCC such as consumption of tobacco and alcohol, exposure to environmental pollutants, and infection with human papillomavirus or Epstein-Barr virus [3]. With an increase in the number of HPV-related HNSCC with favorable prognosis, the overall survival rate of HNSCC is improving [4]. However, the prognosis of HPV-negative HNSCC has not improved, even with multidisciplinary treatments combining surgery, irradiation, chemotherapy, molecular targeted agents, and immunotherapy [3,5]. More than 60% of HNSCC cases are at an advanced stage at the time of the first diagnosis [1]. Treatment that combines chemotherapy with radiation therapy or surgical resection is the first option for locally advanced HNSCC patients [6]. Due to the anatomical characteristics of HNSCC, these treatments have a significant impact on the quality of life. Despite the use of invasive procedures, there Biomedicines 2022, 10, 663 2 of 19 are many cases resistant to treatment and recurrence or metastasis after treatment is not uncommon [7]. To further improve treatment outcomes, it is necessary to elucidate the molecular biological mechanisms that underlie recurrence and metastasis of HNSCC.
MicroRNA (miRNA) is a type of single-strand, noncoding RNA, and its length is only 18-25 nucleotides [8]. miRNA acts as a negative controller of gene expression in a sequence-dependent manner [8,9]. In human cells, a single species of miRNA can control a vast number of genes, and the expression of a single mRNA is subject to numerous miRNAs [8,10]. Bioinformatic analysis suggested that more than 60% of protein-coding genes are controlled by miRNAs [8,11]. Therefore, aberrant expression of miRNAs likely disrupts intracellular RNA networks. In fact, numerous studies have demonstrated that aberrantly expressed miRNAs are involved in human diseases including various types of cancer [12,13].
Recent advances in nanotechnology have led to the development of drug delivery systems that deliver various drugs to target cancer cells [14]. Many attempts to use miRNAs as pharmaceuticals have been reported so far [15,16]. The advantage of miRNAs as drugs is that one type of miRNA has the potential to control many target genes [15]. Recently, exosomes have been attracting attention as a drug delivery system. Exosomes are a type of cell-derived vesicle characterized as extracellular vesicles. Of particular note, some miRNAs are contained within exosomes and migrate between cells through exosomes [14,17]. The development of new therapies that embed tumor-suppressive miRNAs in exosomes and deliver them to cancer cells is very attractive.
Interestingly, some miRNAs are in close proximity within the human genome. These miRNAs are called clustered miRNAs [18,19]. The clustered miRNAs share a number of properties: (a) they are composed of physically adjacent miRNA genes that are transcribed together in the same orientation, (b) no separate transcriptional units exist between the members of the cluster, and (c) there are no miRNAs in opposite directions [19]. Members of miRNA clusters have been shown to exhibit similar expression levels and often regulate genes and biological functions belonging to the same signaling pathway [20]. Clustered miRNAs work more efficiently than single miRNA genes, because they include numerous miRNA-coding genes [19]. Detailed analysis of clustered miRNAs will be an important topic in future miRNA research.
Our analysis revealed that Profilin 2 (PFN2) was directly controlled by miR-1-3p, miR-206, miR-133a-3p, and miR-133b, and its expression was involved in HNSCC pathogenesis. Functional studies demonstrated that aberrant expression of PFN2 facilitated the migratory and invasive abilities in HNSCC cells. Searching for antitumor miRNAs and the target molecules that these miRNAs coordinately control will improve our understanding of HNSCC.

Analysis of miRNA Expression in HNSCC
Expression levels of each miRNA in HNSCC clinical specimens was examined based on the miRNA expression signature (GSE184991) and TCGA-HNSC data (TCGA, Firehose Legacy).
Six cDNA libraries obtained from three paired of cancer and normal tissues were sequenced by Next Seq500 (Illumina, San Diego, CA, USA) for miRNA expression signature. The clinical information of three HNSCC patients used for miRNA sequencing are shown in Table S1. All specimens used for our miRNA signature were derived from surgical resection at Chiba University Hospital. Cancer tissues were collected from each primary tumor, and normal tissues were collected from normal mucosa at least 1 cm away from the margins of the primary tumor.

HNSCC Cell Lines and Cell Culture
SAS and Sa3 were purchased originally from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). These cell lines were cultured in DMEM medium with 10% fetal bovine serum and antibiotics (i.e., penicillin/streptomycin) The cells were grown in a humidified atmosphere of 5% CO 2 and 95% air at 37 • C. The features of the cell lines are shown in Table S2.

Transfection of Mature miRNAs and siRNAs
The protocol used for transient transfection of miRNAs and siRNAs were described in our previous studies [22,28,29]. All miRNA precursors were transfected at 10 nM, and siRNAs were transfected at 5nM into HNSCC cell lines using RNAiMAX (Invitrogen, Carlsbad, CA, USA). Mock was a group without precursors or siRNAs. Control groups were transfected with the negative control precursor. The reagents used in the analysis are shown in Table S3.

Functional Assays (Cell Proliferation, Migration, and Invasion) Conducted in HNSCC Cells
The procedures for functional assays (cell proliferation, migration, and invasion assays) in HNSCC cells have been described previously [22,28,29]. Briefly, in proliferation assay, SAS or Sa3 cells were plated at 3.0 × 10 3 cells per well in 96-well plates. Cell proliferation was examined by XTT assays (Sigma-Aldrich, St. Louis, MO, USA) 72 h after miRNA/siRNA transfection. For migration and invasion assays, SAS or Sa3 cells at 2.5 × 10 5 cells per well were transfected in 6-well plates. After 48 h transfection, SAS or Sa3 at 1.0 × 10 5 cell per well were added into the Corning BioCoat TM cell culture chamber (Corning, Corning, NY, USA) for migration assays or into the Corning BioCoat Matrigel Invasion Chamber for invasion assays. After 48 h, the cells at the bottom of the chamber were counted and analyzed.
Five-year overall survival rates between the groups were analyzed by log-rank test. In addition, the multivariate statistical technique was performed using Cox's proportional hazards model. The ç cases were divided into two groups according to the median value of each gene in OncoLnc (http://www.oncolnc.org; accessed on 20 April 2021) [32].

RNA Extraction and Quantitative Reverse-Transcription PCR (qRT-PCR)
Total RNA was isolated using TRIzol reagent and the PureLink™ RNA Mini Kit (Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA)). Reverse transcription was achieved with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). We performed qRT-PCR using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). GAPDH was used as the normalized control. Taqman assays (Applied Biosystems) used in this report are shown in Table S3.

Western Blotting
The procedures for Western blotting have been described previously [22,29,33]. We incubated the membranes with the anti-PFN2 antibody (1:500) overnight at 4 • C and with the secondary antibody for 1 h at room temperature. GAPDH was used as an internal control. The reagents used in the analysis are shown in Table S3. Full blots are shown in Figures S1 and S2.

Immunostaining
Paraffin sections were obtained from HNSCC cases who received surgical treatment at Chiba University Hospital. The clinical features are shown in Table S4. Specimens were incubated with anti-PFN2 antibody (1:1000) overnight at 4 • C. We incubated samples with secondary antibody for 30 min at room temperature and counterstained them with hematoxylin. The reagents used in the analysis are shown in Table S3.

Dual Luciferase Reporter Assays
PFN2 DNA sequences including or lacking predicted miRNA-binding sequence were inserted into the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Transfection of the purified plasmid vectors into HNSCC cells were performed using Lipofectamine 2000 (Invitrogen) at 50 ng/well. After 48 h of transfection, we conducted dual luciferase reporter assays using the Dual Luciferase Reporter Assay System (Promega). Luminescence data are presented as the Renilla/Firefly luciferase activity ratio.

Gene Set Enrichment Analysis (GSEA)
To investigate the molecular pathways in HNSCC, GSEA was performed. TCGA-HNSC data were divided into high-and low-expression groups according to the Z-score of the PFN2 expression level. We generated a ranked list of genes by the log 2 ratio comparing the expression levels of each gene between the two groups. We uploaded the resultant gene lists into GSEA software [34,35] and applied the Hallmark gene set in The Molecular Signatures Database [34,36].

Statistical Analysis
JMP Pro 15 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses. Comparisons between the two groups were assessed by Welch's t-test. Differences between multiple groups were assessed by Dunnett's test compared to control group. A p-value < 0.05 was considered statistically significant. Significant differences within the figures are expressed as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.: not significant. Quantitative data are presented as the means and standard errors.
Our strategy for searching for common putative target genes is shown in Figure 3. Based on the TargetScan Human database (release 7.2), we identified a total of 896 genes that had putative miR-133a-3p/miR-133b-binding sites in the 3 -UTR, and a total of 711 genes that had putative miR-1-3p/miR-206-binding sites in the 3 -UTR. Ninety-five of these genes were common putative targets of clustered miRNAs.
Our strategy for searching for common putative target genes is shown in Figure 3. Based on the TargetScan Human database (release 7.2), we identified a total of 896 genes that had putative miR-133a-3p/miR-133b-binding sites in the 3'-UTR, and a total of 711 genes that had putative miR-1-3p/miR-206-binding sites in the 3'-UTR. Ninety-five of these genes were common putative targets of clustered miRNAs. Next, we confirmed the expression levels of these genes in HNSCC using TCGA-HNSC data. Among these genes, 28 were upregulated in cancer tissues (n = 518) compared to normal tissues (n = 44). We further analyzed these 28 genes as candidates for common oncogenic targets (Table 1). Next, we confirmed the expression levels of these genes in HNSCC using TCGA-HNSC data. Among these genes, 28 were upregulated in cancer tissues (n = 518) compared to normal tissues (n = 44). We further analyzed these 28 genes as candidates for common oncogenic targets (Table 1).

Clinical Significance of miR-1/miR-133 Clustered miRNAs Targets by TCGA Analysis
We investigated the clinical significance of the 28 common putative targets controlled by miR-133a-3p/miR-133b and miR-1-3p/miR-206 in HNSCC. The expression and 5-year overall survival analysis showed that three genes (PFN2, PSEN1, and SYT1) were significantly upregulated in cancer tissues ( Figure 4A), and increased expression levels of each gene were associated with a poorer prognosis in HNSCC patients (log rank test; p < 0.05 and false discovery rate < 0.05; Figure 4B and Table 1).
In addition, Cox proportional hazards regression analysis was performed for 5-year overall survival rates, using each gene expression level (i.e., PFN2, PSEN1, and SYT1), tumor stage, pathological grade, and age as covariates. The multivariate analysis showed that the expression levels of PFN2 and PSEN1 were independent prognostic factors (PFN2: HR 1.490, p < 0.05; PSEN1: HR 1.444, p < 0,05; Figure 4C). These results suggested that PFN2 and PSEN1 were oncogenes related to molecular pathogenesis and clinical prognosis in HNSCC patients.

Direct Control of PFN2 Expression by All Members of the miR-1/miR-133 Clustered miRNAs in HNSCC Cells
First, qRT-PCR was performed to evaluate whether expression of PFN2 and PSEN1 was controlled by clustered miRNAs (miR-133a-3p/miR-133b and miR-1-3p/miR-206) in HNSCC cells ( Figure 5A,B). We found that the expression levels of PFN2 were significantly suppressed by miR-133a-3p/miR-133b and miR-1-3p/miR-206 in HNSCC cells ( Figure 5A). On the other hand, transfection of miR-133a-3p/miR-133b reduced the expression level of PSEN1, whereas transfection of miR-1-3p/miR-206 did not significantly suppress the expression ( Figure 5B). According to these results, we focused on PFN2 as a common oncogenic target of miR-133a-3p/miR-133b and miR-1-3p/miR-206 in HNSCC. Western blotting revealed that the protein levels of PFN2 were reduced by clustered miRNA transfection ( Figure 6A). To prove that direct binding between PFN2 and clustered miRNAs was sequence dependent, a dual-luciferase reporter assay was conducted. The luciferase activity was significantly reduced following co-transfection with miR-133a-3p/miR-133b and a vector containing the miR-133a-3p/miR-133b-binding site in the 3'-UTR of PFN2 ( Figure 6B). On the other hand, co-transfection with a vector that lacked the sequence of the miR-133a-3p/miR-133b site showed no change in luciferase activity ( Figure  6B). Co-transfection with miR-1-3p/miR-206 and a vector containing the miR-1-3p/miR-206binding site reduced the luciferase activity, but co-transfection with miR-1-3p/miR-206 and a vector lacking the miR-1-3p/miR-206-binding site did not inhibit luciferase activity (Figure 6C). Western blotting revealed that the protein levels of PFN2 were reduced by clustered miRNA transfection ( Figure 6A). To prove that direct binding between PFN2 and clustered miRNAs was sequence dependent, a dual-luciferase reporter assay was conducted. The luciferase activity was significantly reduced following co-transfection with miR-133a-3p/miR-133b and a vector containing the miR-133a-3p/miR-133b-binding site in the 3 -UTR of PFN2 ( Figure 6B). On the other hand, co-transfection with a vector that lacked the sequence of the miR-133a-3p/miR-133b site showed no change in luciferase activity ( Figure 6B). Co-transfection with miR-1-3p/miR-206 and a vector containing the miR-1-3p/miR-206binding site reduced the luciferase activity, but co-transfection with miR-1-3p/miR-206 and a vector lacking the miR-1-3p/miR-206-binding site did not inhibit luciferase activity ( Figure 6C).

Overexpression of PFN2 in HNSCC Clinical Specimens
To confirm expression of the PFN2 protein in HNSCC clinical specimens, immunohistochemical staining was performed. Clinical features of four HNSCC cases used for immunostaining are summarized in Table S1. Whereas there was almost no PFN2 expression in the normal epithelium, high expression of PFN2 was detected in cancer lesions in HNSCC (Figure 7).

Overexpression of PFN2 in HNSCC Clinical Specimens
To confirm expression of the PFN2 protein in HNSCC clinical specimens, immunohistochemical staining was performed. Clinical features of four HNSCC cases used for immunostaining are summarized in Table S1. Whereas there was almost no PFN2 expression in the normal epithelium, high expression of PFN2 was detected in cancer lesions in HNSCC (Figure 7).

Effects of PFN2 Knockdown on the Proliferation, Migration, and Invasion of HNSCC Cells
To assess the role of PFN2 as an oncogene in HNSCC cells, functional knockdown assays using siRNA were performed. First, we confirmed the inhibitory effects of siRNA by performing qRT-PCR and Western blotting. Two different siRNAs targeting PFN2 (i.e., siPFN2-1 and siPFN2-2) were used for this study. The mRNA and protein levels of PFN2 were significantly inhibited after transfection of siRNAs into HNSCC cell lines ( Figure  8A,B).
Then, functional assays using these siRNAs were performed. Knockdown of PFN2 had little effect on cell proliferation in HNSCC cells ( Figure 9A). However, cell migration and invasion were significantly suppressed after transfection of siPFN2 in SAS and Sa3 cells ( Figure 9B,C). Typical images of cells in migration and invasion assays following siPFN2 transfection are shown in Figures S5 and S6.

Effects of PFN2 Knockdown on the Proliferation, Migration, and Invasion of HNSCC Cells
To assess the role of PFN2 as an oncogene in HNSCC cells, functional knockdown assays using siRNA were performed. First, we confirmed the inhibitory effects of siRNA by performing qRT-PCR and Western blotting. Two different siRNAs targeting PFN2 (i.e., siPFN2-1 and siPFN2-2) were used for this study. The mRNA and protein levels of PFN2 were significantly inhibited after transfection of siRNAs into HNSCC cell lines ( Figure 8A,B).
Then, functional assays using these siRNAs were performed. Knockdown of PFN2 had little effect on cell proliferation in HNSCC cells ( Figure 9A). However, cell migration and invasion were significantly suppressed after transfection of siPFN2 in SAS and Sa3 cells ( Figure 9B,C). Typical images of cells in migration and invasion assays following siPFN2 transfection are shown in Figures S5 and S6.

PFN2-Mediated Molecular Pathways in HNSCC Cells
To identify the molecular pathways involving PFN2 in HNSCC, we performed gene set enrichment analysis (GSEA) using TCGA-HNSC RNA-seq data. GSEA analysis revealed that "epithelial-mesenchymal transition" was the most enriched pathway in the PFN2 high expression group ( Figure 10 and Table 2). These results suggest that aberrant expression of PFN2 contributes to the malignant phenotype, including migration and/or invasion of HNSCC, through the epithelial-mesenchymal transition pathway.

PFN2-Mediated Molecular Pathways in HNSCC Cells
To identify the molecular pathways involving PFN2 in HNSCC, we performed gene set enrichment analysis (GSEA) using TCGA-HNSC RNA-seq data. GSEA analysis revealed that "epithelial-mesenchymal transition" was the most enriched pathway in the PFN2 high expression group ( Figure 10 and Table 2). These results suggest that aberrant expression of PFN2 contributes to the malignant phenotype, including migration and/or invasion of HNSCC, through the epithelial-mesenchymal transition pathway.

Discussion
Even when various strategies are used to treat HNSCC patients, their prognosis is still poor due to the high rate of recurrence and metastasis [3,7]. Unfortunately, combination therapy with EGFR inhibitor or PD-L1 inhibitor has not achieved satisfactory therapeutic results [37,38]. With the advent of immune checkpoint inhibitors, treatment options for recurrent or metastatic cases have increased, but their efficacy is limited [39,40]. Despite vigorous RNA sequence analysis using samples from patients with HNSCC, the search for therapeutic target molecules for HNSCC has not fully succeeded [41], with a few exceptions [42,43].
We are continuing tumor-suppressive miRNA-based analysis to explore prognostic markers and therapeutic targets in HNSCC [21,23,29,44]. Our previous studies showed that miR-143 and miR-145 functioned as tumor-suppressive miRNAs in a wide range of cancers [45][46][47][48]. Notably, they are clustered miRNAs on the human chromosome at region 5q31 [45,46]. Our studies demonstrated that Golgi membrane protein 1 (GOLM1) and hexokinase-2 (HK2) were directly controlled by miR-143-3p and miR-145-5p in prostate cancer and renal cell carcinoma, respectively [45,46]. Further study showed that the MET proto-oncogene was coordinately regulated by miR-23b and miR-27b clustered miRNAs in HNSCC cells [49]. Clustered miRNAs regulate one gene through different seed sequences. Continuous and genome-wide analyses of clustered miRNAs are essential to explain the biological implications of clustered miRNAs on the human genome.
Analysis of our original miRNA expression signatures in several cancers, including HNSCC showed that members of the miR-1/133 clustered miRNAs were downregulated in cancer tissues [21,23,50]. Downregulation of each miRNA in the miR-1/miR-133 cluster was confirmed by TCGA-HNSC database analysis. Originally, miR-1/miR-133 clustered miRNAs were discovered through their roles involved in the development of skeletal and cardiac muscles, called "myomiR" [51]. In cancer research, downregulation of miR-1/miR-133 clustered miRNAs were reported in a wide range of cancers, and their functional analyses showed that these miRNAs acted as tumor-suppressive miRNAs [21]. Therefore, the search for oncogenes and oncogenic pathways controlled by each miRNA is being vigorously carried out [52][53][54].
In this study, a total of 28 genes were identified as putative targets of miR-1/miR-133 clustered miRNAs. Among these targets, we identified PFN2 as a gene directly controlled by all members of miR-1/miR-133 clustered miRNAs in HNSCC cells. Our present analysis showed that aberrantly expressed PFN2 facilitated cancer cell migration and invasion and was closely involved in the malignant phenotypes of HNSCC.
Profilin is an actin-binding protein that forms an ATP-actin-PFN complex. It recruits monomeric actin to the barbed end of actin filaments and it contributes to elongation [55]. In mammals, profilin is constituted by four members (e.g., PFN1, PFN2, PFN3, and PFN4); PFN1 and PFN2 are the most common types of profilins [56].
Several studies have reported that PFN2 expression is involved in malignant transformation of cancer cells [57][58][59]. In triple-negative breast cancer (TNBC), high expression of PFN2 was related to a poorer prognosis (10-year overall survival and relapse-free survival) [58]. In esophageal squamous cell carcinoma (ESCC), PFN2 protein expression was markedly increased gradually from low-grade intraepithelial neoplasia to ESCC, and high expression was positively correlated with the depth of invasion and lymph node metastasis [57]. A recent study reported that PFN2 is involved in small cell lung cancer metastasis and angiogenesis through exosomes [60].
Transforming growth factor-β (TGF-β) signaling induces cancer cell development and progression [61]. Expression of PFN2 induced the transactivation of Smad2 and Smad3, and these transmission factors enhanced TGF-β-induced EMT and angiogenesis in lung cancer [59]. PFN2 overexpression reduced epithelial markers and increased mesenchymal markers in several cancers [57,58]. These results suggest that PFN2 promotes tumor aggressiveness via EMT. Our present study showed that PFN2 contributes to the malignant phenotype in HNSCC through the EMT pathway, and these results are consistent with previous reports.

Conclusions
Analysis of miRNA expression signatures of HNSCC showed that all members of the miR-1/miR-133 miRNA cluster (e.g., miR-1-3p, miR-206, miR-133a-3p, and miR-133b) were frequently downregulated in cancer tissues. TCGA database analysis confirmed that these miRNAs were significantly reduced in cancer tissues. Ectopic expression assays demonstrated that these miRNAs acted as antitumor miRNAs in HNSCC cells. A combination of in silico analyses and luciferase reporter assays revealed that PFN2 was directly controlled by all members of the miR-1/miR-133 cluster in HNSCC cells. Expression of PFN2 was closely involved in the prognosis of patients with HNSCC. Moreover, aberrant expression of PFN2 facilitated cancer cell migration and invasion. Those abilities might be controlled by EMT pathways. Our tumor-suppressive miRNA-based strategy thus provides novel insights, contributing to our overall understanding of the molecular pathogenesis of HNSCC.

Institutional Review Board Statement:
The study was conducted according to the Declaration of Helsinki and approved by the Bioethics Committee of Chiba University (approval number: 915, 11 July 2021).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Our expression data were deposited in the GEO database (accession number: GSE189290).