csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis.

BACKGROUND: Clonorchis sinensis (CS) is classified as a group 1 carcinogen and can cause intrahepatic cholangiocarcinoma (ICC). CS extracellular vesicles (CsEVs) play important roles in mediating communication between parasitic helminths and humans. Ferroptosis is a novel cell death mechanism that is mainly induced by lipid peroxidation and iron overload. However, the role of CsEVs in the regulation of ferroptosis in ICC remains unclear. This study aimed to explore the role of CS-secreted miR-96-5p (csi-miR-96-5p) delivered by CsEVs in ICC progression and ferroptosis. METHODS: Tissue samples were collected from ICC patients with CS infection (CS-ICC) or without CS infection (NC-ICC). The levels of csi-miR-96-5p and PTEN gene were determined by quantitative polymerase chain reaction (qPCR) and western blotting, and survival analysis was performed. CsEVs were isolated and identified by ultracentrifugation and transmission electron microscopy. Lentiviruses were used to establish stable cell lines with csi-miR-96-5p mimic expression, PTEN overexpression (PTEN-EXO) and PTEN CRISPR/Cas9-based knockout (PTEN-KO) and their respective negative controls. Cell proliferation was assessed by performing Cell Counting Kit-8 assays in vitro and in a tumor xenograft model in vivo, and cell migration was assessed by performing Transwell assays. Erastin is used to induce ferroptosis. Ferroptosis levels were evaluated using biomarkers. RESULTS: High csi-miR-96-5p and low PTEN expression was observed in CS-ICC tissues and was associated with poor overall survival. csi-miR-96-5p was highly enriched in CsEVs and was taken up by ICC cells. csi-miR-96-5p mimics or PTEN-KO significantly promoted the growth and migration of ICC cells in vitro and in vivo, whereas PTEN-EXO exerted the opposite effect. Mechanistically, csi-miR-96-5p mimics or PTEN-KO inhibited erastin-induced ferroptosis, including reducing the accumulation of Fe2+, lipid reactive oxygen species, and malondialdehyde, increasing the GSH/GSSG ratio and levels of SLC7A11 and GPX4, whereas PTEN-EXOs exerted the opposite effect. CONCLUSIONS: csi-miR-96-5p delivered by CsEVs reduced ferroptosis by regulating the expression of the PTEN/SLC7A11/GPX4 axis, thereby promoting ICC proliferation and migration. For the first time to our knowledge, we found that CS miRNAs could promote tumor development through ferroptosis.

[Serological detection of Cryptosporidium spp. infection in outpatients in Changchun].

CP23 gene of Cryptosporidium parvum was expressed in Escherichia coli, and the recombinant protein was purified. Its immunoreactivity was analyzed by Western blotting. Serum samples were collected from outpatients of different ages from August to November, 2010 in Changchun. Indirect ELISA was established to detect the anti-CP23 IgG in sera. Western blotting analysis indicated that the recombinant CP23 protein was recognized by sera from Cryptosporidium panum-infected calves and positive human sera, but not recognized by sera of mice infected with Schistosoma japonicum, sera from falciparum malaria patients and negative human sera. The overall anti-CP23 IgG positive rate was 3.2% (65/2 046). The seropositive rate was 2.7% (28/1 036) in men and 3.7% (37/1 010) in women (P > 0.05). The seropositive rates were significantly different among age groups (P < 0.05), and the age group of 71-80 had the highest positive rate (8.6%, 13/152).