OBJECTIVEto determine the effects of survivin antisense oligonucleotide (ASODN) on the expression levels of survivin mRNA and human mucoepidermoid carcinoma cell line (highly metastatic Mc3) apoptosis and to explore the feasibility of survivin gene as the mucoepidermoid carcinoma therapeutic targets.

METHODSthe survivin ASODN was designed and synthesized and then respectively transfected into Mc3 cells. The morphological changes of the Mc3 cells were observed 24, 48 and 72 h after transfection by inverted microscope and the apoptosis rate detected by flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the effect of the transfection on cell poliferation, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method for analysis of apoptotic index, and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of survivin.

RESULTSin survivin ASODN transfection group, there was less Mc3 cells than in other groups. The suspended cells dropping from the wall increased and showed typical apoptotic changes and time-dependent. Mc3 cell apoptosis rate in survivin ASODN transfection group transfected for 24, 48, 72 h was 12.96%, 14.43%, 22.69%, respectively, which were significantly higher than in other groups (P < 0.01) and time-dependent (P < 0.05). The inhibitory rate of Mc3 cells in survivin ASODN transfection group was 22.35%, 39.04%, 43.46%, which were significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The apoptosis index (AI) of Mc3 cells in survivin ASODN transfection group was 11.038%, 12.172%, 18.900%, significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The survivin mRNA levels in Mc3 cells were 0.739 ± 0.008, 0.668 ± 0.007, 0.500 ± 0.006, and the relative inhibition rate in these cells was 18.21%, 26.06%, 44.82%, significantly lower than other groups (P < 0.01) and time-dependent manner (P < 0.01).

CONCLUSIONSsurvivin ASODN could inhibit the proliferation of Mc3 cells and induce the apoptosis of Mc3 cells. It also can inhibit the expression of survivin mRNA. Survivin can be used as a gene therapy targets for mucoepidermoid carcinoma.

Apoptosis ; Carcinoma, Mucoepidermoid ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Gene Targeting ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Microtubule-Associated Proteins ; Neoplasm Proteins ; Oligonucleotides, Antisense ; RNA, Messenger ; Salivary Gland Neoplasms ; metabolism ; Transfection

Cannabinoid receptor type 2( CB2 R),a member of the G protein-coupled receptor( GPCR) superfamily,has a variety of biological activities,such as regulating pain response,resisting inflammation and fibrosis,and mediating bone metabolism. Some CB2 R regulators exhibit a good regulatory effect on bone metabolism. Cannabinoids in Cannabis sativa can cause psychoactive effects despite various pharmacological actions they exerted by targeting CB2 R. Therefore,it is of great significance to discover CB2 R regulators in non-Cannabis plants for finding new lead compounds without psychoactive effects and elucidating the action mechanism of plant drugs. The present study clarifies the discovery,structure,and physiological functions of CB2 R,especially its regulatory effects on bone metabolism,summarized CB2 R regulators extracted from non-Cannabis plants,and systematically analyzes the regulatory effects of CB2 R regulators on bone metabolism in animals,osteoblasts,and osteoclasts,to provide a scientific basis for the discovery of new CB2 R regulators and the development of anti-osteoporotic drugs.
Animals ; Cannabinoids/pharmacology* ; Cannabis ; Osteoblasts ; Osteoclasts ; Receptors, Cannabinoid