Molecular Evolution of SNAREs in Vitis vinifera and Expression Analysis under Phytohormones and Abiotic Stress.

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a key role in mediating a variety of plant biological processes. Currently, the function of the SNARE gene family in phytohormonal and abiotic stress treatments in grapevine is currently unknown, making it worthwhile to characterize and analyze the function and expression of this family in grapevine. In the present study, 52 VvSNARE genes were identified and predominantly distributed on 18 chromosomes. Secondary structures showed that the VvSNARE genes family irregular random coils and α-helices. The promoter regions of the VvSNARE genes were enriched for light-, abiotic-stress-, and hormone-responsive elements. Intraspecific collinearity analysis identified 10 pairs collinear genes within the VvSNARE family and unveiled a greater number of collinear genes between grapevine and apple, as well as Arabidopsis thaliana, but less associations with Oryza sativa. Quantitative real-time PCR (qRT-PCR) analyses showed that the VvSNARE genes have response to treatments with ABA, NaCl, PEG, and 4 °C. Notably, VvSNARE2, VvSNARE14, VvSNARE15, and VvSNARE17 showed up-regulation in response to ABA treatment. VvSNARE2, VvSNARE15, VvSNARE18, VvSNARE19, VvSNARE20, VvSNARE24, VvSNARE25, and VvSNARE29 exhibited significant up-regulation when exposed to NaCl treatment. The PEG treatment led to significant down-regulation of VvSNARE1, VvSNARE8, VvSNARE23, VvSNARE25, VvSNARE26, VvSNARE31, and VvSNARE49 gene expression. The expression levels of VvSNARE37, VvSNARE44, and VvSNARE46 were significantly enhanced after exposure to 4 °C treatment. Furthermore, subcellular localization assays certified that VvSNARE37, VvSNARE44, and VvSNARE46 were specifically localized at the cell membrane. Overall, this study showed the critical role of the VvSNARE genes family in the abiotic stress response of grapevines, thereby providing novel candidate genes such as VvSNARE37, VvSNARE44, and VvSNARE46 for further exploration in grapevine stress tolerance research.

The Yunnan national medicine Maytenus compound inhibits the proliferation of hepatocellular carcinoma (HCC) by suppressing the activation of the EGFR-PI3K-AKT signaling pathway.

Objective: To investigate the effects of Maytenus compound on the proliferation of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to explore the underlying mechanism. Methods: The half maximal inhibitory concentration (IC50) values of Maytenus compound in HepG2 and BEL-7402 cells were determined by the MTS assay. HepG2 and BEL-7402 cells were treated with different concentrations of Maytenus compound. MTS assays, colony formation assays and cell cycle analyses were performed to clarify the inhibitory effect of Maytenus compound on the proliferation of HepG2 and BEL-7402 cells in vitro. After subcutaneous injection of HepG2 cells, nude mice were randomly divided into a vehicle control group and a drug intervention group, which were intragastrically administered ddH2O or Maytenus compound, respectively. The inhibitory effect of Maytenus compound on the proliferation of HepG2 cells in vivo was analyzed using subcutaneous tumor growth curves, tumor weight, the tumor growth inhibition rate and the immunohistochemical detection of BrdU-labeled cells in S phase. The organ toxicity of Maytenus compound was initially evaluated by comparing the weight difference and organ index of the two groups of nude mice. The main proteins in the EGFR-PI3K-AKT signaling pathway were detected by Western blot after Maytenus compound intervention in vivo and in vitro. Results: Maytenus compound showed favorable antiproliferation activity against HepG2 and BEL-7402 cells with IC50 values of 79.42±11.71 µg/mL and 78.48±8.87 µg/mL, respectively. MTS assays, colony formation assays and cell cycle analyses showed that Maytenus compound at different concentration gradients within the IC50 concentration range significantly suppressed the proliferation of HepG2 and BEL-7402 cells in vitro and inhibited cell cycle progression from G1 to S phase. Additionally, Maytenus compound, at an oral dose of 2.45 g/kg, dramatically inhibited, without obvious organ toxicity, the proliferation of subcutaneous tumors formed by HepG2 cells in nude mice. In addition, the tumor growth inhibition rate for Maytenus compound was 66.94%. Furthermore, Maytenus compound inhibited the proliferation of liver orthotopic transplantation tumors in nude mice. Western blot analysis showed that Maytenus compound significantly downregulated the expression of p-EGFR, p-PI3K, and p-AKT and upregulated the expression of p-FOXO3a, p27, and p21 in vivo and in vitro. Conclusion: Maytenus compound significantly inhibited the proliferation of HCC cells in vitro and in vivo. The downregulation of the EGFR-PI3K-AKT signaling pathway and subsequent inhibition of cell cycle progression from G1 to S phase is one of the possible mechanisms. Maytenus compound has a high tumor growth inhibition rate and has no obvious organ toxicity, which may make it a potential anti-HCC drug, but the results from this study need to be confirmed by further clinical trials in HCC patients.