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EMT confers increased metastatic potential and the resistance to chemotherapies to cancer cells. However, the precise mechanisms of EMT-related chemotherapy resistance remain unclear. c-Src-mediated caspase 8 phosphorylation essential for EMT in lung adenocarcinoma cell lines preferentially occurs in cells with the mesenchymal phenotype, resulting in chemoresistance to cisplatin plus paclitaxel in patients with resectable lung adenocarcinoma and a significantly worse 5-year PFS. Cisplatin killed lung adenocarcinoma cells regardless of caspase 8. Paclitaxel-triggered necroptosis in lung adenocarcinoma cells was dependent on the phosphorylation or deficiency of caspase 8, during which FADD interacted with RIPK1 to activate the RIPK1/RIPK3/MLKL signaling axis. Accompanied with c-Src-mediated caspase 8 phosphorylation to trigger EMT, a novel lncRNA named lncCRLA was markedly upregulated and inhibited RIPK1-induced necroptosis by impairing RIPK1RIPK3 interaction via binding to the intermediate domain of RIPK1. Dasatinib mitigated c-Src-mediated phosphorylation of caspase 8-induced EMT and enhanced necroptosis in mesenchymal-like lung adenocarcinoma cells treated with paclitaxel, while c-FLIP knockdown predominantly sensitized the mesenchymal-like lung adenocarcinoma cells to paclitaxel+dasatinib. c-Srccaspase 8 interaction initiates EMT and chemoresistance via caspase 8 phosphorylation and lncCRLA expression, to which the dasatinib/paclitaxel liposome+siFLIP regimen was lethal. [ABSTRACT FROM AUTHOR]

EMT confers increased metastatic potential and the resistance to chemotherapies to cancer cells. However, the precise mechanisms of EMT-related chemotherapy resistance remain unclear. c-Src-mediated caspase 8 phosphorylation essential for EMT in lung adenocarcinoma cell lines preferentially occurs in cells with the mesenchymal phenotype, resulting in chemoresistance to cisplatin plus paclitaxel in patients with resectable lung adenocarcinoma and a significantly worse 5-year PFS. Cisplatin killed lung adenocarcinoma cells regardless of caspase 8. Paclitaxel-triggered necroptosis in lung adenocarcinoma cells was dependent on the phosphorylation or deficiency of caspase 8, during which FADD interacted with RIPK1 to activate the RIPK1/RIPK3/MLKL signaling axis. Accompanied with c-Src-mediated caspase 8 phosphorylation to trigger EMT, a novel lncRNA named lncCRLA was markedly upregulated and inhibited RIPK1-induced necroptosis by impairing RIPK1RIPK3 interaction via binding to the intermediate domain of RIPK1. Dasatinib mitigated c-Src-mediated phosphorylation of caspase 8-induced EMT and enhanced necroptosis in mesenchymal-like lung adenocarcinoma cells treated with paclitaxel, while c-FLIP knockdown predominantly sensitized the mesenchymal-like lung adenocarcinoma cells to paclitaxel+dasatinib. c-Srccaspase 8 interaction initiates EMT and chemoresistance via caspase 8 phosphorylation and lncCRLA expression, to which the dasatinib/paclitaxel liposome+siFLIP regimen was lethal.

Objective: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique., Methods: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method., Results: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice., Conclusions: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.