circMTDH.4/miR-630/AEG-1 axis participates in the regulation of proliferation, migration, invasion, chemoresistance, and radioresistance of NSCLC.

Astrocyte elevated gene-1 (AEG-1) plays a critical role in the development, progression, and metastasis of a variety of cancers, including non-small-cell lung cancer (NSCLC). The objective of the current study is to unravel the upstream signaling of AEG-1. A cohort of 28 NSCLC tissues and 30 normal tissues were collected. Quantitative reverse transcription-polymerase chain reaction and Western blotting were used to examine AEG-1, migration, and invasion related markers in NSCLC cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay coupled with colony formation assay were conducted to monitor cell growth. Transwell assay was performed to determine cell migration and invasion. Apoptotic cells were detected by costaining with Annexin-V-fluorescein isothiocyanate and propidium iodide. Immunofluorescent staining was used to observe the levels of migration and invasion related markers. Xenograft models were used to investigate tumor formation in vivo. Dual-luciferase reporter assay and RNA immunoprecipitation were carried out to determine the interaction between circMTDH.4 and miR-630, as well as the associated between miR-630 and AEG-1. AEG-1 was highly expressed in NSCLC tissues and cell lines. Silencing of AEG-1 inhibited cell proliferation, migration, invasion, and chemoresistance/radioresistance in NCI-H1650 and A549 cells. circMTDH.4 regulated AEG-1 expression via sponging miR-630. Knockdown of circMTDH.4 and/or overexpression of miR-630 inhibited chemoresistance and radioresistance in NSCLC cells, whereas overexpression of AEG-1 or knockdown of miR-630 exerted rescue effects. circMTDH.4/miR-630/AEG-1 axis is responsible for chemoresistance and radioresistance in NSCLC cells.

Carnosol inhibits cerebral ischemia-reperfusion injury by promoting AMPK activation.

BACKGROUND: Carnosol is a phytopolyphenol (diterpene) found and extracted from plants of Mediterranean diet, which has anti-tumor, anti-inflammatory and antioxidant effects. However, its role in ischemic stroke has not been elucidated. METHODS: Primary neurons subjected to oxygen-glucose deprivation (OGD) was used to investigate the effect of carnosol in vitro. A mouse MCAO model was used to evaluate the effect of carnosol on ischemic stroke in vivo. The mRNA level of inflammatory and apoptosis-related genes was determined by RT-PCR. The protein level of total and phosphorylated AMPK was determined by WB. H&E and Immunofluorescent assay was used to investigate the necrosis, inflammation and apoptosis in brain tissue. RESULTS: Carnosol protected the activity of primary neurons subjected to oxygen-glucose deprivation (OGD) in vitro, as well as inhibited inflammation and apoptosis. Furthermore, carnosol could significantly reduce the infarct and edema volume and protect against neurological deficit in vivo, and had a significant inhibitory effect on brain neuroinflammation and apoptosis. Mechanically, carnosol could activate AMPK, and the effect of carnosol on cerebral ischemia-reperfusion injury cell model could be abolished by AMPK phosphorylation inhibitor. CONCLUSION: Carnosol has a protective effect on ischemic stroke, and this effect is achieved through AMPK activation. Our study demonstrates the protective effect of carnosol on cerebral ischemia-reperfusion injury and provides a new perspective for the clinical treatment of ischemic stroke.

[Effects of PI3K/Akt/NF-κB signal pathway on FSH facilitation on cell proliferation and invasion by human epithelial ovarian cancer].

OBJECTIVE: To explore the effects of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor-kappa B (NF-κB) signal pathway on the process of follicle-stimulating hormone (FSH) facilitating cell proliferation and invasion in human epithelial ovarian cancer. METHODS: Ovarian cancer cell lines SKOV3 and 3AO were cultured to exponential phase, then assigned to control group, FSH group, LY294002 group and FSH + LY294002 group, respectively. Cells were treated with different concentration of FSH and LY294002, respectively. The effects of FSH on cell proliferation were observed by methylthiazolyl tetrazolium (MTT). Morphological changes were observed by phase contrast microscope. The ability of cell invasion was investigated by transwell invasion assay. The expression of FSH receptor (FSHR), Akt1/2, phosphorylated-Akt (p-Akt) and NF-κB p65 protein were detected by western blot. RESULTS: (1) FSH could promote the proliferation of SKOV3 and 3AO cells. When the cells were treated with 40 U/L FSH for 48 hours (SKOV3) and 24 hours (3AO), compared with those in control groups, they reached the highest proliferation rate (P < 0.05), respectively. (2) The morphology of SKOV3 and 3AO cells in four groups:in control group, SKOV3 cells were short spindle and 3AO cells were long spindle, the nuclei of them were both roundness or oval, the cytoplasm were bright. In FSH group, the cells changed to slightly longer or polygonal, they were full in shape, meanwhile, the cell intensity were higher than control group. In LY294002 group, some cells changed from spindle to round, and began to shrink. The cell intensity diminished. The morphology of FSH + LY294002 group was similar with control group, but the cell intensity was lower than that in FSH group. (3) The number of SKOV3 cell that passed through the membrane in control group, FSH group, LY294002 group and FSH + LY294002 group was (26 ± 6), (118 ± 19), (18 ± 5) and (38 ± 7), respectively. The number of 3AO cell was (19 ± 4), (134 ± 20), (12 ± 3) and (58 ± 11), respectively. The results showed that the number of cells in FSH group was significantly higher than that in control group (P < 0.05), while the number of cell in FSH + LY294002 group was significantly fewer than that in FSH group (P < 0.05). (4) There was no significant difference in the expression of FSHR and Akt1/2 between FSH group and control group (P > 0.05), but FSH increased the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm in SKOV3 and 3AO cells, there were significant differences compared with control group (P < 0.05). LY294002 reversed the effects of FSH on increasing the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm, there were significant differences among LY294002 group, FSH + LY294002 group and FSH group (P < 0.05). CONCLUSION: The effects of FSH on proliferation and invasion of ovarian cancer cell lines SKOV3 and 3AO may be realized by regulating the activity of NF-κB in PI3K/Akt signal pathway.

A Nematode Calreticulin, Rs-CRT, Is a Key Effector in Reproduction and Pathogenicity of Radopholus similis.

Radopholus similis is a migratory plant-parasitic nematode that causes severe damage to many agricultural and horticultural crops. Calreticulin (CRT) is a Ca2+-binding multifunctional protein that plays key roles in the parasitism, immune evasion, reproduction and pathogenesis of many animal parasites and plant nematodes. Therefore, CRT is a promising target for controlling R. similis. In this study, we obtained the full-length sequence of the CRT gene from R. similis (Rs-crt), which is 1,527-bp long and includes a 1,206-bp ORF that encodes 401 amino acids. Rs-CRT and Mi-CRT from Meloidogyne incognita showed the highest similarity and were grouped on the same branch of the phylogenetic tree. Rs-crt is a multi-copy gene that is expressed in the oesophageal glands and gonads of females, the gonads of males, the intestines of juveniles and the eggs of R. similis. The highest Rs-crt expression was detected in females, followed by juveniles, eggs and males. The reproductive capability and pathogenicity of R. similis were significantly reduced after treatment with Rs-crt dsRNA for 36 h. Using plant-mediated RNAi, we confirmed that Rs-crt expression was significantly inhibited in the nematodes, and resistance to R. similis was significantly improved in transgenic tomato plants. Plant-mediated RNAi-induced silencing of Rs-crt could be effectively transmitted to the F2 generation of R. similis; however, the silencing effect of Rs-crt induced by in vitro RNAi was no longer detectable in F1 and F2 nematodes. Thus, Rs-crt is essential for the reproduction and pathogenicity of R. similis.