Expression of Salivary miR-203a-3p Was Related with Oral Health-Related Quality of Life in Healthy Volunteers.

Oral health-related quality of life (OHRQoL) is a multidimensional construct that involves subjective evaluation of an individual's oral health. Although it is difficult to evaluate OHRQoL biologically, recently, it has been reported that circulating microRNAs (miRNAs) in several body fluids could reflect various health conditions. The aim of this pilot study was to investigate whether salivary miRNAs expression differs according to OHRQoL in healthy volunteers. Forty-six volunteers (median age, 23.0 years) were recruited, and their OHRQoL was assessed using the Japanese version of the Oral Health Impact Profile (OHIP-J). Then, we compared salivary microRNA profiles of the high-OHRQoL group (≤25th percentile score of OHIP-J) and the low-OHRQoL group (≥75th percentile score of OHIP-J) using the polymerase chain reaction (PCR) array and the quantitative real-time PCR. There were no significant differences between the two groups in terms of oral health status. In the PCR array, miR-203a-3p and miR-30b-5p were significantly more expressed in the low-OHRQoL group (p < 0.05). Quantitative real-time PCR assay also showed that miR-203a-3p was more highly expressed in the low-OHRQoL group than in the high-OHRQoL group (p < 0.05). These observations suggest that expression of salivary miR-203a-3p was related with OHRQoL in healthy volunteers.

MicroRNAs as serum biomarkers for periodontitis.

AIM: Studies demonstrated that periodontitis modulates microRNA (miRNAs) expression rates in periodontal tissue. However, the relationship between periodontitis and miRNAs profile in circulation remains unclear. In this study, we investigated the effects of periodontitis on serum miRNAs profile in a rat model. MATERIAL AND METHODS: Male Wistar rats (n = 32, 8 weeks old) were divided into four groups of eight rats each. The control groups received no treatment for 2 or 4 weeks. In the other two groups, periodontitis was ligature induced for 2 or 4 weeks. Serum miRNAs expression profiles of each group were compared. RESULTS: Ligation around teeth induced periodontal inflammation at 2 weeks and periodontal tissue destruction at 4 weeks. Microarray results showed that 25 miRNAs were expressed with a <0.5 or >2 difference between the control and periodontitis groups at 4 weeks. Results of real-time PCR revealed that the periodontitis group up-regulated expression rates of serum miR-207 and miR-495 at 2 weeks, and miR-376b-3p at 4 weeks (p < 0.05). CONCLUSION: Serum miRNAs (miR-207, miR-495, and miR-376b-3p) could be valuable biomarkers for periodontitis.

MicroRNAs in Salivary Exosome as Potential Biomarkers of Aging.

The aim of this study was to examine whether salivary exosomal miRNAs could be identified as aging biomarkers. Fifteen young healthy volunteers (median age, 21.0 years) and 13 old individuals (median age, 66.0 years) were recruited. Unstimulated whole saliva was collected, salivary exosomes were isolated, and total RNA was extracted. In a microarray, 242 miRNAs were commonly detected in these two mixed samples. Based on the cut-off values of 2- or 0.5-fold changes (FC) and regulatory power for aging process, six candidate miRNAs (miR-24-3p, miR-371a-5p, miR-3175, miR-3162-5p, miR-671-5p, and miR-4667-5p) were selected. After comparing each total RNA obtained by the 15 young and 13 old individuals to validate the FC values using quantitative real-time PCR, miR-24-3p was identified as a novel candidate aging biomarker. This pilot study suggested that salivary exosomal miRNAs could be identified as candidate aging biomarkers. To confirm whether miR-24-3p in salivary exosomes are suitable biomarkers of aging, further validation research is required.

Factors related to the formation of buccal mucosa ridging in university students.

OBJECTIVE: Buccal mucosa ridging (BMR) is known as a clinical sign of clenching, which is one of the major manifestations of bruxism. However, there are few reports about the formation of BMR and no data regarding the association between BMR and factors such as malocclusion. The purpose of the current study was to investigate the relationship between BMR and factors such as the number of teeth present, gender, body mass index (BMI), occlusion and clenching habit in university students. MATERIALS AND METHODS: A total of 2101 students (1164 males, 937 females), aged 18-29 years old, were included in the study. BMR and the number of teeth present were recorded and malocclusion was defined using a modified version of the Index of Orthodontic Treatment Need. Additional information regarding gender, clenching habit and BMI was collected via a questionnaire. RESULTS: Forty-six per cent of the subjects had BMR and the prevalence of BMR in females was significantly higher than that of males (chi square test, p < 0.001). According to logistic regression analysis, the probability of BMR was significantly associated with female gender (OR = 1.501, 95% CI = 1.259-1.790, p < 0.001), crowding (OR = 2.102, 95% CI = 1.706-2.590, p < 0.001) and overjet (OR = 0.585, 95% CI = 0.418-0.818, p = 0.002). On the other hand, BMR was not associated with awareness of clenching habit and BMI. CONCLUSIONS: Gender, crowding and overjet were related to the formation of BMR in university students. When evaluating BMR as a clinical sign of clenching, one might have to take factors such as gender and crowding into consideration.

Gut-derived Endotoxin-TLR4 Signaling Drives MYC-Ig Translocation to Promote Lymphoproliferation through c-JUN and STAT3 Activation.

Synergism between obesity and virus infection promotes the development of B-cell lymphoma. In this study, we tested whether obesity-associated endotoxin release induced activation-induced cytidine deaminase (AID). TLR4 activation in turn caused c-JUN-dependent and STAT3-dependent translocations of MYC loci to suppress transactivation of CD95/FAS. We used viral nucleocapside Core transgenic (Tg) mice fed alcohol Western diet to determine whether oncogenesis arising from obesity and chronic virus infection occurred through TLR4-c-JUN-STAT3 pathways. Our results showed B cell-specific, c-Jun and/or Stat3 disruption reduced the incidence of splenomegaly in these mice. AID-dependent t(8;14) translocation was observed between the Ig promoter and MYC loci. Comparison with human B cells showed MYC-immunoglobulin (Ig) translocations after virus infection with lipopolysaccharide stimulation. Accordingly, human patients with lymphoma with virus infections and obesity showed a 40% incidence of MYC-Ig translocations. Thus, obesity and virus infection promote AID-mediated translocation between the Ig promoter and MYC through the TLR4-c-JUN axis, resulting in lymphoproliferation. Taken together, preventative treatment targeting either c-JUN and/or STAT3 may be effective strategies to prevent tumor development. IMPLICATIONS: Obesity increases gut-derived endotoxin which induces Toll-like receptor-mediated MYC-Ig translocation via c-JUN-STAT3, leading to lymphoproliferation.

Profiling of Circulating Tumor Cells for Screening of Selective Inhibitors of Tumor-Initiating Stem-Like Cells.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells.

Chemoresistance and plasticity of tumor-initiating stem-like cells (TICs) promote tumor recurrence and metastasis. The gut-originating endotoxin-TLR4-NANOG oncogenic axis is responsible for the genesis of TICs. This study investigated mechanisms as to how TICs arise through transcriptional, epigenetic, and post-transcriptional activation of oncogenic TLR4 pathways. Here, we expressed constitutively active TLR4 (caTLR4) in mice carrying pLAP-tTA or pAlb-tTA, under a tetracycline withdrawal-inducible system. Liver progenitor cell induction accelerated liver tumor development in caTLR4-expressing mice. Lentiviral shRNA library screening identified histone H3K4 methylase SETD7 as central to activation of TLR4. SETD7 combined with hypoxia induced TLR4 through HIF2 and NOTCH. LIN28 post-transcriptionally stabilized TLR4 mRNA via de-repression of let-7 microRNA. These results supported a LIN28-TLR4 pathway for the development of HCCs in a hypoxic microenvironment. These findings not only advance our understanding of molecular mechanisms responsible for TIC generation in HCC, but also represent new therapeutic targets for the treatment of HCC.

c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity.

Metabolic syndrome is associated with obesity, insulin resistance, and the risk of cancer. We tested whether oncogenic transcription factor c-JUN metabolically reprogrammed cells to induce obesity and cancer by reduction of glucose uptake, with promotion of the stemness phenotype leading to malignant transformation. Liquid alcohol, high-cholesterol, fat diet (HCFD), and isocaloric dextrin were fed to wild-type or experimental mice for 12 months to promote hepatocellular carcinoma (HCC). We demonstrated 40% of mice developed liver tumors after chronic HCFD feeding. Disruption of liver-specific c-Jun reduced tumor incidence 4-fold and improved insulin sensitivity. Overexpression of c-JUN downregulated RICTOR transcription, leading to inhibition of the mTORC2/AKT and glycolysis pathways. c-JUN inhibited GLUT1, 2, and 3 transactivation to suppress glucose uptake. Silencing of RICTOR or c-JUN overexpression promoted self-renewal ability. Taken together, c-JUN inhibited mTORC2 via RICTOR downregulation and inhibited glucose uptake via downregulation of glucose intake, leading to self-renewal and obesity.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.

Clinical efficacy of naftopidil on lower urinary tract symptoms after radical prostatectomy.

OBJECTIVE: The management of lower urinary tract symptoms that persist after radical prostatectomy remains to be established. We investigated whether an alpha1-blocker, naftopidil, improves LUTS in patients >or=1 year after radical prostatectomy. METHODS: A total of 29 male patients received 25 mg/day of naftopidil for the first week, then 75 mg/day for 4 weeks. The frequency-volume chart, international prostate symptom score and quality of life index (QOL) were examined before and at the end of the 5-week administration in all subjects. RESULTS: Total international prostate symptom score (I-PSS) and I-PSS subtotals associated with voiding symptoms and storage symptoms were significantly decreased at 5 weeks compared with baseline (P < 0.001 each). QOL index was significantly improved with naftopidil for 5 weeks (P < 0.001). From analyses of the frequency-volume chart, mean and maximum volume/void were significantly increased (P < 0.05 each). CONCLUSION: Lower urinary tract symptoms detected in patients >or=1 year after radical prostatectomy were markedly improved with administration of naftopidil at 75 mg/day. These symptoms could represent a novel target for medical treatment by improved understanding of the symptom pathology in the near future.

Entinostat reverses P-glycoprotein activation in snail-overexpressing adenocarcinoma HCC827 cells.

Epithelial-to-mesenchymal transition (EMT) in cancer cells facilitates tumor progression by promoting invasion and metastasis. Snail is a transcriptional factor that induces EMT, while P-glycoprotein (P-gp) is an efflux transporter involved in anticancer drug resistance, and P-gp efflux activity is stimulated in Snail-overexpressing lung cancer cells with EMT characteristics. Since the histone deacetylase (HDAC) inhibitor entinostat (Ent) reverses EMT features, our aim in this study was to determine whether Ent also suppresses P-gp activation in Snail-induced cells. First, we confirmed that Ent treatment reduced migration activity, downregulated E-cadherin and upregulated vimentin at the mRNA level in Snail-overexpressing cells, thus inhibiting EMT. Efflux and uptake assays using rhodamine123 (Rho123), a fluorescent P-gp substrate, showed that Ent also inhibited Snail-induced activation of P-gp. Moreover, P-gp activity was more strongly inhibited by Ent in Snail-overexpressing cells than in Mock cells. When we evaluated the uptakes of Rho123 by LLC-PK1 cells and P-gp-overexpressing LLC-GA5COL150 cells, Rho123 accumulation in LLC-GA5COL150 cells was significantly decreased compared with that in LLC-PK1 cells. Coincubation with Ent had no effect on Rho123 accumulation in either of the cell lines. Thus, Ent appears to be an inhibitor, but not a substrate, of P-gp at low concentration. Our results suggest that Ent treatment might suppress not only Snail-induced cancer malignant alteration, but also P-gp-mediated multidrug resistance.

Association between periodontitis and prognosis of pancreatobiliary tract cancer: A pilot study.

Several studies have indicated that periodontitis is a risk factor for cancer. However, the association between periodontitis and the prognosis of pancreatobiliary tract cancer remains unclear. The aim of this pilot study was to investigate the association between periodontitis and prognosis of pancreatobiliary tract cancer. A total of 22 patients diagnosed with pancreatobiliary tract cancer were analyzed. Oral health status, including severity of periodontitis, general health status and biochemical serum markers were evaluated. The Kaplan-Meier method and Cox proportional hazards model were used to assess factors affecting the prognosis of pancreatobiliary tract cancer. The Kaplan-Meier analysis demonstrated that low body mass index, high concentration of serum C-reactive protein (CRP) and severe periodontitis were significant prognostic factors for survival rate. The Cox proportional hazards model revealed that serum carbohydrate antigen 19-9 concentration [hazard ratio (HR)=1.002; 95% confidence interval (CI): 1.000-1.004] and serum CRP concentration (HR=2.57; 95% CI: 1.15-5.74) were significantly associated with the prognosis of pancreatobiliary tract cancer. In addition, cancer patients with severe periodontitis had higher serum CRP concentrations compared with those without severe periodontitis. Therefore, severe periodontitis indirectly affected the prognosis of pancreatobiliary tract cancer through promoting systemic inflammation.

Preventive Effects of Drinking Hydrogen-Rich Water on Gingival Oxidative Stress and Alveolar Bone Resorption in Rats Fed a High-Fat Diet.

Obesity induces gingival oxidative stress, which is involved in the progression of alveolar bone resorption. The antioxidant effect of hydrogen-rich water may attenuate gingival oxidative stress and prevent alveolar bone resorption in cases of obesity. We examined whether hydrogen-rich water could suppress gingival oxidative stress and alveolar bone resorption in obese rats fed a high-fat diet. Male Fischer 344 rats (n = 18) were divided into three groups of six rats each: a control group (fed a regular diet and drinking distilled water) and two experimental groups (fed a high-fat diet and drinking distilled water or hydrogen-rich water). The level of 8-hydroxydeoxyguanosine was determined to evaluate oxidative stress. The bone mineral density of the alveolar bone was analyzed by micro-computerized tomography. Obese rats, induced by a high-fat diet, showed a higher gingival level of 8-hydroxydeoxyguanosine and a lower level of alveolar bone density compared to the control group. Drinking hydrogen-rich water suppressed body weight gain, lowered gingival level of 8-hydroxydeoxyguanosine, and reduced alveolar bone resorption in rats on a high-fat diet. The results indicate that hydrogen-rich water could suppress gingival oxidative stress and alveolar bone resorption by limiting obesity.

[A case of non-traumatic rupture of varicocele].

We report a case of a non-traumatic rupture of varicocele. A 28-year-old man visited our hospital complaining of left scrotal swelling with severe spontaneous pain of sudden onset after straining for defecation. MRI revealed a dilated spermatic cord with scrotal hematoma surrounding the left testis, which leads to the diagnosis of varicocele rupture. Conservative treatment with oral analgesics for a couple of weeks relieved the swelling and pain. Subinguinal microscopic ligation of left spermatic veins was performed 4 months later.

[5 years intravesical instillation with mitomycin-C and pirarubicin as a prophylactic treatment for superficial bladder cancer].

PURPOSE: We studied prophylactic intravesical instillation of mitomycin C (MMC) and pirarubicin (THP) following transurethral resection of bladder tumor (TUR-Bt) for superficial bladder cancer. MATERIALS AND METHODS: Forty-six evaluable patients were administered intravesically 20 mg of MMC dissolved in 20 ml saline on day 1 and 20 mg of THP dissolved 20 ml 5% dextrose on day 2. The patients were followed up by cystscopy and urinary cytology. Intravesical instillations were performed once a month and continued for 5 years. RESULTS: The non-recurrence rates at 1, 3 and 5 years were 88.8%, 79.5% and 67.0%, respectively. No significant differences were observed between grade 1-2 and 3, male and female, and solitary and multiple tumors. Although the side effects were relatively mild, 6 patients were stopped intravesical instillation. CONCLUSIONS: Because non-recurrence rates of our report is not better than previous reports with shorter treatment periods, intravesical MMC and THP instillation for 5 years is not beneficial to the patients with superficial bladder cancer.

miR­1246 and miR­4644 in salivary exosome as potential biomarkers for pancreatobiliary tract cancer.

Pancreatobiliary tract cancer is a highly fatal cancer. Detection of pancreatobiliary tract cancer is difficult because it lacks typical clinical symptoms and because of its anatomical location. Biomarker discovery is therefore important to detect pancreatobiliary tract cancer in its early stage. A study demonstrated that expression levels of miR­1246, miR­3976, miR­4306, and miR­4644 in serum exosomes were higher in pancreatic cancer patients than these levels in healthy control participants. Supposing that microRNA (miRNA) expression profiles in saliva are similar to those in serum, four miRNAs (miR­1246, miR­3976, miR­4306, and miR­4644) in salivary exosomes may also be useful for detection of pancreatobiliary tract cancer. In this study, it was examined whether these miRNAs could be used as biomarkers for pancreatobiliary tract cancer. Twelve pancreatobiliary tract cancer patients and 13 healthy control participants were analyzed as a cancer and a control group, respectively. Unstimulated whole saliva was collected, salivary exosomes were isolated, and total RNA was extracted. Using quantitative real­time PCR (RT­qPCR), the relative expression ratios of miR­1246 and miR­4644 were significantly higher in the cancer group than these ratios in the control group. Receiver operating characteristic (ROC) curves were constructed to analyze the discrimination power of these miRNAs. For miR­1246, the results yielded an area under the curve (AUC) of 0.814 (P=0.008). For miR­4644, the results yielded an AUC of 0.763 (P=0.026). For the combination of miR­1246 and miR­4644, the results yielded an increased AUC of 0.833 (P=0.005). This pilot study suggests that miR­1246 and miR­4644 in salivary exosomes could be candidate biomarkers for pancreatobiliary tract cancer.

[Bone loss in men under androgen-deprivation therapy for prostate cancer].

Bone is a major target tissue for sex hormones and hypogonadism is a known cause of male osteoporosis. Androgen-deprivation therapy (ADT) for prostate cancer yields hypogonadotropic status and accelerates age-related decrease in bone mineral density. The risk for osteoporotic fractures is also increased which may lead to a shorter life expectancy for prostatic cancer patients. Baseline and follow up bone density measurement is desired for all men beginning ADT and it is advisable to take calcium and vitamin D as well as maintain a moderate exercise. Bisphosphonate is a possible treatment for those in whom osteoporosis develops. More data are desirable to make a guideline to prevent and treat male osteoporosis under ADT.

[A case of non-functioning adrenocortical adenoma associated with renal cell carcinoma].

Left renal and left adrenal masses were incidentally found by computerized tomography (CT) in a 56-year-old man who was admitted to our hospital for treatment of upper digestive tract hemorrhage. Apparently no clinical signs suggestive of Cushing's syndrome existed. The renal tumor was diagnosed as renal cell carcinoma based on the findings on enhanced CT. 131I-adosterol uptake in the examination of adrenal scintigraphy under dexamethasone suppression was definitely increased in the left adrenal gland, although hormonal examinations of serum and urine for adrenal functions were within the normal range. Plasma adrenocorticotropic hormone (ACTH) and serum cortisol were suppressed by administration of 2 mg dexamethasone for 2 days. The left kidney was radically removed by surgery together with the left adrenal gland. Histological diagnoses were left renal cell carcinoma and adrenocortical adenoma.

[A case of giant perineal epidermal cyst: a case report].

A 33-year-old man was admitted to our hospital with the chief complaint of a giant perineal mass causing difficulty in walking. Magnetic resonance imaging revealed cystic tumor with a diameter of 13 cm. We performed tumor resection. Histopathological diagnosis of the tumor was epidermal cyst. To our knowledge, this is the sixth case of perineal epidermal cyst reported in Japan.