High frequency of bone recurrence as an initial recurrence site after radical surgery in T1N3 gastric cancer: a propensity score matching analysis.

PURPOSE: T1 gastric cancer (GC) with seven or more metastatic lymph nodes is extremely rare, and very few clinical studies have been conducted to evaluate the clinicopathological features of their recurrence. METHODS: We retrospectively analyzed the outcomes of T1 GC and T2-4 GC patients who had multiple nodal metastases after radical surgery from 2006 to 2020. Propensity score matching was performed to compare the two groups of patients. RESULTS: After propensity score matching, 18 of 22 patients in the T1 group and 36 of 144 patients in the T2-4 group were selected. Recurrence occurred in six patients (33.3%) in the T1 group. In the T1 group, the most common site of initial recurrence was bone (15.0%). The prevalence of bone recurrence was significantly higher in the T1 group than in the T2-4 group (P = 0.02). The median interval time between radical surgery and bone recurrence was 24 months, and the median survival time after bone recurrence was 14 months. CONCLUSION: Bone recurrence was more frequently identified as an initial recurrence site in T1 GC cases with multiple metastases after radical surgery compared with that in T2-4 GC cases. Careful attention should be paid to postoperative bone recurrence in the long-term postoperative course of these patients.

Protective effect of dimethyl fumarate for the development of pressure ulcers after cutaneous ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) is associated with various pathogenic conditions, and there has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. Several studies demonstrated that oxidative stress is a key player in I/R injury, and the inhibition of oxidative stress may be capable of protecting tissue damage after I/R injury in various organs including skin. Dimethyl fumarate (DMF) approved by the Food and Drug Administration is Nrf2 activator, and recent studies revealed the antioxidative and anti-inflammatory effects of DMF on I/R injury in animal models. Our objective was to assess the effects of oral administration of DMF on the development of PUs after cutaneous I/R injury in mice. We found that DMF administration significantly decreased the size of PUs after cutaneous I/R. Cutaneous I/R-induced oxidative stress was also significantly inhibited by DMF in OKD48 mice, in which oxidative stress can be visually assessed. In addition, DMF treatment decreased hypoxic area, the numbers of apoptotic cells, and vascular loss in I/R area. DMF treatment suppressed the infiltration of MPO+ neutrophils and the production of proinflammatory cytokines in I/R site after cutaneous I/R injury. in vitro experiments, DMF treatment suppressed the production of reactive oxygen species in pericyte-like cells. These results suggest that DMF treatment might prevent the formation of PUs induced by cutaneous I/R injury via suppressing oxidative stress and subsequent inflammation. DMF treatment during the early phase of decubitus ulcers might protect against further progression.

Topical betamethasone butyrate propionate exacerbates pressure ulcers after cutaneous ischemia-reperfusion injury.

Ischaemia-reperfusion (I/R) is involved in the development of various organ diseases. There has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. However, there is no evidence-based treatment for early-stage PUs. Our objective was to assess the effects of topical steroid on the development of PUs after cutaneous I/R injury in mice. Cutaneous I/R was performed by trapping the dorsal skin between two magnetic plates for 12 h, followed by plate removal. Topical application of betamethasone butyrate propionate (BBP) in I/R areas significantly increased the size of PUs after I/R. The number of thromboses was increased, and CD31(+) vessels were decreased in the I/R area treated with topical BBP. The number of oxidative stress-associated DNA-damaged cells and apoptotic cells in the I/R area was increased by topical BBP treatment. In addition, the mRNA level of NADPH oxidase 4 (Nox4), the essential enzyme that produces reactive oxygen species, was significantly increased and that of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates the expression of antioxidant proteins, was inhibited in the I/R area treated by BBP. The number of CD68(+) macrophages and the level of transforming growth factor-beta in lesional skin were also decreased by BBP. These results suggest that a topical steroid might accelerate the formation of PUs induced by cutaneous I/R injury by aggravating oxidative stress-induced tissue damage. Topical steroids might not be recommended for the treatment of acute-phase decubitus ulcers.

Increased susceptibility to oxidative stress- and ultraviolet A-induced apoptosis in fibroblasts in atypical progeroid syndrome/atypical Werner syndrome with LMNA mutation.

Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a disorder of premature ageing caused by mutation of the lamin A gene, the same causal gene involved in Hutchinson-Gilford syndrome (HGS). We previously reported the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). Recently, it has been reported that UVA induced abnormal truncated form of lamin A, called progerin, as well as HGS-like abnormal nuclear structures in normal human fibroblasts, being more frequent in the elderly, suggesting that lamin A may be involved in the regulation of photoageing. The objective of this study was to elucidate the sensitivity to cell damage induced by oxidative stress or UVA in fibroblasts from APS/AWS patient. Using immunofluorescence staining and flow cytometry analysis, the amount of early apoptotic cells and degree of intra-cellular reactive oxygen species (ROS) generation were higher in H2 02 - or UVA-treated APS/AWS fibroblasts than in normal fibroblasts, suggesting that repeated UV exposure may induce premature ageing of the skin in APS/AWS patients and that protecting against sunlight is possibly important for delaying the emergence of APS/AWS symptoms. In addition, we demonstrated that H2 O2 -, or UVA-induced apoptosis and necrosis in normal and APS/AWS fibroblasts were enhanced by farnesyltransferase inhibitor (FTI) treatment, indicating that FTI might not be useful for treating our APS/AWS patient.

MFG-E8 regulates angiogenesis in cutaneous wound healing.

Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF-PDGFRß signaling mediated by integrin-growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31(+) blood vessels, and MFG-E8 colocalized with PDGFRß(+), αSMA(+), and NG2(+) pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.

Pathogenesis of multiple lentigines in LEOPARD syndrome with PTPN11 gene mutation.

LEOPARD syndrome (LS) is an autosomal dominant condition with multiple anomalies, including multiple lentigines. LS is caused by mutations in PTPN11, encoding the protein tyrosine phosphatase, SHP-2. We report here 2 unrelated Japanese cases of LS with different PTPN11 mutations (p.Y279C and p.T468P). To elucidate the pathogenesis of multiple lentigines in LS, ultrastructural and immunohistochemical analyses of lentigines and non-lesional skin were performed. Numerous mature giant melanosomes in melanocytes and keratinocytes were observed in lentigines. In addition, the levels of expression of endothelin-1 (ET-1), phosphorylated Akt, mTOR and STAT3 in the epidermis in lentigines were significantly elevated compared with non-lesional skin. In in vitro assays, melanin synthesis in human melanoma cells expressing SHP-2 with LS-associated mutations was higher than in cells expressing normal SHP-2, suggesting that LS-associated SHP-2 mutations might enhance melanin synthesis in melanocytes, and that the activation of Akt/mTOR signalling may contribute to this process.

Role of endothelin-1/endothelin receptor signaling in fibrosis and calcification in nephrogenic systemic fibrosis.

Nephrogenic systemic fibrosis (NSF) is characterized by systemic fibrosis and abnormal calcification in patients with severe renal dysfunction. It is considered that gadolinium (Gd)-containing contrast agents used for magnetic resonance imaging trigger the development of NSF. However, the causative role of Gd and the mechanism of Gd-induced fibrosis and calcification in NSF are unknown. Recently, it has been known that endothelin-1 (ET-1)/ET receptor (ETR) signalling regulates fibrosis and calcification. The objective was to elucidate the role of ET-1/ETR signalling in Gd-induced fibrosis and calcification in NSF. First, we demonstrated that Gd enhanced proliferation and calcification of human adipose tissue-derived mesenchymal stem cells (hMSC) in vitro. Next, we examined the expression of ET-1 and ETR-A in hMSC using proliferation or calcification assay. ET-1 and ETR-A expression in hMSC treated with Gd were elevated. ET-1/ETR signalling inhibitor, bosentan, inhibited Gd-induced proliferation and calcification of hMSC. In addition, bosentan inhibited Gd-induced phosphorylation of ERK and Akt in hMSC. Plasma ET-1 levels of the patients were significantly higher than these of normal individuals and systemic sclerosis patients. In immunofluorescence staining, the expression of ETR-A in fibroblasts in dermal fibrosis lesion of NSF was increased. We conclude that Gd induces proliferation and calcification of hMSC via enhancement of ET-1/ETR signalling. Our results contribute to understand the pathogenesis of NSF.

Laparoscopy-Assisted Distal Gastrectomy for the Eldest Elderly Patients with Gastric Cancer.

BACKGROUND/AIMS: The number of the eldest elderly (aged 85 years and older) patients with gastric cancer has been rising in Japan. Laparoscopy-assisted distal gastrectomy (LADG) has been accepted as a less invasive treatment for gastric cancer. The purpose of this study is to evaluate the efficacy and safety of LADG for eldest elderly patents. METHODOLOGY: From January 2006 to July 2010, 262 patients underwent LADG for gastric cancer. Of these, 9 patients were 85 years old and over (eldest elderly group) and the remaining 253 patients were younger than 85 years (control group). Clinicopathological characteristics and operative outcomes were analyzed. RESULTS: Among clinicopathological characteristics analyzed in this study (gender, body mass index, co-morbidity, American Society of Anesthesiologists physical status and tumor status), only gender showed a significant difference between the eldest elderly and the control groups. There were no significant differences in operation time, blood loss, postoperative complication and postoperative hospital stay between the 2 groups. No serious complications or mortality were found in the eldest elderly group. CONCLUSIONS: It is suggested that LADG is a safe and efficient procedure for the treatment of gastric cancer, even in eldest elderly patients.

[A case of squamous cell carcinoma of the nipple skin successfully treated with S-1 alone].

Squamous cell carcinoma of the breast is uncommon, but that of the nipple skin is rarer. The effect of chemotherapy in these cases is yet to reach consensus. We report a rare case in which primary squamous cell carcinoma of the nipple skin was successfully treated with S-1 alone. A 64-year-old woman was admitted to our hospital because of a granulomatous tumor mass over the right nipple, which she was aware of for 10 years; the tumor showed a rapid increase in growth before admission. The tumor was approximately 4 cm at the first visit, and was diagnosed as squamous cell carcinoma by incisional biopsy. We administered preoperative systemic chemotherapy owing to the presence of metastasis in an axillary lymph node. After 2 courses of chemotherapy with oralS -1 at 100mg/day for 28 days followed by a 14-day resting period, the primary tumor and metastatic lymph node showed a remarkable reduction in size. The patient subsequently underwent a radical operation and is currently healthy without any recurrence.

Real-world effectiveness and safety of abrocitinib in 12 Japanese patients with atopic dermatitis and transcriptome analysis with peripheral blood.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by recurrent, pruritic, and localized eczema. Various types of new drugs have been recently investigated for treating AD. The efficacy and safety of abrocitinib in treating AD has been reported in clinical trials, but the real-world data from Japan has not been reported. Herein, we analyzed 12 Japanese patients with AD treated with 100 mg of abrocitinib using our real-world data. We also performed transcriptome analysis with peripheral blood to investigate the effects of abrocitinib on cytokine expressions and inflammatory pathways in AD from three patients. This study included patients with moderate to severe AD treated with abrocitinib at Gunma University Hospital, Japan. All patients were systemic treatment-naïve. All patients received a 100-mg dose of abrocitinib daily, and used strong or very strong topical steroids and moisturizers. The Eczema Area and Severity Index (EASI) response analysis revealed that after 4 weeks, 25% (three of 12) of the cases reached a 75% reduction in the EASI score (EASI-75) and a 90% reduction in the EASI score (EASI-90). After 12 weeks, 83.3.% (10 of 12), 41.6% (five of 12), and 16.7% (two of 12) of the patients reached EASI-50, a 75% reduction in the EASI score (EASI-75), and EASI-90. Peak Pruritus Numerical Rating Scale was achieved in nine patients (75%) at week 12. The most frequent adverse reaction was acne (six cases [50%]). Gene Ontology pathway analysis using Differentially expressed genes from RNA sequencing analysis revealed attenuation of defense responses to biotic stimulus, virus, and cytokines. Th2 cytokine expression was not suppressed, but several chemokines, especially CXCL1, were suppressed by abrocitinib treatment. Our results indicate abrocitinib as a fast-acting and highly antipruritic agent that is effective for moderate skin eruptions.

Keratinocyte-Specific SOX2 Overexpression Suppressed Pressure Ulcer Formation after Cutaneous Ischemia-Reperfusion Injury via Enhancement of Amphiregulin Production.

Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing the transcription factor SOX2 promotes cutaneous wound healing through EGFR signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well-characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes (KCs) in cutaneous I/R injury. SOX2 was transiently expressed in KCs after cutaneous I/R injury. Ulcer formation was significantly suppressed in KC-specific SOX2-overexpressing mice. SOX2 in skin KCs significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokine expression were suppressed, and antioxidant stress factors and amphiregulin were elevated by SOX2 induction in skin KCs. Recombinant amphiregulin administration suppressed pressure ulcer development after cutaneous I/R injury in mice and suppressed oxidative stress-induced ROS production and apoptosis in vitro. These findings support that SOX2 in KCs might regulate cutaneous I/R injury through amphiregulin production, resulting in oxidative stress suppression. Recombinant amphiregulin can be a potential therapeutic agent for cutaneous I/R injury.

A case of rapidly progressive, fatal mycosis fungoides presenting as a haematoma-like lesion.

Mycosis fungoides usually follows an indolent clinical course. We report here a rapidly progressive case of mycosis fungoides with peculiar clinical and histological features, presenting as a haematoma-like mass on the thigh accompanied by multiple reddish brown erythematous lesions on the trunk and extremities. Histopathologically, the erythematous lesions showed epidermotropism of atypical T lymphocytes expressing CD4 and prominent syringotropism without syringometaplasia. The haematoma-like lesion consisted of diffuse and dense infiltrates of medium-to-large-sized pleomorphic lymphocytic cells expressing CD30, suggesting that CD30+ large-cell transformation had occurred. Mycosis fungoides presenting as a haematoma-like lesion is rare and may be a poor prognostic sign.

Real-world effectiveness and safety of bosentan in Japanese patients with systemic sclerosis: A single-center retrospective study.

Patients with systemic sclerosis (SSc) develop various vascular disorders, including digital ulcers (DUs), which are sometimes intractable. Bosentan is a dual endothelin receptor antagonist expected to suppress the development of new DUs. The objective of this study was to analyze retrospectively Japanese SSc patients treated with bosentan and investigate its efficacy and safety. We analyzed 40 patients who visited our department from 2009 to 2022 and were treated with bosentan. Of the 25 patients who were able to continue bosentan, 64% (16 patients) were cured by 16 weeks . New DUs occurred in 5.9% (2/34) of patients and the number of new DUs per person was 0.1. Adverse events occurred in 45% (18/40), and hepatic dysfunction was occurred most frequently at 32.5% (13/40). In univariate analysis, hepatic dysfunction was significantly high in patients with low modified Rodnan total skin thickness score. Antimitochondria-antibody-positive patients were more likely to develop liver dysfunction. Hepatic dysfunction was improved without the reduction or discontinuation, dose reduction, discontinuation, or concomitant use of ursodeoxycholic acid. These results suggest that bosentan can be selected as an additional treatment for DU, which is difficult to treat with existing therapies, while carefully monitoring hepatic function.

Kaempferol therapy improved MC903 induced-atopic dermatitis in a mouse by suppressing TSLP, oxidative stress, and type 2 inflammation.

BACKGROUND: Atopic dermatitis is a common skin disease caused by genetic susceptibility, environmental factors, immune response, and skin barrier dysfunction. Kaempferol is a natural flavonoid widely found in tea, vegetables, and fruits and has been reported to have excellent anti-inflammation activity. However, the therapeutic effect of kaempferol on atopic dermatitis is unclear. OBJECTIVE: This study aimed to elucidate the effect of kaempferol on skin inflammation in atopic dermatitis. METHODS: The suppressive effect of kaempferol administration on skin inflammation was examined using MC903-induced atopic dermatitis-like skin inflammation mouse model. Quantification of skin dermatitis and transepidermal water loss was performed. A histopathological study was performed to examine thymic stromal lymphopoietin expression, cornified envelope proteins such as filaggrin, loricrin, and involucrin, and the numbers of infiltrating inflammatory cells, including lymphocytes, macrophages, and mast cells in the dermatitis area. The expressions of IL-4 and IL-13 were investigated by qPCR and flow cytometry analysis using skin tissues. The expression of HO-1 was investigated by western blot and qPCR. RESULTS: Kaempferol therapy significantly suppressed MC903-induced dermatitis, TEWL, TSLP, and HO-1 expression, and infiltration of inflammatory cells. Kaempferol therapy improved the decreased expressions of filaggrin, loricrin, and involucrin in MC903-induced dermatitis skin site. The expressions of IL-4, and IL-13 were partially decreased in kaempferol-treated mice. CONCLUSION: Kaempferol might improve MC903-induced dermatitis via suppression of type 2 inflammation and improvement of barrier dysfunction by inhibition of TSLP expression and oxidative stress. Kaempferol might have the potential to be a new treatment for atopic dermatitis.

Case report: further delineation of AEBP1-related Ehlers-Danlos Syndrome (classical-like EDS type 2) in an additional patient and comprehensive clinical and molecular review of the literature.

The Ehlers-Danlos Syndromes (EDS), a group of hereditary connective tissue disorders, were classified into 13 subtypes in the 2017 International Classification. Recently, a new subtype of EDS called classical-like EDS type 2 (clEDS2), which is caused by biallelic variants in the adipocyte enhancer binding protein 1 (AEBP1) gene, was identified. We describe the 11th patient (9th family) with clEDS2, who was complicated by a critical vascular event (superior mesenteric artery aneurysm and rupture). A next-generation sequencing panel-based analysis revealed compound heterozygous variants in AEBP1: NM_001129.5:c.[2296G>T]; [2383dup], p.[(Glu766*)]; [(Glu795Glyfs*3)]. Light microscopic analyses showed increased interfibrillar spaces in the reticular dermis, a disorganized arrangement of collagen fibers, and decreased collagen content. An electron microscopic analysis showed the presence of collagen fibrils with irregular contours (flower-like appearance) and small collagen fibrils. A biochemical analysis showed reduced secretion of type I and type III procollagen. Clinical and molecular features of the current patient and all previously reported patients were reviewed comprehensively. Manifestations noted in most cases (>80%) included skin features (hyperextensibility, atrophic scars, easy bruising, excessive skin/skin folding, delayed wound healing, translucency, piezogenic papules), skeletal features (generalized joint hypermobility, dislocations/subluxations, pes planus), dental abnormalities, and neuromuscular abnormalities. Critical complications, each occurring in a single case, included superior mesenteric artery multiple aneurysm and rupture, aortic root dilation requiring surgery, and bowel rupture. Most AEBP1 variants were predicted or experimentally confirmed to lead to nonsense-mediated mRNA decay, whereas one variant resulted in a protein that was retained intracellularly and not secreted. Clinical, molecular, pathological, and biochemical features of the current patient, as well as a review of all previously reported patients, suggest the importance of the aortic carboxypeptidase-like protein encoded by AEBP1 in collagen fibrillogenesis.

TRPV4 Regulates the Development of Psoriasis by Controlling Adenosine Triphosphate Expression in Keratinocytes and the Neuroimmune System.

TRPV4 is a calcium ion channel that is widely expressed in various cells. It is also involved in physiological and pathological processes. However, the role of TRPV4 in psoriasis remains unknown. We aimed to investigate the role of TRPV4 in psoriasis using human psoriasis skin samples and an imiquimod-induced psoriasis-like mouse model. Keratinocytes in human psoriasis skin had high TRPV4 expression. Trpv4-knockout mice had less severe dermatitis than wild-type mice in the imiquimod-induced mouse model. Knockout mice had significantly reduced epidermal thickness and a low number of infiltrated CD3+ T cells and CD68+ macrophages on the basis of histopathological studies and decreased mRNA expression of Il17a, Il17f, and Il23, as detected through qPCR. Furthermore, knockout mice had a significantly low expression of neuropeptides and the neuron marker PGP9.5. Adenosine triphosphate release was significantly suppressed by TRPV4 knockdown in both human and mouse keratinocytes in vitro. Finally, treatment with TRPV4 antagonist was significantly effective in preventing the progression of psoriasis-like dermatitis. In conclusion, TRPV4 mediates the expression of keratinocyte-derived adenosine triphosphate and increases the secretion of neuropeptides, resulting in the activation and amplification of IL-23/Th17 responses. Hence, TRPV4 can serve as a novel therapeutic target in psoriasis.

Transient receptor potential vanilloid 4 promotes cutaneous wound healing by regulating keratinocytes and fibroblasts migration and collagen production in fibroblasts in a mouse model.

BACKGROUND: Transient receptor potential vanilloid 4 (TRPV4), a cation ion channel, is expressed in different cells, and it regulates the development of different diseases. We recently found a high TRPV4 expression in the wounded skin area. However, the role of TRPV4 in cutaneous wound healing is unknown. OBJECTIVE: To investigate the role of TRPV4 in cutaneous wound healing in a mouse model. METHODS: Skin wound healing experiment and histopathological studies were performed between WT and TRPV4 KO mice. The effect of TRPV4 antagonist and agonist on cell migration, proliferation, and differentiation were examined in vitro. RESULTS: TRPV4 expression was enhanced in wounded area in the skin. TRPV4 KO mice had impaired cutaneous wound healing compared with the WT mice. Further, they had significantly suppressed re-epithelialization and formation of granulation tissue, amount of collagen deposition, and number of α-SMA-positive myofibroblasts in skin wounds. qPCR revealed that the KO mice had decreased mRNA expression of COL1A1 and ACTA2 in skin wounds. In vitro, treatment with selective TRPV4 antagonist suppressed migrating capacity, scratch stimulation enhanced the expression of phospho-ERK in keratinocytes, and TGF-ß stimulation enhanced the mRNA expression of COL1A1 and ACTA2 in fibroblasts. Selective TRPV4 agonist suppressed cell migration in keratinocytes, and did not enhance proliferation and migration, but promoted differentiation in fibroblasts. CONCLUSION: TRPV4 mediates keratinocytes and fibroblasts migration and increases collagen deposition in the wound area, thereby promoting cutaneous wound healing.

Association of microRNA-21 expression with its targets, PDCD4 and TIMP3, in pancreatic ductal adenocarcinoma.

Since the discovery of small non-coding RNAs, the analyses of microRNA (miRNA) expression patterns in human cancer have provided new insights into cancer biology. miRNA-21 has been suggested to be one of the miRNAs that have an important role in the development or biological behavior of a variety of malignancies, including pancreatic cancer. This study was conducted to evaluate the relationship between the expression of miRNA-21 and that of its molecular targets, programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinase (TIMP3), in pancreatic ductal adenocarcinoma. The study included 65 pancreatic ductal adenocarcinomas and 5 normal pancreatic tissue specimens for comparison. The miRNA expression profiling of five selected pancreatic ductal adenocarcinomas and five normal pancreatic specimens was performed using a microarray platform, and was evaluated by a hierarchical clustering analysis. The miRNA most highly expressed in pancreatic ductal adenocarcinomas (ie, miRNA-21) was further assessed by quantitative real-time reverse transcription PCR (RT-PCR) assays in the 65 pancreatic ductal adenocarcinoma cases. The expression pattern of its molecular targets (eg, PDCD4 and TIMP3) in pancreatic ductal adenocarcinoma was examined immunohistochemically. In the microarray analyses, 28 miRNAs were upregulated in pancreatic ductal adenocarcinoma compared with normal pancreatic tissue, whereas 48 miRNAs were downregulated. miRNA-21 was the most significantly overexpressed miRNA in the pancreatic ductal adenocarcinomas analyzed, and was also highly expressed in 75% of the 65 pancreatic ductal adenocarcinomas examined by real-time RT-PCR. High miRNA-21 expression was correlated with a worse prognosis in the pancreatic ductal adenocarcinoma patients (P=0.045). The immunohistochemical expression patterns of PDCD4 (reduced nuclear staining pattern) and TIMP3 (downregulated expression) were significantly associated with both the upregulated miR-21 expression (P<0.05) and the poor survival of the patients (P<0.001 and P=0.001, respectively). Our data suggest that an overexpression of miRNA-21 is, therefore, associated with the biological behavior of pancreatic ductal adenocarcinoma via the downregulation of the expression of tumor suppressors, PDCD4 and TIMP3, thus resulting in tumor progression and the adverse clinical course of pancreatic ductal adenocarcinoma.