MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Animals ; Antigens, Polyomavirus Transforming/blood ; Case-Control Studies ; Down-Regulation ; Female ; Humans ; Liver Cirrhosis/*blood/diagnosis ; Male ; Mice ; Mice, Inbred C57BL ; Middle Aged ; RNA, Small Nuclear/*blood ; Reference Values ; Sepsis/*blood/diagnosis

BACKGROUND: Circulating cell-free nucleic acids are known to be a noninvasive diagnostic tool for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, a nuclear core complex, is overexpressed in early stage lung cancer. We intended to evaluate the usefulness of plasma hnRNP B1 mRNA in differentiating non-small cell lung cancer (NSCLC) from other benign lung diseases, especially pulmonary tuberculosis, which is highly prevalent in Korea and often difficult to distinguish from lung cancer. METHODS: Plasma RNA was extracted from 30 patients with NSCLC, 30 patients with benign lung diseases including pulmonary tuberculosis, and 10 healthy controls. Plasma hnRNP B1 mRNA was measured by TaqMan Gene Expression Assay (Applied Biosystems, USA), and pre-developed beta-actin (ACTB) mRNA was used for normalization. We analyzed the relative gene expression data using the delta-delta Ct method. RESULTS: Plasma hnRPN B1 mRNA was measurable in 93.3% (28/30) of NSCLC patients. Normalized 2-DeltaDeltaCt of plasma hnRPN B1 mRNA was 62.2 (95%Cl, 6.4-210.1) in NSCLC patients and 2.7 (95%Cl, 0.5-13.6) in benign lung disease patients (P<0.001, Mann-Whitney U test). CONCLUSIONS: Plasma hnRNP B1 mRNA was significantly increased in patients with lung cancer compared with that in patients with other benign lung diseases. Plasma hnRNP B1 mRNA may be useful as a potential marker for the detection of NSCLC.
Adult ; Aged ; Carcinoma, Non-Small-Cell Lung/*genetics ; Female ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/*blood/genetics ; Humans ; Lung Diseases/blood/genetics ; Lung Neoplasms/*genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; RNA, Messenger/*blood/metabolism ; Tumor Markers, Biological

OBJECTIVE: To investigate the effect of pSUPER/CD147siRNA on the formation, proliferation, and angiogenesis of malignant melanoma in nude mice. METHODS: The nude mouse subcutaneous xenotransplantation models of malignant melanoma were constructed and observed using morphological analysis. The protein expression of PCNA and CD31 in tumors was detected using immunohistochemical technique. Tumoral proliferative activity in the nude mice was quantitatively evaluated by proliferation cell nuclear antigen (PCNA). Microvessel density( MVD) was counted based on the endothelial cells positively stained with anti-CD31 antibody. RESULTS: The subcutaneous tumors appeared in all the nude mice 5 days later after the transplantation, but the volume of malignant melanoma in the experiment group significantly decreased compared with the control group (P<0.01). PCNA expression and MVD were significantly lower in the experiment group than those in the control group ( P<0.01). CONCLUSION CD147 siRNA inhibits the growth and angiogenesis of malignant melanoma in vivo, suggesting that CD147 might be a new gene therapy target molecule for malignant melanoma.
Animals ; Basigin ; genetics ; Cell Line, Tumor ; Humans ; Melanoma ; blood supply ; genetics ; pathology ; Mice ; Mice, Nude ; Microvessels ; Neovascularization, Pathologic ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Xenograft Model Antitumor Assays