Regulation mechanism of miR-494-3p on endometrial receptivity in mice via PI3K/AKT/mTOR pathway.

Successful implantation requires endometrial receptivity. To investigate the mechanisms of miR-494-3p on endometrial receptivity, GnRHa's superovulation scheme was designed to reduce endometrial receptivity, and the pregnant mice were injected with miR-494-3p antagomir. The regulatory role of miR-494-3p was identified by RT-qPCR, uterine blastocyst count, scanning electron microscopy, hematoxylin-eosin (HE) staining, and Western blot. Results indicated that miR-494-3p antagomir increased uterine blastocysts numbers, promoted the pinocytosis expressions, and increased endometrial thickness. Besides, miR-494-3p antagomir significantly increased leukemia inhibitory factor (LIF), Ang-2 and VEGF protein expressions, and up-regulated p-AKT/AKT and p-mTOR/mTOR protein ratios in endometrium. Luciferase assay confirmed that LIF was a potential target of miR-494-3p. Subsequently, human endometrial epithelial cells (hEECs) were transfected with miR-494-3p inhibitor and PI3K inhibitor (LY294002). The role of miR-494-3p was identified by RT-qPCR, CCK-8 assay, transwell assay and flow cytometry. Results indicated that miR-494-3p inhibitor significantly increased proliferation and invasion, and significantly inhibited apoptosis in hEECs, while LY294002 reversed its biological function. Overall, these results suggested that miR-494-3p is the key regulator of endometrial receptivity in mice, regulating this complex process through the PI3K/AKT/mTOR pathway. Understanding the role of miR-494-3p in endometrial receptivity is of great significance for exploring new targets for the diagnosis and treatment of early pregnancy failure, and improving the success rates of artificial reproduction.

Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.