Long noncoding RNA MIRG induces osteoclastogenesis and bone resorption in osteoporosis through negative regulation of miR-1897.

OBJECTIVE: To investigate the expression of long noncoding RNA (LncRNA) MIRG and its potential functions in regulating osteoclastogenesis and bone resorption function through modulating miR-1897 in bone marrow macrophages (BMMs). MATERIALS AND METHODS: qRT-PCR was performed to detect the expressions of MIRG and its co-expression mRNA NFATc1 at different stages during osteoclastogenesis. The CCK-8 assay was performed to evaluate cell proliferation and differentiation. The correlation between miR-1897 and MIRG was detected by statistical analysis. Bioinformatics and luciferase assay were performed to explore the interaction and binding site of MIRG and miR-1897. We also cloned the mice NFATc1 3'-UTR into the luciferase reporter vector and constructed miR-1897 binding mutants to validate the inhibited regulation of miR-1897 to the expression of NFATc1. RESULTS: Results showed that expressions of MIRG and NFATc1 were upregulated during osteoclastogenesis. qRT-PCR and CCK-8 assay showed that MIRG expression is associated with osteoclastogenesis and bone resorption. The bioinformatics prediction and luciferase assay suggested that by interacting with miR-1897, MIRG acts as a molecular sponge for the miR-1897 target NFATc1, to partly modulate the inhibitory effect of miR-1897 on NFATc1. CONCLUSIONS: We found that lncRNA-MIRG was upregulated in osteoclasts, which could promote osteoclastogenesis and bone resorption function as a molecular sponge by modulating the inhibitory effect of miR-1897 on NFATc1.

Down-expression of miR-373 predicts poor prognosis of glioma and could be a potential therapeutic target.

OBJECTIVE: MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including glioma. The main objective of this work was to investigate the expression level of miR-373 and its clinical significance in glioma. PATIENTS AND METHODS: The expression levels of miR-373 in glioma tissues and non-neoplastic brain tissues were measured by the qRT-PCR assay. Patients were divided into two groups based on the median miR-373 expression. The probability of differences in overall and progression-free survival as a function of time was ascertained by use of the Kaplan-Meier method. Cox regression analysis of factors potentially associated with survival was conducted to identify independent factors. RESULTS: In clinical gastric cancer samples, we found that miR-373 expression was significantly down-regulated in glioma tissues compared with non-neoplastic brain tissues (p<0.01). Reduced expression of miR-373 was associated with serum WHO grade (p=0.015) and KPS score (p=0.001). Kaplan-Meier analysis indicated that patients with low level of miR-373 expression had poorer overall survival (OS) and progression-free survival (PFS). Multivariate survival analysis verified that miR-373 expression level was an independent predictor of both OS and PFS for glioma patients. CONCLUSIONS: Our study showed miR-373 was associated to progression in glioma, and suggested it as a potential predictive factor for the prognosis of glioma.

FPR1 interacts with CFH, HTRA1 and smoking in exudative age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE: To determine the genetic association of an inflammation-related gene, formyl peptide receptor 1 (FPR1), in exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS: The coding region of FPR1 gene was sequenced in 554 unrelated Chinese individuals: 155 exudative AMD patients, 179 PCV patients, and 220 controls. Interactions and combined effects of FPR1 with complement factor H (CFH), high temperature requirement factor A1 (HTRA1), and smoking were also investigated. RESULTS: A total of 28 polymorphisms in FPR1 were identified. Single nucleotide polymorphisms (SNP) rs78488639 increased the risk to exudative AMD (P=0.043) and PCV (P=0.016), whereas SNP rs867229 decreased the risk to exudative AMD (P=0.0026), but not PCV. Homozygous G allele of rs1042229 was associated with exudative AMD (P=0.0394, odds ratio (OR)=2.27, 95% confident interval: 1.08-4.74), but not with PCV. Exudative AMD, but not PCV, was associated with the heterozygous genotypes of rs2070746 (P=0.019, OR=0.57) and rs867229 (P=0.0082, OR=0.54). Significantly, interactions were identified among FPR1 rs78488639, CFH rs800292, and HTRA1 rs11200638 in both exudative AMD and PCV. Combined heterozygous risk alleles of CFH rs800292 GA and FPR1 rs78488639 CA were posed to PCV (P=2.22 × 10(-4), OR=10.47), but not exudative AMD. Furthermore, FPR1 rs78488639 CA combining with HTRA1 rs11200638 and smoking was also predisposed risks to exudative AMD and PCV. CONCLUSION: FPR1 is associated with exudative AMD and PCV in a Hong Kong Chinese cohort. FPR1 rs78488639 interacted with CFH rs800292, HTRA1 rs11200638, and smoking, enhancing risk to exudative AMD and PCV.

Detection of Klinefelter syndrome by G-banding analysis combined with primed in situ labeling technique.

Primed in situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We employed triple-color PRINS technique to detect chromosomal abnormalities in Klinefelter syndrome patients diagnosed by G-banding karyotype analysis. Among 1034 infertile male patients, 134 were found to be cytogenetically abnormal, including 70 with chromosomal number abnormalities and 64 with chromosomal structure abnormalities. Among these cytogenetically abnormal patients, 56 were diagnosed as having Klinefelter syndrome. PRINS technique was used on cultured lymphocyte metaphase cells of the Klinefelter syndrome patients; the same result was obtained with G-banding karyotype analysis. PRINS proved to be a rapid and reliable method to detect numerical chromosome abnormalities in peripheral blood lymphocytes in metaphase.

Primed in situ labeling for detecting single-copy genes.

In order to analyze male sterility caused by deletion of SRY and DAZ, we examined the accuracy and cost-effectiveness of a modified primed in situ labeling (PRINS) technique for detection of single-copy genes. Peripheral blood samples were collected from 50 healthy men; medium-term cultured lymphocytes from these samples were suspended in fixative solution and then spread on clean slides. We used four primers homologous to unique regions of the SRY and DAZ regions of the human Y-chromosome and incorporated reagents to increase polymerase specificity and to enhance the hybridization signal. PRINS of SRY and DAZ gave bands at Yp11.3 and Yq11.2, respectively, in all 50 metaphase spreads. The PRINS SRY signals were as distinct as those obtained using traditional fluorescence in situ hybridization (FISH). This new method is ideal for rapid localization of single-copy genes or small DNA segments, making PRINS a cost-effective alternative to FISH. Further enhancement of PRINS to increase its speed of implementation may lead to its wide use in the field of medical genetics.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.