Generation of bone-specific lysyl hydroxylase 2 knockout mice and their phenotypes.

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of type I collagen. This modification is critical for the formation of stable hydroxylysine-aldehyde derived collagen cross-links, thus, for the stability of collagen fibrils. Though dysfunction of LH2 causes Bruck syndrome, recessive osteogenesis imperfecta with joint contracture, the molecular mechanisms by which LH2 affects bone formation are still not well understood. Since the Plod2 knockout mice are embryonically lethal, we generated bone-specific LH2 conditional knockout mice (bsLH2-cKO) using the osteocalcin-Cre/loxP system, and evaluated phenotypes of femurs. LH2 mRNA and protein levels assessed by qPCR, immunohistochemistry and Data Independent Acquisition proteomics were all markedly low in bsLH2-cKO femurs when compared to controls. Lysine hydroxylation of both carboxy- and amino-terminal telopeptides of an α1(I) chain were significantly diminished resulting in reduction of the hydroxylysine-aldehyde derived cross-links. The collagen fibrils in bsLH2-cKO appeared to be thicker, often fused and irregular when compared to controls. In addition, bone mineral density and mechanical properties of bsLH2-cKO femurs were significantly impaired. Taken together, these data demonstrate that LH2-catalyzed modification and consequent cross-linking of collagen are critical for proper bone formation and mechanical strength.

Synthetic Circular RNA for microRNA-1269a Suppresses Tumor Progression in Oral Squamous Cell Carcinoma.

microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.

Radiotherapy Plus Cetuximab for Squamous Cell Carcinoma of the Oral Cavity: A Multicenter Retrospective Study of 79 Patients in Japan.

There are a few reports that focus on radiotherapy (RT) and cetuximab (CET) therapy exclusively for oral cancer. This retrospective study aimed to investigate the efficacy and safety of RT and CET therapy for locally advanced (LA) or recurrent/metastatic (R/M) oral squamous cell carcinoma (OSCC). Seventy-nine patients from 13 hospitals who underwent RT and CET therapy for LA or R/M OSCC between January 2013 and May 2015 were enrolled in the study. Response, overall survival (OS), disease-specific survival (DSS), and adverse events were investigated. The completion rate was 62/79 (78.5%). The response rates in patients with LA and R/M OSCC were 69% and 37.8%, respectively. When only completed cases were examined, the response rates were 72.2% and 62.9%, respectively. The 1- and 2-year OS were 51.5% and 27.8%, respectively (median, 14 months), for patients with LA OSCC, and 41.5% and 11.9% (median, 10 months) for patients with R/M OSCC. The 1- and 2-year DSS were 61.8% and 33.4%, respectively (median, 17 months), for patients with LA OSCC, and 76.6% and 20.4% (median, 12 months) for patients with R/M OSCC. The most common adverse event was oral mucositis (60.8%), followed by dermatitis, acneiform rash, and paronychia. The completion rate was 85.7% in LA patients and 70.3% in R/M patients. The most common reason for noncompletion was an inadequate radiation dose due to worsening general conditions in R/M patients. Although the standard treatment for LA or R/M oral cancer is concomitant RT with high-dose cisplatin (CCRT) and the efficacy of RT and CET therapy for oral cancer is not considered to be as high as that for other head and neck cancers, it was thought that RT and CET therapy could be possible treatments for patients who cannot use high-dose cisplatin.

A novel cell-based transplantation method using a Rho kinase inhibitor and a specific catheter device for the treatment of salivary gland damage after head and neck radiotherapy.

Radiotherapy (RT) for head and neck cancer results in irreversible damage to salivary glands (SGs) and decreases saliva production, leading to a dry mouth. To date, there are no satisfactory therapies to solve this problem. We recently established a novel culturing method using a Rho kinase inhibitor (RI), Y-27632, that maintained cellular morphology and function for a prolonged period of time. In the present study, we investigated whether cell-based transplantation using our culturing method ameliorated the dysfunction of irradiated SGs. First, rat SG cells were cultured in a medium with RI. Cells were characterized by morphological findings and mRNA expression analysis. We also assessed features of SG cells in three-dimensional (3-D) culture by scanning electron microscopy and immunohistochemistry (IHC). The RI-containing medium led to higher cell proliferation of rat SG cells with preservation of cell morphology and higher alpha-amylase (AMY) expression in both 2-D and 3-D culture systems. To establish the atrophic-SG models, external RT at a dose of 15 Gy was delivered to the head and neck fields of nude rats. The SG cells derived from GFP-rats were cultured in medium with RI, after which they were transplanted into the submandibular glands of atrophic-SG rats using a catheter placed into Wharton's duct. IHC and salivary flow rate (SFR) analyses were measured 12 weeks after the transplantation. Following transplantation, donor cells (GFP-SG cells) were primarily located in the ductal region of the SG, and AMY expression in SGs and the SFR were increased in the SG cell transplantation group compared with the control. Those data indicated that cell-based therapy using RI-treated SG cells could restore salivary hypofunction of irradiated SGs by direct integration of the donor cells in the duct of SGs. We propose that these data support future clinical plans in which SG cells would be excised from the labial minor SGs of the patients with head and neck cancers prior to RT, cultured during RT, and auto-transplanted into SGs using a catheter into the Wharton's duct. We believe that our culturing and transplantation methods can be applied to SG cells, constituting a therapeutic approach for the treatment of patients with dry mouth after not only RT but also aging and Sjögren's syndrome.

Genome-Wide Super-Enhancer-Based Analysis: Identification of Prognostic Genes in Oral Squamous Cell Carcinoma.

Advanced-stage oral squamous cell carcinoma (OSCC) patients are treated with combination therapies, such as surgery, radiation, chemotherapy, and immunotherapy. However, OSCC cells acquire resistance to these treatments, resulting in local recurrence and distant metastasis. The identification of genes involved in drug resistance is essential for improving the treatment of this disease. In this study, we applied chromatin immunoprecipitation sequencing (ChIP-Seq) to profile active enhancers. For that purpose, we used OSCC cell lines that had been exposed to cetuximab for a prolonged period. In total, 64 chromosomal loci were identified as active super-enhancers (SE) according to active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) ChIP-Seq. In addition, a total of 131 genes were located in SE regions, and 34 genes were upregulated in OSCC tissues by TCGA-OSCC analysis. Moreover, high expression of four genes (C9orf89; p = 0.035, CENPA; p = 0.020, PISD; p = 0.0051, and TRAF2; p = 0.0075) closely predicted a poorer prognosis for OSCC patients according to log-rank tests. Increased expression of the four genes (mRNA Z-score ≥ 0) frequently co-occurred in TCGA-OSCC analyses. The high and low expression groups of the four genes showed significant differences in prognosis, suggesting that there are clear differences in the pathways based on the underlying gene expression profiles. These data indicate that potential stratified therapeutic strategies could be used to overcome resistance to drugs (including cetuximab) and further improve responses in drug-sensitive patients.

Identification of Antitumor miR-30e-5p Controlled Genes; Diagnostic and Prognostic Biomarkers for Head and Neck Squamous Cell Carcinoma.

Analysis of microRNA (miRNA) expression signatures in head and neck squamous cell carcinoma (HNSCC) has revealed that the miR-30 family is frequently downregulated in cancer tissues. The Cancer Genome Atlas (TCGA) database confirms that all members of the miR-30 family (except miR-30c-5p) are downregulated in HNSCC tissues. Moreover, low expression of miR-30e-5p and miR-30c-1-3p significantly predicts shorter survival of HNSCC patients (p = 0.0081 and p = 0.0224, respectively). In this study, we focused on miR-30e-5p to investigate its tumor-suppressive roles and its control of oncogenic genes in HNSCC cells. Transient expression of miR-30e-5p significantly attenuated cancer cell migration and invasive abilities in HNSCC cells. Nine genes (DDIT4, FOXD1, FXR1, FZD2, HMGB3, MINPP1, PAWR, PFN2, and RTN4R) were identified as putative targets of miR-30e-5p control. Their expression levels significantly predicted shorter survival of HNSCC patients (p < 0.05). Among those targets, FOXD1 expression appeared to be an independent factor predicting patient survival according to multivariate Cox regression analysis (p = 0.049). Knockdown assays using siRNAs corresponding to FOXD1 showed that malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities) of HNSCC cells were significantly suppressed. Overexpression of FOXD1 was confirmed by immunostaining of HNSCC clinical specimens. Our miRNA-based approach is an effective strategy for the identification of prognostic markers and therapeutic target molecules in HNSCC. Moreover, these findings led to insights into the molecular pathogenesis of HNSCC.

Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma.

Recently, our studies revealed that some passenger strands of microRNAs (miRNAs) were closely involved in cancer pathogenesis. Analysis of miRNA expression signatures showed that the expression of miR-30e-3p (the passenger strand of pre-miR-30e) was significantly downregulated in cancer tissues. In this study, we focused on miR-30e-3p (the passenger strand of pre-miR-30e). We addressed target genes controlled by miR-30e-3p that were closely associated with the molecular pathogenesis of head and neck squamous cell carcinoma (HNSCC). Ectopic expression assays demonstrated that the expression of miR-30e-3p attenuated cancer cell malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities). Our analysis of miR-30e-3p targets revealed that 11 genes (ADA, CPNE8, C14orf126, ERGIC2, HMGA2, PLS3, PSMD10, RALB, SERPINE1, SFXN1, and TMEM87B) were expressed at high levels in HNSCC patients. Moreover, they significantly predicted the short survival of HNSCC patients based on 5-year overall survival rates (p < 0.05) in The Cancer Genome Atlas (TCGA). Among these targets, SERPINE1 was found to be an independent prognostic factor for patient survival (multivariate Cox regression; hazard ratio = 1.6078, p < 0.05). Aberrant expression of SERPINE1 was observed in HNSCC clinical samples by immunohistochemical analysis. Functional assays by targeting SERPINE1 expression revealed that the malignant phenotypes (e.g., proliferation, migration, and invasion abilities) of HNSCC cells were suppressed by the silencing of SERPINE1 expression. Our miRNA-based approach will accelerate our understanding of the molecular pathogenesis of HNSCC.

Assay for transposase-accessible chromatin with high-throughput sequencing reveals radioresistance-related genes in oral squamous cell carcinoma cells.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.

Aberrant GIMAP2 expression affects oral squamous cell carcinoma progression by promoting cell cycle and inhibiting apoptosis.

GTPases of immunity-associated protein 2 (GIMAP2) is a GTPase family member associated with T cell survival. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unknown. Therefore, the present study aimed to elucidate the possible role of GIMAP2 in OSCC development by investigating its expression levels and molecular mechanisms in OSCC. Reverse transcription quantitative PCR, immunoblotting and immunohistochemistry indicated that GIMAP2 expression was significantly upregulated (P<0.05) in OSCC-derived cell lines and primary OSCC specimens compared with that in their normal counterparts. GIMAP2-knockdown OSCC cells exhibited decreased cell growth, which was associated with cyclin-dependent kinase (CDK)4, CDK6 and phosphorylated Rb downregulation and p53 and p21 upregulation. In addition to cell cycle arrest, GIMAP2 affected anti-apoptotic functions in GIMAP2-knockdown cells by upregulating Bcl-2 and downregulating Bax and Bak. These findings indicated that GIMAP2 may significantly influence OSCC development and apoptosis inhibition and thus is a potential biomarker of OSCC.

Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration.

Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.

Decrease of lysyl hydroxylase 2 activity causes abnormal collagen molecular phenotypes, defective mineralization and compromised mechanical properties of bone.

Lysyl hydroxylase 2 (LH2) is an enzyme that catalyzes the hydroxylation of lysine (Lys) residues in fibrillar collagen telopeptides, a critical post-translational modification for the stability of intermolecular cross-links. Though abnormal LH2 activities have been implicated in various diseases including Bruck syndrome, the molecular basis of the pathologies is still not well understood. Since LH2 null mice die at early embryonic stage, we generated LH2 heterozygous (LH2+/-) mice in which LH2 level is significantly diminished, and characterized collagen and bone phenotypes using femurs. Compared to the wild-type (WT), LH2+/- collagen showed a significant decrease in the ratio of hydroxylysine (Hyl)- to the Lys-aldehyde-derived collagen cross-links without affecting the total number of aldehydes involved in cross-links. Mass spectrometric analysis revealed that, in LH2+/- type I collagen, the extent of hydroxylation of all telopeptidyl Lys residues was significantly decreased. In the helical domain, Lys hydroxylation at the cross-linking sites was either unaffected or slightly lower, but other sites were significantly diminished compared to WT. In LH2+/- femurs, mineral densities of cortical and cancellous bones were significantly decreased and the mechanical properties of cortical bones evaluated by nanoindentation analysis were compromised. When cultured, LH2+/- osteoblasts poorly produced mineralized nodules compared to WT osteoblasts. These data provide insight into the functionality of LH2 in collagen molecular phenotype and its critical role in bone matrix mineralization and mechanical properties.

Ginkgolide B Regulates CDDP Chemoresistance in Oral Cancer via the Platelet-Activating Factor Receptor Pathway.

The platelet-activating factor receptor (PAFR) is a key molecule that participates in intracellular signaling pathways, including regulating the activation of kinases. It is involved in cancer progression, but the detailed mechanism of its chemosensitivity is unknown. The purpose of the current study was to elucidate the mechanism regulating cisplatin (CDDP) sensitivity through PAFR functions in oral squamous cell carcinoma (OSCC). We first analyzed the correlation between PAFR expression and CDDP sensitivity in seven OSCC-derived cell lines based upon cell viability assays. Among them, we isolated 2 CDDP-resistant cell lines (Ca9-22 and Ho-1-N-1). In addition to conducting PAFR-knockdown (siPAFR) experiments, we found that ginkgolide B (GB), a specific inhibitor of PAFR, enhanced both CDDP chemosusceptibility and apoptosis. We next evaluated the downstream signaling pathway of PAFR in siPAFR-treated cells and GB-treated cells after CDDP treatment. In both cases, we observed decreased phosphorylation of ERK and Akt and increased expression of cleaved caspase-3. These results suggest that PAFR is a therapeutic target for modulating CDDP sensitivity in OSCC cells. Thus, GB may be a novel drug that could enhance combination chemotherapy with CDDP for OSCC patients.

Identification of miR-199-5p and miR-199-3p Target Genes: Paxillin Facilities Cancer Cell Aggressiveness in Head and Neck Squamous Cell Carcinoma.

Our previous study revealed that the miR-199 family (miR-199a-5p/-3p and miR-199b-5p/-3p) acts as tumor-suppressive miRNAs in head and neck squamous cell carcinoma (HNSCC). Furthermore, recent studies have indicated that the passenger strands of miRNAs are involved in cancer pathogenesis. The aim of this study was to identify cancer-promoting genes commonly regulated by miR-199-5p and miR-199-3p in HNSCC cells. Our in silico analysis and luciferase reporter assay identified paxillin (PXN) as a direct target of both miR-199-5p and miR-199-3p in HNSCC cells. Analysis of the cancer genome atlas (TCGA) database showed that expression of PXN significantly predicted a worse prognosis (5-year overall survival rate; p = 0.0283). PXN expression was identified as an independent factor predicting patient survival according to multivariate Cox regression analyses (p = 0.0452). Overexpression of PXN was detected in HNSCC clinical specimens by immunostaining. Functional assays in HNSCC cells showed that knockdown of PXN expression attenuated cancer cell migration and invasion, suggesting that aberrant expression of PXN contributed to HNSCC cell aggressiveness. Our miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Molecular Pathogenesis of the Coronin Family: CORO2A Facilitates Migration and Invasion Abilities in Oral Squamous Cell Carcinoma.

In humans, the coronin family is composed of seven proteins containing WD-repeat domains that regulate actin-based cellular processes. Some members of the coronin family are closely associated with cancer cell migration and invasion. The Cancer Genome Atlas (TCGA) analysis revealed that CORO1C, CORO2A, and CORO7 were significantly upregulated in oral squamous cell carcinoma (OSCC) tissues (p < 0.05). Moreover, the high expression of CORO2A was significantly predictive of the 5-year survival rate of patients with OSCC (p = 0.0203). Overexpression of CORO2A was detected in OSCC clinical specimens by immunostaining. siRNA-mediated knockdown of CORO2A suppressed cancer cell migration and invasion abilities. Furthermore, we investigated the involvement of microRNAs (miRNAs) in the molecular mechanism underlying CORO2A overexpression in OSCC cells. TCGA analysis confirmed that tumor-suppressive miR-125b-5p and miR-140-5p were significantly downregulated in OSCC tissues. Notably, these miRNAs bound directly to the 3'-UTR of CORO2A and controlled CORO2A expression in OSCC cells. In summary, we found that aberrant expression of CORO2A facilitates the malignant transformation of OSCC cells, and that downregulation of tumor-suppressive miRNAs is involved in CORO2A overexpression. Elucidation of the interaction between genes and miRNAs will help reveal the molecular pathogenesis of OSCC.

Tumor Suppressive Circular RNA-102450: Development of a Novel Diagnostic Procedure for Lymph Node Metastasis from Oral Cancer.

Circular RNAs (circRNAs), which form as covalently closed loop structures, have several biological functions such as regulation of cellular behavior by adsorbing microRNAs. However, there is limited information of circRNAs in oral squamous cell carcinoma (OSCC). Here, we aimed to elucidate the roles of aberrantly expressed circRNAs in OSCC. CircRNA microarray showed that circRNA-102450 was down-regulated in OSCC cells. Clinical validation of circRNA-102450 was performed using highly sensitive droplet digital PCR in preoperative liquid biopsy samples from 30 OSCC patients. Interestingly, none of 16 studied patients with high circRNA-102450 had regional lymph node metastasis (RLNM), whereas 4 of 14 studied patients (28.5%) with low expression had pathologically proven RLNM. Overexpressed circRNA-102450 significantly inhibited the tumor metastatic properties of cell proliferation, migration, and invasion. Furthermore, circRNA-102450 directly bound to, and consequently down-regulated, miR-1178 in OSCC cells. Taken together, circRNA-102450 has a tumor suppressive effect via the circRNA-102450/miR-1178 axis and may be a novel potential marker of RLNM in OSCC patients.

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma.

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.

Identification of Tumor Suppressive Genes Regulated by miR-31-5p and miR-31-3p in Head and Neck Squamous Cell Carcinoma.

We identified the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) tissues by RNA sequencing, in which 168 miRNAs were significantly upregulated, including both strands of the miR-31 duplex (miR-31-5p and miR-31-3p). The aims of this study were to identify networks of tumor suppressor genes regulated by miR-31-5p and miR-31-3p in HNSCC cells. Our functional assays showed that inhibition of miR-31-5p and miR-31-3p attenuated cancer cell malignant phenotypes (cell proliferation, migration, and invasion), suggesting that they had oncogenic potential in HNSCC cells. Our in silico analysis revealed 146 genes regulated by miR-31 in HNSCC cells. Among these targets, the low expression of seven genes (miR-31-5p targets: CACNB2 and IL34; miR-31-3p targets: CGNL1, CNTN3, GAS7, HOPX, and PBX1) was closely associated with poor prognosis in HNSCC. According to multivariate Cox regression analyses, the expression levels of five of those genes (CACNB2: p = 0.0189; IL34: p = 0.0425; CGNL1: p = 0.0014; CNTN3: p = 0.0304; and GAS7: p = 0.0412) were independent prognostic factors in patients with HNSCC. Our miRNA signature and miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Engineered exosomes delivering specific tumor-suppressive RNAi attenuate oral cancer progression.

Exosomes are involved in a wide range of biological processes in human cells. Considerable evidence suggests that engineered exosomes (eExosomes) containing therapeutic agents can attenuate the oncogenic activity of human cancer cells. Despite its biomedical relevance, no information has been available for oral squamous cell carcinoma (OSCC), and therefore the development of specific OSCC-targeting eExosomes (octExosomes) is urgently needed. We demonstrated that exosomes from normal fibroblasts transfected with Epstein-Barr Virus Induced-3 (EBI3) cDNA were electroporated with siRNA of lymphocyte cytoplasmic protein 1 (LCP1), as octExosomes, and a series of experiments were performed to evaluate the loading specificity/effectiveness and their anti-oral cancer cell activities after administration of octExosomes. These experiments revealed that octExosomes were stable, effective for transferring siLCP1 into OSCC cells and LCP1 was downregulated in OSCC cells with octExosomes as compared with their counterparts, leading to a significant tumor-suppressive effect in vitro and in vivo. Here we report the development of a new valuable tool for inhibiting tumor cells. By engineering exosomes, siLCP1 was transferred to specifically suppress oncogenic activity of OSCC cells. Inhibition of other types of human malignant cells merits further study.

Distribution of PAPP-A and total hCG between 11 and 13 weeks of gestation in Japanese pregnant women.

Objectives: To establish the reference values for PAPP-A and total hCG between 11 and 13 weeks of gestation for the use of risk assessment of fetal aneuploidy in Japanese pregnant women.Methods: A multicenter prospective study was conducted. The subjects included only Japanese pregnant women with viable singleton who requested the first trimester combined (nuchal translucency and maternal serum marker) screening for fetal aneuploidy. Reference values of PAPP-A and total hCG in Japanese population were made and compared with them in Caucasian.Results: Overall 1,751 Japanese pregnant women were analyzed. Median vales of maternal serum concentration in Japanese pregnant women from 11 + 0-13 + 6 weeks' gestation were ranged from 3.01 to 9.51 mIU/mL for PAPP-A and from 70.2 to 58.3 IU/mL for total-hCG, respectively. Regression curve of median maternal serum PAPP-A and total-hCG concentration against gestational days are significantly higher in Japanese comparing with Caucasian. At most distant values, Japanese serum concentration indicated 1.45 MoM for total-hCG and 1.70 MoM for PAPP-A based on Caucasian regression curves.Conclusion: A modification of the equations by specific reference values is necessary for Japanese pregnant women at the risk assessment of chromosomal abnormalities using the first trimester maternal serum marker.

SYT12 plays a critical role in oral cancer and may be a novel therapeutic target.

Synaptotagmin12 (SYT12) has been well characterized as the regulator of transmitter release in the nervous system, however the relevance and molecular mechanisms of SYT12 in oral squamous cell carcinoma (OSCC) are not understood. In the current study, we investigated the expression of SYT12 and its molecular biological functions in OSCC by quantitative reverse transcriptase polymerase chain reaction, immunoblot analysis, and immunohistochemistry. SYT12 were up-regulated significantly in OSCC-derived cell lines and primary OSCC tissue compared with the normal counterparts (P<0.05) and the SYT12 expression levels were correlated significantly with clinical indicators, such as the primary tumoral size, lymph node metastasis, and TNM stage (P<0.05). SYT12 knockdown OSCC cells showed depressed cellular proliferation, migration, and invasion with cell cycle arrest at G1 phase. Surprisingly, we found increased calcium/calmodulin-dependent protein kinase 2 (CAMK2) inhibitor 1 (CAMK2N1) and decreased CAMK2-phosphorylation in the knockdown cells. Furthermore, treatment with L-3, 4-dihydroxyphenylalanine (L-dopa), a drug approved for Parkinson's disease, led to down-regulation of SYT12 and similar phenotypes to SYT12 knockdown cells. Taken together, we concluded that SYT12 plays a significant role in OSCC progression via CAMK2N1 and CAMK2, and that L-dopa would be a new drug for OSCC treatment through the SYT12 expression.