miR-520d-5p can reduce the mutations in hepatoma cancer cells and iPSCs-derivatives.

BACKGROUND: Human microRNAs (miRNAs) have diverse functions in biology, and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS: To examine whether there were any changes in mutation status in cells treated with 520d-5p, we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS: 520d-5p induced a shift towards a wild type or non-malignant phenotype, which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further, 520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS: Cancer cell genomic mutations cannot be repaired in most contexts. However, these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine.

Tumor-suppressive effects of atelocollagen-conjugated hsa-miR-520d-5p on un-differentiated cancer cells in a mouse xenograft model.

BACKGROUND: We previously demonstrated that hsa-miR-520d-5p can convert cancer cells into induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) via a demethylation process and p53 upregulation in vivo. Additionally, we have reported the non-tumorigenic effect of miR-520d-5p on normal human cells, including fibroblasts. METHODS: We used atelocollagen-conjugated miR-520d-5p (520d/atelocollagen) to confirm the possibility of a therapeutic effect on cancer cells. We traced the size and signal intensity of GFP-expressing tumors in mice each week, beginning 4 weeks after subcutaneous inoculation. RESULTS: 520d/atelocollagen treatment suppressed tumor growth by greater than 80 % each week relative to controls and resulted in an approximately 30 % disappearance of tumors. In mice whose tumors disappeared, the existence of human genomic material at the injection site was examined by quantitative Alu-PCR, and we confirmed the co-existence of both species-derived cells. In every site where a tumor disappeared in immunodeficient mice, GFP protein was expressed in the connective tissues, and approximately 0.1 % of the extracted DNA contained human genomic material. We could not identify any adverse effects in vivo. CONCLUSIONS: This is the first report to confirm an inhibitory effect of 520d/atelocollagen on cancer cells in vivo. The development of optimized modifications of this carrier is expected to enhance the efficiency of entry into tumor cells and the induction of its inhibitory effect.

Dipeptidyl peptidase-4 inhibitor-associated bullous pemphigoid, likely triggered by scabies, in a hemodialysis patient with human leukocyte antigen-DQB1*03:01.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal bullous diseases. Autoantibodies against hemidesmosomal adhesion proteins might be involved in the developing process. BP usually affects the elderly with high mortality whereas the drug-induced BP is often improved and rarely relapses after the withdrawal of the suspected drug. An accumulated evidence suggests that dipeptidyl peptidase-4 inhibitor (DPP-4I), which has been widely used as the antidiabetic drug improves glycemic control with little risk for hypoglycemia, could be an inducer of DPP-4I-associated BP (DPP-4I-BP). While the precise mechanism remains unclear, a unique immunological profile with human leukocyte antigen (HLA)-DQB1*03:01 could be a biomarker of genetic susceptibility to DPP-4I-BP. Here, we encountered an interesting case of DPP-4I-BP with HLA-DQB1*03:01, which was likely triggered by scabies. A 56-year-old Japanese male with type 2 diabetes on hemodialysis was referred to our hospital due to worsened blisters. Prior to his admission, he had been on linagliptin, a DPP-4I, for 5 months. He then suffered from scabies 2 weeks before his admission while the treatment with ivermectin failed to improve his symptom. Based on his clinical symptom, positive for anti-BP180 autoantibody in serum, and the pathological alterations of skin biopsy specimens, he was diagnosed with DPP-4I-BP. Importantly, he also carried an HLA-DQB1*03:01 allele. Oral prednisolone was subsequently administered after the discontinuation of linagliptin, and his symptom gradually disappeared. Given the fact that the DPP-4I-BP could be a life-threating disease, we should be cautious of prescribing DPP-4I in hemodialysis patients, whose immune system could be impaired.

Pharmacokinetic Interaction Study of Ranitidine and Daijokito in Healthy Volunteers.

BACKGROUND: Ranitidine is a histamine 2 receptor antagonist, and daijokito is a Kampo (Chinese herbal medicine as practiced in Japan) formula, which is traditionally used for treating constipation and digestive trouble. Previous study demonstrated that daijokito significantly affected the pharmacokinetics of ranitidine in rats; however, the doses of ranitidine and daijokito in that study were higher than in clinical practice. Therefore, we examined the pharmacokinetic interaction between ranitidine and daijokito in clinical practice doses in healthy volunteers. METHODS: This was a randomized, open label, two-period crossover study in healthy volunteers (n = 7). Volunteers received administrations of either a single dose of ranitidine 300 mg, or ranitidine 300 mg in combination with daijokito extract granules 2.5 g. Plasma concentrations of ranitidine were measured over 12 h by LC/MS/MS method. RESULTS: Plasma concentrations of ranitidine were lower with co-administration of daijokito compared with ranitidine alone. Co-administration of daijokito significantly decreased ranitidine area under the plasma concentration-time curve from 0 to 12 h (AUC0-12) and maximum plasma concentration (Cmax) with geometric mean (GM) ratio [90% confidence interval (CI)] for AUC0-12 of 0.609 (0.449, 0.826) and Cmax of 0.515 (0.345, 0.771). CONCLUSION: Co-administration of ranitidine with daijokito resulted in a significant decrease in plasma level of ranitidine in healthy volunteers.

Hsa-miR-520d-5p promotes survival in human dermal fibroblasts exposed to a lethal dose of UV irradiation.

We previously reported that hsa-miR-520d-5p is functionally involved in the induction of the epithelial-mesenchymal transition and stemness-mediated processes in normal cells and cancer cells, respectively. On the basis of the synergistic effect of p53 upregulation and demethylation induced by 520d-5p, the current study investigated the effect of this miRNA on apoptotic induction by ultraviolet B (UVB) light in normal human dermal fibroblast (NHDF) cells. 520d-5p was lentivirally transfected into NHDF cells either before or after a lethal dose of UVB irradiation (302 nm) to assess its preventive or therapeutic effects, respectively. The methylation level, gene expression, production of type I collagen and cell cycle distribution were estimated in UV-irradiated cells. NHDF cells transfected with 520d-5p prior to UVB irradiation had apoptotic characteristics, and the transfection exerted no preventive effects. However, transfection with 520d-5p into NHDF cells after UVB exposure resulted in the induction of reprogramming in damaged fibroblasts, the survival of CD105-positive cells, an extended cell lifespan and prevention of cellular damage or malfunction; these outcomes were similar to the effects observed in 520d-5p-transfected NHDF cells (520d/NHDF). The gene expression of c-Abl (Abelson murine leukemia viral oncogene homolog 1), ATR (ataxia telangiectasia and Rad3-related protein), and BRCA1 (breast cancer susceptibility gene I) in transfectants was transcriptionally upregulated in order. These mechanistic findings indicate that ATR-dependent DNA damage repair was activated under this stressor. In conclusion, 520d-5p exerted a therapeutic effect on cells damaged by UVB and restored them to a normal senescent state following functional restoration via survival of CD105-positive cells through c-Abl-ATR-BRCA1 pathway activation, p53 upregulation, and demethylation.

Components of Boiogito Suppress the Progression of Hypercholesterolemia and Fatty Liver Induced by High-Cholesterol Diet in Rats.

BACKGROUND: Ogi, one main component of boiogito (BOT), is reported to have an effect on hypercholesterolemia and NAFLD. In this experiment, we examined effects of ogi on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats and compared with the effects of ogi combined with ginger or hesperidin. METHODS: Hypercholesterolemia and fatty liver were induced by a high cholesterol diet in rats. Extract of ogi, ogi with hesperidin, and ogi with ginger were added to the high-cholesterol diet, respectively. Ezetimibe was also added to the high-cholesterol diet as a positive control. After 6 and 12 weeks, body, liver and adipose tissue weights, blood chemistry, lipid-related and inflammatory-related factors were examined. RESULTS: The high cholesterol diet increased body, liver and adipose tissue weights, and serum cholesterol concentrations. Ogi, ogi with hesperidin or ginger and ezetimibe improved them. In the histological examinations, we observed a significant improvement after treatment. The lipid-related factors (RBP4, HFABP and CFABP) were improved by treatment. Biomarkers of cholesterol synthesis (lathosterol) and absorption (campesterol, beta-sitosterol) were lower in the treatment groups. Inflammatory-related factors (MCP1, CCR2 and TNF-alpha) and ICAM-1 were ameliorated after treatment, especially by ogi with ginger. CONCLUSION: Ogi, ogi with hesperidin or ginger have a similar effect of BOT and ezetimibe on hypercholesterolemia and fatty liver. Ogi with ginger reveals a stronger additive effect with no significant difference. However, as for the anti-inflammatory (MCP1, CCR2 and TNF-alpha) and anti-arteriosclerotic (ICAM-1) effects, additive effects of ogi with ginger are more potent than that of ogi alone or ezetimibe.

Comparative study of ¹8F-FDG-PET/CT imaging and serum hTERT mRNA quantification in cancer diagnosis.

We have reported on the clinical usefulness of human telomerase reverse transcriptase (hTERT) mRNA quantification in sera in patients with several cancers. Positron emission tomography-computed tomography (PET/CT) using ¹8F-fluorodeoxyglucose (FDG) has recently become an excellent modality for detecting cancer. We performed a diagnostic comparative study of FDG-PET/CT and hTERT mRNA quantification in patients with cancer. Four hundred seventy subjects, including 125 healthy individuals and 345 outpatients with cancer who had received medical treatments for cancer in their own or other hospitals, were enrolled. The subjects were diagnosed by FDG-PET/CT, and we measured their serum hTERT mRNA levels using real-time RT-PCR, correlating the quantified values with the clinical course. In this prospective study, we statistically assessed the sensitivity and specificity, and their clinical significance. hTERT mRNA and FDG-PET/CT were demonstrated to be correlated with the clinical parameters of metastasis and recurrence (P < 0.001), and of recurrence and tumor number in cancer compared with noncancer patients, respectively. A multivariate analysis showed a significant difference in the detection by FDG-PET/CT, ¹8F-FDG uptake, the detection by hTERT mRNA, and age. The use of both FDG-PET/CT and hTERT mRNA resulted in a positivity of 94.4% (221/234) for the detection of viable tumor cells. FDG-PET/CT is superior to hTERT mRNA quantification in the early detection of cancer and combinative use of FDG-PET/CT and hTERT mRNA may improve the diagnostic accuracy of cancer.

Hsa-miR-520d converts fibroblasts into CD105+ populations.

BACKGROUND: We have previously shown that hsa-miR-520d-5p can convert cancer cells into induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) via a dedifferentiation by a demethylation mechanism. METHODS: We tested the effect of miR-520d-5p on human fibroblasts to determine whether it could be safely used in normal cells for future clinical therapeutic applications. After we transfected the microRNA into fibroblasts, we analyzed the phenotypic changes, gene expression levels, and stemness induction in vitro, and we evaluated tumor formation in an in vivo xenograft model. RESULTS: The transfected fibroblasts turned into CD105+ cell populations, survived approximately 24 weeks, and exhibited increases in both the collagen-producing ability and in differentiation. Combinatorial transfection of small interfering RNAs for miR-520d-5p target genes (ELAVL2, GATAD2B, and TEAD1) produced similar results to miR-520d-5p transfection. These molecules converted normal cells into MSCs and not iPSCs. CONCLUSIONS: In vitro data indicate the potent usefulness of this small molecule as a therapeutic biomaterial in normal cells and cancer cells because CD105+ cells never converted to iPSCs despite repeated transfections and all types of transfectants lost their tumorigenicity. This maintenance of a benign state following miR-520d-5p transfection appears to be caused by p53 upregulation. We conclude that miR-520d-5p may be a useful biomaterial at an in vitro level.