MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

mRNA expression of the DNA replication-initiation proteins in epithelial dysplasia and squamous cell carcinoma of the tongue.

BACKGROUND: The tongue squamous cell carcinomas (SCCs) are characterized by high mitotic activity, and early detection is desirable. Overexpression of the DNA replication-initiation proteins has been associated with dysplasia and malignancy. Our aim was to determine whether these proteins are useful biomarkers for assessing the development of tongue SCC. METHODS: We analyzed the mRNA expression of CDC6, CDT1, MCM2 and CDC45 in formalin-fixed, paraffin-embedded benign and malignant tongue tissues using quantitative real-time PCR followed by statistical analysis. RESULTS: We found that the expression levels are significantly higher in malignant SCC than mild precancerous epithelial dysplasia, and the expression levels in general increase with increasing grade of precancerous lesions from mild, moderate to severe epithelial dysplasia. CDC6 and CDC45 expression is dependent of the dysplasia grade and lymph node status. CDT1 expression is higher in severe dysplasia than in mild and moderate dysplasia. MCM2 expression is dependent of the dysplasia grade, lymph node status and clinical stage. The expression of the four genes is independent of tumor size or histological grade. A simple linear regression analysis revealed a linear increase in the mRNA levels of the four genes from the mild to severe dysplasia and SCC. A strong association was established between CDC6 and CDT1, and between MCM2 and CDC45 expression. The nonparametric receiver operating characteristic analysis suggested that MCM2 and CDC45 had a higher accuracy than CDC6 and CDT1 for distinguishing dysplasia from tongue SCC. CONCLUSION: These proteins can be used as biomarkers to distinguish precancerous dysplasia from SCC and are useful for early detection and diagnosis of SCC as an adjunct to clinicopathological parameters.

Expression of Mcm7 and Cdc6 in oral squamous cell carcinoma and precancerous lesions.

BACKGROUND: The present study aimed at evaluating the clinical importance of Mcm7 and Cdc6 expression in oral squamous cell carcinoma (OSCC) and precancerous lesions. MATERIALS AND METHODS: RT-PCR and immunohistochemistry analysis were performed on 47 frozen samples and 98 paraffin-embedded samples to evaluate the mRNA and protein expressions of Mcm7 and Cdc6. RESULTS: RT-PCR and immunohistochemistry indicated positive expressions of Mcm7 mRNA and protein in normal oral mucosa, precancerous lesions and OSCC. Significant differences were found between all the groups. Cdc6 mRNA and protein had low expressions in normal oral mucosa but were highly expressed in precancerous lesions and OSCC. Mcm7 and Cdc6 expressions in the lymph node metastasis cases were significantly higher than those of the nonmetastatic carcinomas. CONCLUSION: High expressions of Mcm7 and Cdc6 are correlated with the development and metastasis of OSCC and may become a molecular marker for the early diagnosis and prognosis prediction for OSCC.

Experimental study on evaluation and optimization of tilt angle of parallel-plate electrodes using electrocoagulation device for oily water demulsification.

Tilt angle of parallel-plate electrodes (APE) is very important as it improves the economy of diffusion controlled Electrocoagulation (EC) processes. This study aimed to evaluate and optimize APE of a self-made EC device including integrally rotary electrodes, at a fixed current density of 120 Am-2. The APEs investigated in this study were selected at 0°, 30°, 45°, 60°, 90°, and a special value (α(d)) which was defined as a special orientation of electrode when the upper end of anode and the lower end of cathode is in a line vertical to the bottom of reactor. Experiments were conducted to determine the optimum APE for demulsification process using four evaluation indexes, as: oil removal efficiency in the center between electrodes; energy consumption and Al consumption, and besides, a novel universal evaluation index named as evenness index of oil removal efficiency employed to fully reflect distribution characteristics of demulsification efficiency. At a given plate spacing of 4 cm, the optimal APE was found to be α(d) because of its potential of enhancing the mass transfer process within whole EC reactor without addition, external mechanical stirring energy, and finally the four evaluation indexed are 97.07%, 0.11 g Al g-1 oil, 2.99 kwhkg-1 oil, 99.97% and 99.97%, respectively.

[Prokaryotic expression of SARS coronavirus spike protein and construction of its DNA vaccine].

OBJECTIVE: To study the immunological characteristics of the spike (S) protein of SARS coronavirus (SARS-CoV) and analyze the feasibility of using this protein as the component for SARS vaccine development. METHODS: The two truncated fragments of S gene were separately cloned into the prokaryotic expression vector pET-15b and expressed in E.coli. The resulting recombinant proteins, rS(a) and rS(b), were purified by affinity chromatography. The full-length S gene was cloned into the eukaryotic expression plasmid pSecTagB to prepare recombinant plasmid pSecS as the DNA vaccine to immunize BALB/c mice for inducing the secretion of anti-SARS-CoV protein. The immunological effect of anti-SARS-CoV antibody was tested with purified rS(a) and rS(b) proteins by enzyme-linked immunosorbent assay (ELISA). RESULTS: Both the truncated recombinant proteins were expressed in soluble forms and reacted specifically with the sera from immunized pSecS mice and clinically diagnosed SARS patients. The prokaryotically expressed recombinant truncated S protein had similar antigenicity with SARS-CoV S protein. CONCLUSION: The recombinant protein could be used as an antigen for detecting the serum of SARS CoV-infected patients. The SARS-CoV S gene vaccine could induce the production of specific antibody, which offers clues for the research of SARS DNA vaccine.

Enhanced peroxisomal ß-oxidation metabolism in visceral adipose tissues of high-fat diet-fed obesity-resistant C57BL/6 mice.

This study aimed to investigate the potential mechanisms of natural resistance to high-fat diet-induced obesity. Four-week-old C57BL/6 mice were fed a high-fat diet for 6 weeks and were then designated as high-fat diet-fed obesity-prone (HOP) and obesity-resistant (HOR) animals. Their blood biochemistry was evaluated, and visceral adipose tissue samples were subjected to proteomic, Western blot and quantitative real-time PCR (q-PCR) analyses. The HOR mice showed reduced visceral fat weight and size, as well as lowered serum lipid and leptin levels. Proteomic analysis showed that enoyl coenzyme A hydratase 1, peroxisomal (Ech1) expression was significantly increased in their visceral adipose tissues. Moreover, other proteins, such as α-tropomyosin, myosin light chain, urine-nucleoside phosphorylase and transgelin, were also significantly increased. Furthermore, q-PCR analysis showed that the expression of acyl-CoA oxidase 1 palmitoyl, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase and 3-oxoacyl-CoA thiolase responsible for peroxisomal ß-oxidation was also up-regulated in the visceral adipose tissues of the HOR mice. The expression of peroxisome proliferator-activated receptor α (PPARα) was increased in the HOR mice as shown by Western blot analysis. Obesity-resistant animals show enhanced peroxisomal ß-oxidation metabolism and reduced fat accumulation in visceral adipose tissues by up-regulating the expression of Ech1, peroxisomal or other related peroxisomal ß-oxidation marker genes, which may be driven or enhanced by the up-regulation of the expression of PPARα. However, further validation in future studies is required.

miRNA-mediated functional changes through co-regulating function related genes.

BACKGROUND: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation. RESULTS: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1. CONCLUSIONS: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.