Toosendanin (TSN) is a tetracyclic triterpenoid extracted from Melia toosendan Sieb, et Zucc, which primarily grows in specific areas of China. Although toosendanin (TSN) exerts antitumoral effects on various human cancer cells, its influence on gastric cancer (GC) is remains to be elucidated. MicroRNAs (miRNAs/miRs) serve crucial roles in apoptosis and proliferation of cancer cells. miR‑23a‑3p has been shown to be associated with human GC; however, the specific function of miR‑23a‑3p in GC remains unclear. Therefore, the present study aimed to elucidate the role of miR‑23a‑3p in the regulation of GC cell proliferation and apoptosis induced in vitro by TSN treatment. Subsequently, apoptosis‑related genes expression levels were quantified by reverse transcription‑quantitative PCR and western blot analysis, respectively, and the target relationship between miR‑23a‑3p and BCL2 was determined by luciferase reporter gene analysis. Additionally, cell proliferation and apoptosis experiments were carried out. The results indicated that TSN inhibited proliferation and induced apoptosis in MKN‑45 cells. Moreover, it upregulated the expression of miR‑23a‑3p. B‑cell lymphoma‑2 (BCL2) was identified as a potential target gene of miR‑23a‑3p, which was demonstrated to bind to the 3'‑untranslated region of BCL2 mRNA, as detected by the luciferase reporter assay. Further studies revealed that BCL2 expression was downregulated following overexpression of miR‑23a‑3p. In addition, the overexpression of the miR‑23a‑3p inhibited proliferation, induced G1 arrest and increased apoptosis in MKN‑45 cells. The results of the present study demonstrated that miR‑23a‑3p inhibited proliferation and induced apoptosis of GC cells, which may be attributable to its direct targeting of BCL2. These results may provide a novel insight into the apoptosis of GC cells, and may lead to investigations into the mechanisms of the effects of TSN.

The human gut microbiota play essential roles in metabolism and human health, especially by enzymatically utilizing dietary fiber that the host cannot directly digest and releasing functional components including short-chain fatty acids (SCFAs) and hydroxycinnamic acids (e.g., ferulic acid). In our previous study, seven potential feruloyl esterase (FAE) genes were identified from the gut microbiota. In the current work, one of the genes encoding a novel FAE (DfFAE) from Dorea formicigenerans of Firmicutes was bacterially expressed, purified and characterized. The 30.5 kDa type-A DfFAE has an optimum pH and temperature of 8.4 and 40 °C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five α-helices cap domain that shaped an unusually narrow and deep active site pocket containing a specific substrate access tunnel in DfFAE. Furthermore, rational design strategy was subjected to the active site pocket in an aim of improving its enzymatic activities. The mutants V252A, N156A, W255A, P149A, and P186A showed 1.8 to 5.7-fold increase in catalytic efficiency toward pNPA, while W255A also exhibited altered substrate preference toward long-chain substrate pNPO (45.5-fold). This study highlighted an unusual active site architecture in DfFAE that influenced its substrate selectivity and illustrated the applicability of rational design for enhanced enzymatic properties.

Dryopteris fragrans (L.) Schott is a fern in the family Dryopteridaceae that shows a good curative efficacy against several skin diseases, such as psoriasis and athlete’s foot. One of the substances that are effective is a flavonoid. The enzyme 4-Coumaric acid coenzyme A ligase (4CL) plays a key role in the phenylpropanoid metabolism pathway, which controls the metabolic direction of phenylpropanoids and is associated with the synthesis of flavonoids and lignin. In this paper, we examined the role of 4CL1 gene of D. fragrans (Df4CL1) by transferring it to the model plant tobacco. Then its gene function was analyzed through tissue expression, determination of protein and enzyme activity, and assessment of flavonoids and lignin production. The analysis showed that overexpression of Df4CL1 in the transgenic tobacco (TT) had higher protein content, 4CL activity, and content of flavonoids and lignin compared with wild type tobacco (WT). The tissue expression of Df4CL1 in TT was positively correlated with the content of flavonoids and lignin. Therefore, we conclude that Df4CL1 was associated with both flavonoids and lignin biosynthesis by controlling the relative abundance of flavonoids and lignin during normal development.

RNAi-based Knockdown of Multidrug Resistance-associated Protein 1 is Sufficient to Reverse Multidrug Resistance of Human Lung Cells Multidrug resistance;MRP1;ShRNA;protein expression;tumor cells; Up-regulation of multidrug resistance-associated protein 1 (MRP1) is regarded as one of the main causes for multidrug resistance (MDR) of tumor cells, leading to failure of chemotherapy-based treatment for a multitude of cancers. However, whether silencing the overexpressed MRP1 is sufficient to reverse MDR has yet to be validated. This study demonstrated that RNAi-based knockdown of MRP1 reversed the increased efflux ability and MDR efficiently. Two different short haipin RNAs (shRNAs) targeting MRP1 were designed and inserted into pSilence-2.1-neo. The shRNA recombinant plasmids were transfected into cis-dichlorodiamineplatinum-resistant A549 lung (A549/DDP) cells, and then shRNA expressing cell clones were collected and maintained. Real time PCR and immunofluorescence staining for MRP1 revealed a high silent efficiency of these two shRNAs. Functionally, shRNA-expressing cells showed increased rhodamine 123 retention in A549/DDP cells, indicating reduced efflux ability of tumor cells in the absence of MRP1. Consistently, MRP1-silent cells exhibited decreased resistance to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DDP, suggesting reversal of MDR in these tumor cells. Specifically, MRP1 knockdown increased the DDP-induced apoptosis of A549/DDP cells by increased trapping of their cell cycling in the G2 stage. Taken together, this study demonstrated that RNAi-based silencing of MRP1 is sufficient to reverse MDR in tumor cells, shedding light on possible novel clinical treatment of cancers.