[Effects of Targeting lncRNA HOTAIR on the Invasion and Nude Mouse Tumorigenicity of Epithelial Ovarian Cancer Cells].

OBJECTIVE: To explore the effects of shRNA-mediated downregulating lncRNA HOTAIR on the invasion,nude mouse tumorigenicity and snail expression of epithelial ovarian cancer SKOV3 cells. METHODS: The expression of lncRNA HOTAIR was detected by RT-PCR in SKOV3 cells. The shRNA targeting the lncRNA HOTAIR gene was cloned into RNA interference plasmid. The construction shHOTAIR vector was transfected into ovarian cancer SKOV3 cells by lipofectamine 2000,and the stably transfected cells were isolated by G418 and single clone selection. The downregulating expression of lncRNA HOTAIR was detected by quantitative real time PCR(qRT-PCR). The characteristics of shHOTAIR transfected SKOV3 cells were analyzed from the assays of invasion and nude mouse tumorigenicity,as well as the expression of snail and E-cadherin mRNA detected by qRT-PCR,and snail detected by immunohistochemistry and Western blot methods in xenograft tumor,respectively. RESULTS: The lncRNA HOTAIR expression was proved by RT-PCR in SKOV3 cells. The lncRNA HOTAIR expression in shHOTAIR transfected SKOV3 cells was significantly lower than the scramble control (P<0.01). The shHOTAIR transfected SKOV3 cells show that the invasion ability was significantly decreased compared with the scramble control (P<0.01). The nude mouse tumorigenicity,including tumorigenicity mouse number and tumor volume,was significantly decreased compared with the control group (P<0.01). The snail protein expression detected by IHC and Western blot in shHOTAIR-SKOV3 xenograft tumor was significantly decreased compared with the control scramble- SKOV3 group (P<0.05). The lncRNA HOTAIR low expression resulted in increasing E-cadherin and decreasing snail expression detected by qRT-PCR in the shHOTAIR transfected SKOV3 cells of xenograft tumor,compared with the scramble control (P<0.05). CONCLUSION: Targeting lncRNA HOTAIR expression in SKOV3 cells with RNA interference can decrease snail,increase E-cadherin and significantly reduce the invasion and tumorigenicity of epithelial ovarian cancer SKOV3 cells. These results suggest that the lncRNA HOTAIR could be an effective therapeutic target for human epithelial ovarian caner treatment.

[Effect of cordycepin on apoptosis and autophagy of tongue cancer cells in vitro and the molecular mechanism].

OBJECTIVE: To study the effect of cordycepin on cell cycle, apoptosis and autophagy of human tongue cancer TCA-8113 cells and explore the mechanism of cordycepin for inhibiting the occurrence of tongue cancer. METHODS: CCK-8 method was used to assess the inhibitory effect of cordycepin on TCA-8113 cell proliferation in vitro. The cell cycle and cell apoptosis of TCA-8113 cells treated with different concentrations of cordycepin were analyzed using flow cytometry. The expressions of apoptosis-related genes caspase-3, caspase-9, Bcl-2, and Bax were examined using quantitative real-time PCR and Western blotting, and immunohistochemistry was used to detect the expressions of autophagy-related proteins LC-3ß, P62, p-mTOR, and AMPK. RESULTS: CCK-8 assay showed that cordycepin significantly inhibited the proliferation of TCA-8113 cells in a concentration-dependent manner with an IC50 of 3.548 mg/mL at 24 h and an IC50 of 1.185 mg/mL at 48 h. Flow cytometric analysis showed that cordycepin caused cell cycle arrest at S phase and dose-dependently increased the apoptotic rate of TCA-8113 cells. Treatment of the cells with cordycepin enhanced the expressions of Bax, caspase-3 and caspase-9 at both the mRNA and protein levels and inhibited the expression of the antiapoptotic gene Bcl-2. Immunohistochemistry demonstrated that cordycepin promoted the expression of LC-3ß and AMPK and inhibited the expression of P62 and p-mTOR. CONCLUSION: Cordycepin inhibits the proliferation and induces apoptosis of HCT-116 cells through the mitochondrial pathway and induces autophagy via the AMPK/mTOR pathway.

[High mobility group box-1 stimulates proinflammatory cytokine production in endothelial cells via MAP kinases].

OBJECTIVE: To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release. METHODS: HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation. RESULTS: The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng

[Construction of a eukaryotic expression vector for alpha-1-antitrypsin and the localization of the expression product in NIH 3T3 cells].

OBJECTIVE: To construct a eukaryotic expression vector for alpha-1-antitrypsin (AAT) and detect its expression and localization in NIH 3T3 cells. METHODS: The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences for mouse AAT (GenBank accession No. NM_009243) were amplified by RT-PCR and cloned into hemagglutinin (HA)-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, which were observed under fluorescence microscope. RESULTS: The recombinant plasmid was verified by PCR, enzyme digestion and sequence analysis, and the fusion protein was highly expressed in NIH 3T3 cells. Under fluorescence microscope, the fusion protein was found to distribute mainly in the cytoplasm. CONCLUSION: The expression vector for AAT-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells to allow future functional study of AAT in mammalian cells.