miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression.

MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3'-untranslated region (3'-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.

Isolating RNAs from rat facial motor neurons with laser capture microdissection after facial-facial anastomosis.

Both reinnervated and dormant neurons can be observed in facial nucleus following facial-facial anastomosis. Although they may play different roles in the remodeling mechanism of facial nucleus during facial nerve injury and regeneration, comprehensive gene profiling analysis of these two neuron types has never been performed due to the difficulty in isolating specific neuron populations and extracting sufficient amount of RNAs from the heterogeneous facial nucleus. In this study, we developed a method to isolate the Fluoro-Ruby (FR) retrogradely labeled reinnervated facial motor neurons and the Nissl stained dormant neurons in two steps with laser capture microdissection (LCM) technology. The quality and yield of RNAs extracted from these isolated neurons were confirmed with Agilent 2100 Bioanalyzer. This modified LCM based neuron isolation method will facilitate the studies of the roles and interactions of different neurons that co-exist in the same facial nucleus after peripheral injury.

[Adenovirus-mediated delivery of bcl-2 gene attenuates cisplatin-induced degeneration of cultured spiral ganglion cells.].

OBJECTIVE: To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC). METHODS: SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software. RESULTS: SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration. CONCLUSIONS: Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

[Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells].

OBJECTIVE: To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro. METHODS: Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR. RESULTS: The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC. CONCLUSIONS: The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.