Mechanism of miRNA-based Aconitum leucostomum Worosch. Monomer inhibition of bone marrow-derived dendritic cell maturation.

Delvestidine (DLTD) is a monomeric compound isolated from Aconitum leucostomum Worosch, a widely used medicine for local treatment of rheumatoid arthritis (RA). Studies have shown that Aconitum leucostomum Worosch. can inhibit maturation of bone marrow-derived dendritic cells (BMDCs). Further, microRNAs (miRNAs) have regulatory effects on DC maturity and function. However, the mechanism underlying DLTD effects on DC maturity and RA remains to be elucidated. This study investigated whether DLTD-mediated inhibition of DC maturation is regulated by miRNAs. LPS-induced mature BMDCs were treated with DLTD for 48 h. CD80 and CD86 expression on BMDCs was detected by flow cytometry, and levels of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α were detected by ELISA and PCR. Further, gene expression and miRNA expression profiles were investigated by bioinformatics analysis and verified by PCR. DLTD was found to inhibit CD80 and CD86 expression on the surface of BMDCs and secretion of inflammatory factors IL-6, IL-23, IL-1ß, and TNF-α. In total, 54 differentially expressed miRNAs were detected, including 29 up-regulated and 25 down-regulated miRNAs after DLTD treatment. Analysis of biological information revealed that the differentially expressed target genes mainly regulated biological processes, including cell differentiation, cell cycle, and protein kinase complexes. Additionally, miR-511-3p downstream targets Calcr, Fzd10, and Eps8, were closely related to BMDCs maturation. DLTD may induce BMDCs maturity through regulation of miRNAs that affect Calcr, Fzd10, and Eps8 gene signals.

miR-429 is involved in regulation of NF-κBactivity by targeting IKKß and suppresses oncogenic activity in cervical cancer cells.

Dysregulation of microRNAs (miRNAs) can contribute to tumorigenesis in cancers. In this study, we found that miR-429 was downregulated in cervical cancer (CC) tissues and suppressed cell viability and proliferation while promoting apoptosis in CC cells. IKKß was a novel target gene of miR-429 and ectopic expression of IKKß abrogated the phenotypes induced by miR-429. When IKKß was downregulated by miR-429, nuclear factor κB (NF-κB) pathway activation, interleukin-6 (IL-6), and interferon-ß (IFN-ß) production were decreased in CC cells. These findings indicate that miR-429 is involved in regulation of the NF-κB pathway by targeting IKKß and functions as a tumor suppressor in cervical carcinogenesis.

miR-23a promotes IKKα expression but suppresses ST7L expression to contribute to the malignancy of epithelial ovarian cancer cells.

BACKGROUND: Dysregulation of microRNAs (miRNAs) has been found in human epithelial ovarian cancer (EOC). However, the role and mechanism of action of miR-23a in EOC remain unclear. METHODS: The roles of miR-23a, IKKα, and ST7L in EOC were determined by MTT, colony formation, wounding healing, transwell, flow cytometry, immunofluorescence, RT-qPCR, and western blotting experiments. miR-23a target genes were validated by EGFP reporter assays, RT-qPCR, and western blotting analysis. RESULTS: miR-23a is upregulated and promotes tumorigenic activity by facilitating the progress of cell cycle and EMT and repressing apoptosis in EOC cells. miR-23a enhances the expression of IKKα but suppresses the expression of ST7L by binding the 3'UTR of each transcript in EOC cells. The proliferation, migration, and invasion of EOC cells are increased by IKKα and inhibited by ST7L. Furthermore, miR-23a activates NF-κB by upregulating IKKα and WNT/MAPK pathway by downregulating ST7L. CONCLUSIONS: miR-23a functions as an oncogene by targeting IKKα and ST7L, thus contributing to the malignancy of EOC cells.

Development and clinical evaluation of a new gold-immunochromatographic assay for the detection of antibodies against field strains of pseudorabies virus.

An immunochromatographic strip (ICS) was developed for the detection of swine antibodies against glycoprotein E (gE) in Pseudorabies Virus (PRV). In this test, Staphylococcal Protein A (SPA) labeled with colloidal gold was dispensed on a conjugate pad as the detector. Purified PRV-gE and pig-IgG were blotted on a nitrocellulose membrane for the test (T) and control lines (C), respectively. If the tested serum contains IgG antibodies against PRV-gE, the IgG will interact with the colloidal gold-SPA to form a complex (gold-SPA-swine IgG). The complex will react with the immobilized PRV-gE on the T line and the Pig-IgG in the C line of the ICS to form two visible red bands. If there is no IgG antibody against PRV-gE in the sample serum, only the C line will be visible. The ICS was capable of specifically detecting PRV-gE antibody within 5 min, and its stability and reproducibility were quite good after storage at 4°C and use within 4 months. Using an IDEXX Pseudorabies Virus gE Antibody Test Kit (IDEXX PRV gE Ab test) as a reference, the relative specificity and sensitivity of the ICS were determined to be 81.6% and 90.7%, respectively. Furthermore, there was a good agreement between the results obtained by the commercial product and the ICS (kappa=0.7289).

DNA methylation-mediated repression of miR-941 enhances lysine (K)-specific demethylase 6B expression in hepatoma cells.

MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. However, their underlying mechanisms of action in hepatocellular carcinoma (HCC) are poorly understood. Recent evidence suggests that epigenetic silencing of miRNAs through tumor suppression by CpG island hypermethylation may be a common hallmark of human tumors. Here, we demonstrated that miR-941 was significantly down-regulated in HCC tissues and cell lines and was generally hypermethylated in HCC. The overexpression of miR-941 suppressed in vitro cell proliferation, migration, and invasion and inhibited the metastasis of HCC cells in vivo. Furthermore, the histone demethylase KDM6B (lysine (K)-specific demethylase 6B) was identified as a direct target of miR-941 and was negatively regulated by miR-941. The ectopic expression of KDM6B abrogated the phenotypic changes induced by miR-941 in HCC cells. We demonstrated that miR-941 and KDM6B regulated the epithelial-mesenchymal transition process and affected cell migratory/invasive properties.

Efficacy and safety of combination therapy with vildagliptin and metformin versus metformin up-titration in Chinese patients with type 2 diabetes mellitus: study design and rationale of the vision study.

BACKGROUND AND AIM: Limitations of the currently recommended stepwise treatment pathway for type 2 diabetes mellitus (T2DM), especially the failure of monotherapies to maintain good glycemic control, have prompted use of early, more aggressive combination therapies.The VISION study is designed to explore the efficacy and safety of vildagliptin as an add-on to metformin therapy compared with up-titration of metformin monotherapy in Chinese patients with T2DM. METHODS: VISION, a 24-week, phase 4, prospective, randomized, multicenter, open-label, parallel-group study, will include 3312 Chinese T2DM patients aged ≥18 years who are inadequately controlled (6.5% >HbA1c ≤9%) by metformin (750-1000 mg/day). Eligible patients will be randomized to receive either vildagliptin plus metformin or up-titration of metformin monotherapy (5:1). Patients will also be subgrouped (1:1:1:1) based on their age and body mass index (BMI): <60 years and <24 kg/m²; <60 years and ≥24 kg/m²; ≥60 years and <24 kg/m²; and ≥60 years and ≥24 kg/m². CONCLUSION: The VISION study will test the hypothesis that early use of combination therapy with vildagliptin and metformin will provide good glycemic control and will be better tolerated than up-titration of metformin monotherapy. The study will also correlate these benefits with age and BMI.

Separation and identification of differentially expressed nuclear matrix proteins in breast carcinoma forming.

BACKGROUND: Breast carcinoma is one of most prevalent malignant tumors occurring in women. Short of prevention, detection of breast carcinoma at an early, still curable stage would offer the best route to decrease its mortality rates. This highlights the urgent need for suitable biomarkers for early diagnosis and a better understanding of the disease pathogenesis. MATERIAL AND METHODS: NMPs were extracted from normal human breast tissue (Group I), from hyperplastic mammary tissue specimens (Group II), from atypical epithelial hyperplasia specimens (Group III), and from breast carcinoma (Group IV) tissue. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The different NMPs were analyzed in the National Center for Biotechnology Information (NCBI) database with Mascot software. RESULTS: Well-resolved, reproducible 2-DE profiles of human breast tissues were obtained. Average protein spots were 904 +/- 58, 912 +/- 51, 931 +/- 63, 944 +/- 70 in Group I, Group II, Group III, and Group IV, respectively. Several different proteins were analyzed using mass spectrometry and bioinformation. Of these, 12 were well characterized. Compared to Group I, three proteins were up-regulated in Groups II, III, and IV, including Hsp27, prohibitin, and laminA/C. Upregulation was confirmed using Western blotting and immunohistochemical analysis. The correlation of prohibitin expression with clinicopathological features was also investigated. DISCUSSION: The proteins identified in this study may potentially prove to be useful markers for breast carcinoma diagnosis.

Effects of fish CYP inducers on difloxacin N-demethylation in kidney cell of Chinese idle (Ctenopharyngodon idellus).

A drug-drug interaction occurs when the effect of one drug is altered by the presence of another drug which is generally associated with the induction of cytochrome P450s (CYPs) activity. Thus, unexpected treatment failures often happen resulting from inappropriate coadministration in fisheries. However, little information is available about CYP induction in fish. The reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR) belongs to N-demethylation catalyzed mainly by CYP(s). In order to supply useful information on CYP induction, the present study assessed the effects of fish-specific CYP inducers on DIF N-demethylation and enzyme kinetics in kidney cell of Chinese idle (CIK; grass carp (Ctenopharyngodon idellus)) by RP-HPLC. Results demonstrated that the amounts of SAR formation and enzymatic parameters Clint and Vmax were significantly increased due to beta-naphthoflavone (BNF) pretreatment. Therefore, we suggest that CYP1A may be involved in DIF N-demethylation in CIK. This study provides instructive information to ensure treatment success via avoiding CYP induction in fisheries.

The correlations between HPV16 infection and expressions of c-erbB-2 and bcl-2 in breast carcinoma.

Breast carcinoma (BC) is a prevalent malignant tumour occurring in women. Many studies have indicated the role of human papilloma virus type 16 (HPV16) in the pathogenesis of BC; however, the correlations of HPV16 infection with the clinicopathologic features of BC and the expressions of c-erbB-2 and bcl-2 have not yet been elucidated. In this study, HPV16 was detected by amplifying the HPV16 E6 gene by the polymerase chain reaction method, and the expressions of c-erbB-2 and bcl-2 in 40 BCs and 20 normal breast tissue samples, obtained from Shaanxi Province, were examined using the streptavidin-peroxidase method with monoclonal antibodies specific to c-erbB-2 and bcl-2. The infection rate of HPV16 E6 and the positive expression rate of c-erbB-2 were significantly higher in the BCs than in the normal tissues (HPV16 E6: 60% vs. 5%; c-erbB-2: 42.5% vs. 5%, P < 0.05). However, the positive expression rate of bcl-2 was significantly lower in the BCs than in the normal tissues (67.5% vs. 95%, P < 0.05). The infection rate of HPV16 did not correlate with any of the pathological features observed (P > 0.05). HPV16 infection correlated with bcl-2 expression (P = 0.015) but not with c-erbB-2 expression (P = 0.747) in the BCs. Interestingly, HPV16 infection correlated with bcl-2 expression in grade I BCs (P = 0.018) but not in grade II-III BCs (P = 0.633). Our data suggest that HPV16 infection is correlated with bcl-2 expression in BCs.

Renal involvement and followup of 130 patients with primary Sjögren's syndrome.

OBJECTIVE: To identify the clinical characteristics, pathological changes, and outcome of patients with primary Sjögren's syndrome (pSS). METHODS: All patients with pSS and renal involvement who were admitted to Ruijin Hospital from April 1993 to December 2006 were included. All the data of clinical features and pathological changes were retrospectively analyzed. Forty-one patients underwent renal biopsies. RESULTS Our study included 130 patients with pSS: 122 women and 8 men. Ages ranged from 16 to 68 years (mean 44.1 +/- 11.52). Ninety-five patients (73.1%) developed renal tubular acidosis (RTA); 91 were found to have distal RTA. Nine patients presented with hypokalemic paralysis. Four patients developed Fanconi syndrome and 3 were proved to have nephrogenic diabetes insipidus. Twenty-seven of 130 patients (20.8%) developed tubular proteinuria and 18/130 (13.8%) presented glomerular involvement. Thirty-five patients (27.7%) developed renal failure (serum creatinine > 115 micromol/l). Most patients (70.8%) had increased serum IgG levels. The incidence of chronic interstitial nephritis was 80.5% among all the biopsy materials. Immunofluorescent staining was negative in most renal tissue. Ninety-six patients were treated with corticosteroids and/or immunosuppressant. Eighteen recovered renal function. CONCLUSION: Patients with pSS commonly present with renal impairment, mainly from renal tubular dysfunction. The combination of corticosteroids and immunosuppressors significantly improves the renal function of patients with pSS. There is a correlation between hypergammaglobulinemia and distal RTA. The renal acidification capacity for patients with hypergammaglobulinemia. should be monitored.

Propylthiouracil-associated antineutrophil cytoplasmic autoantibody-positive vasculitis: retrospective study of 19 cases.

OBJECTIVE: To investigate the features, followup data, and outcomes of patients with propylthiouracil (PTU)-associated antineutrophil cytoplasmic autoantibody (ANCA)-positive vasculitis. METHODS: Nineteen patients with PTU-associated ANCA-positive vasculitis diagnosed in our hospital from 2000 to 2006 were analyzed retrospectively. RESULTS: Our data showed a female predominance among the patients. Eleven patients had involvement of more than one organ. Renal involvement was the most common manifestation. Fourteen patients underwent renal biopsy. Four patients had focal proliferative glomerulonephritis with crescent formation. Two had necrotizing glomerulonephritis with crescent formation. Two patients had minor glomerular abnormalities, 2 had IgA nephropathy, one had membranous nephropathy, one had focal proliferative glomerulonephritis, one had granulomatous interstitial nephritis, and the remaining one had focal segmental glomerular sclerosis. Immune complex glomerulonephritis was found in 3 patients. On indirect immunofluorescence, 17 patients were perinuclear-pattern ANCA-positive, one was positive for atypical ANCA, and one was positive for cytoplasmic-pattern-ANCA. By ELISA, 4 patients were positive for both myeloperoxidase (MPO)-ANCA and proteinase-3 (PR3)-ANCA, one was positive for PR3-ANCA only, and the others were positive for MPO-ANCA only. For the treatment of vasculitis, 5 patients received prednisone alone, 10 received prednisone and cyclophosphamide, and the remaining 4 did not receive prednisone or cyclophosphamide. During followup, 15 patients achieved remission, 3 patients died, and one patient depended on dialysis. In general, MPO-ANCA concentration did not correlate with disease progression, and a delayed decrease of MPO-ANCA concentration was found in most patients who achieved remission. CONCLUSION: Most patients with PTU-associated ANCA-positive vasculitis had good outcomes; however, severe cases existed. We suggest early recognition and adequate treatment are necessary to improve outcome.

[Evaluation of IL-6 level in serum and bronchoalveolar lavage fluid of patients with non-small cell lung cancer (NSCLC)].

AIM: To study the relationship between the interleukin-6(IL-6) level in serum and broncheoalveolar lavage fluid(BALF) and non-small cell lung cancer(NSCLC). METHODS: The concentration of IL-6 in serum or BALF of 62 NSCLC patients, 36 patients of pulmonary inflammatory diseases (PID) and 25 normal controls were measured by ELISA. RESULTS: (1)The concentration of IL-6 in BALF of NSCLC patients was significantly higher than that of PID patients and normal controls (P<0.01). The concentration of IL-6 in sera of NSCLC patients was higher than that of normal controls (P<0.01) but not statistically different from that of PID patients. (2)In patients with NSCLC, the concentration of IL-6 in BALF was slightly higher than that of serum IL-6, but this difference was not statistically significant (P>0.05). (3)Serum IL-6 level was higher in NSCLC of advanced stages (stage IIIa, IIIb and IV), as compared with that in NSCLC patients of early stages (stage Ia-IIb). BALF IL-6 level of NSCLC patients at different stages was not different. (4)IL-6 (level in serum but not in BALF) of PID patients correlated with the serum concentration of C reactive protein (r=0.74). CONCLUSION: IL-6 in BALF might be used as a marker for NSCLC. Serum IL-6 level might be an indicator for the stage of NSCLC. The serum IL-6 can also reflect the severity of acute lung inflammatory response.

Study on the relationship between level of CD58 expression in peripheral blood mononuclear cell and severity of HBV infection.

BACKGROUND: As one of the intercellular adhesion molecules, CD58 plays important roles in promotion of the adhesion between T cells and target cells, hyperplasia, activation of T cells and natural killer cells, and balance between Th1 and Th2. We studied the relationship between the levels of CD58 expression in peripheral blood mononuclear cells (PBMCs) and severity of HBV infection. METHODS: The levels of CD58 mRNA in PBMCs were detected using quantitative reverse transcription PCR. The percentage of CD58 positive cells was detected by flow cytometry in patients and healthy controls. RESULTS: The levels of CD58 mRNA and the percentage of CD58 positive cells in patients infected with HBV were significantly higher than that in the control. Based on severity of HBV infection, the patients were classified into four groups. The expression of CD58 increased significantly in an order from mild chronic, moderate chronic, severe chronic to severe hepatitis groups. The levels of CD58 mRNA and the percentage of CD58 positive cells in PBMCs from patients with HBV infection were both positively correlated with serum levels of ALT and AST. CONCLUSION: The level of CD58 expression is related with the severity of HBV infection and the degree of liver damage.

[Construction of sense and antisense eukaryotic expression vector of novel gene Collectrin and its function in cell growth].

OBJECTIVE: To construct sense and antisense eukaryotic expression vector of novel gene Collectrin and identify its function in cell growth. METHODS: The open reading frame of Collectrin was amplified by PCR and inserted into pcDNA3.1/V5-His plasmid. The recombinant plasmid was identified by restriction enzyme analysis and sequencing analysis. The recombinant plasmid was transfected into M-1 cell by using lipofectin mediation after being identified by restriction enzyme analysis and sequencing analysis. RT-PCR and Western blot were performed to identify the expression of Collectrin. Beta-Gal staining was used to define the effect of transfection. The growth of M-1 cells was examined by MTT and cell counting. RESULTS: Compared with control group, the expression of Collectrin was decreased significantly at both nucleotide and protein levels transfected by antisense vector, but elevated in sense group. The cell growth was blocked after being transfected by antisense plasmid. CONCLUSION: The sense and antisense eukaryotic expression vector of novel gene Collectrin was successfully constructed. Collectrin was one of basic factors in cell growth.