miR-132 weakens proliferation and invasion of glioma cells via the inhibition of Gli1.

OBJECTIVE: Transcriptional factor Gli1 in Hedgehog signal pathway facilitates epithelial mesenchymal transition (EMT) and is associated with invasion or proliferation of multiple tumor cells. The previous study showed the correlation between miR-132 down-regulation and glioma pathogenesis. We investigated the role of miR-132 in mediating Gli1 expression and in affecting proliferation or invasion of glioma cells. PATIENTS AND METHODS: Dual luciferase reporter gene assay was used to confirm the targeted regulation between miR-132 and Gli1. Tumor tissues at different pathological grades (grade II, III and IV) were collected from glioma patients, in parallel with brain tissues from contusion surgery. The expression of miR-132 and Gli1 was measured by RT-PCR. Glioma cell line U251 was treated with miR-132 or si-Gli1 followed by measuring the expression of Gli1, E-cadherin, Vimentin and Cyclin D1. In addition, flow cytometry and transwell assay were performed to evaluate cell invasion potency. RESULTS: Bioinformatics analysis showed the complementary binding sites between miR-132 and 3'-UTR of Gli1 mRNA. Transfection of miR-132 mimic significantly reduced luciferase activity, indicating the targeted regulatory relationship between miR-132 and Gli1 mRNA. Compared with control group, miR-132 expression was decreased and Gli1 level was elevated in glioma tissues, both of which were correlated with the pathological grade. Transfection of miR-132 mimic or si-Gli1 remarkably suppressed the expression of Gli1, Vimentin or Cyclin D1 in U251 cells, up-regulated E-cadherin expression, suppressed cell proliferation and invasion. CONCLUSIONS: Our data indicated that over-expression of miR-132 could inhibit proliferation or invasion of glioma cells via targeted inhibition of Gli1 expression.

Conjugated linoleic acid depresses the delta9 desaturase index and stearoyl coenzyme A desaturase enzyme activity in porcine subcutaneous adipose tissue.

Conjugated linoleic acid (CLA) has been shown to have an effect on subcutaneous fatty acid composition and has been reported to decrease stearoyl coenzyme A desaturase (SCD) activity by decreasing mRNA expression and(or) catalytic activity in rodents and rodent cell lines. This investigation was designed to study the effects of CLA, corn oil, or beef tallow supplementation on s.c. adipose tissue fatty acid composition, adiposity, SCD enzyme activity, and the delta9 desaturase index in piglets. Eighteen crossbred barrows 16 to 18 d of age were adapted to diet for 1 wk and then assigned randomly to one of three treatments: 1.5% added CLA, 1.5% added corn oil, or 1.5% added beef tallow. Barrows were penned individually and fed the supplemental oils for 35 d (to 25.6 +/- 0.6 kg BW). Subcutaneous adipose tissue samples were obtained after slaughter. Fatty acid composition of the s.c. adipose tissue differed for each fatty acid measured due to diet with the exception of 18:3. The concentrations of CLA trans-10, cis-12 and cis-9, trans-11 were elevated from nondetectable to 1.62 and 2.52 g/100 g lipid, respectively (P < 0.001 for both isomers). Conjugated linoleic acid decreased the delta9 desaturase index (P < 0.01) and SCD enzyme activity, expressed as nanomoles of palmitate converted to palmitoleate/(7 min x g of tissue) (P = 0.075) and nanomoles of palmitate converted to palmitoleate/(7 min 105 cells) (P= 0.056). Tallow-fed pigs had a greater proportion of large adipocytes (> 700 pL) and the greatest SCD activity. These data provide the first direct evidence that dietary CLA depresses SCD enzyme activity in porcine adipose tissue, which may in part be responsible for the depression of adiposity by CLA observed by others in market weight pigs.

Green fluorescent protein as a reporter for gene transfer studies in the cochlea.

This study examined the 'humanized, red-shifted' version of the jellyfish Aequorea victoria green fluorescent protein (hrGFP) as a novel reporter for in vivo gene transfer studies in the cochlea using adeno-associated virus (AAV) vectors. Approximately 10(5) AAV vectors containing the hrGFP reporter gene were infused over 2 days or 1 week into the cochlea of the guinea pig via an osmotic minipump. Saline infused, non-infused, as well as AAV-beta-galactosidase infused guinea pigs served as the negative controls. The hrGFP transgene expression was detected as moderate intensity fluorescence easily distinguished from the background. Increased fluorescence was seen in the spiral ganglion, spiral ligament, spiral limbus, organ of Corti, and Reissner's membrane of the AAV-hrGFP infused animals. Control animals showed minimal fluorescence throughout the cochlea. Comparison of the 2 day and 1 week AAV-hrGFP infused animals showed qualitatively increased fluorescence in the 2 day animals. Background autofluorescence in the stria vascularis was noted in both the experimental and the control animals. In addition, fluorescence was detected in the contralateral cochlea of the AAV-hrGFP infused animals. Subsequent PCR analysis confirmed the presence of viral particles in the AAV-hrGFP infused cochlea as well as in the brain and the contralateral cochlea. This finding has important implications for the eventual implementation of cochlear gene therapy. The results not only reinforce the need to assess the introduction and expression of foreign genes in the target cochlea but also consider issues of viral spread, safety, and modes of gene delivery. This study establishes hrGFP as an effective reporter of gene transfer and transgene expression in the cochlea. GFP's small gene size, stability, ease of detection, and potential for diverse biological applications will be invaluable for a variety of future gene transfer and expression studies in the cochlea.