The Role and Mechanism of JAK2 in Regulating Macrophage M2 Activation in Cervical Cancer Microenvironment
Objective To examine the impact of the Janus kinase 2(JAK2)small molecule inhibitor AG490 on autophagy,apoptosis,and desiccation properties in cervical cancer cells following treatment with TAM-derived exosomes.Methods PMA and human IL-4 were applied to differentiate human peripheral blood mononuclear cells THP-1 into M2 type TAMs;exosomes were isolated by differential centrifugation,and TEM and nanoparticle tracking technologies were used to analyze exosome morphology and size distribution;Western blot was used to determine whether exosomal marker proteins were expressed;Dil in conjunction with PKH67 staining was utilized to ascertain the internalization of TAM-derived exosomes by the HeLa cervical cancer cell line.The HeLa cells were segregated into 3 experimental groups:control,Exo,and Exo+AG490.Subsequently,the HeLa cells were exposed to TAM-derived exosomes and AG490 in accordance with their respective groups.An apoptosis rate was demonstrated via flow cytometry,and autophagy was quantified via double-fluorescence mRFP-GFP-LC3 assay.Western blot analysis was utilized to evaluate the stemness of cancer cells by measuring the protein levels of autophagy-and apoptosis-associated indicators.Further,the levels of tumor stem cell-associated proteins CD 133,OCT4,and SOX2 were also assessed.Results The induction of PMA and IL-4 resulted in significant alterations in the morphology of THP-1 cells,characterized by a decrease in the expression ratio of CD86 and an increase in the expression ratio of CD206(P＜0.05).Approximately 140 nm-sized spherical vesicles were found in the individual particles,with significant expression of exosomal marker proteins Alix,CD63,and TSG101.These findings suggested successful isolation of M2-type TAM-derived exosomes,which were found to be internalized by HeLa cells.In comparison to the control group,apoptosis rate,fluorescence intensity of GFP and mRFP,LC3-Ⅱ/LC3-1 ratio,and the expression levels of Beclin-1,Bax,and caspase-3 were significantly decreased in the Exo group(P＜0.05);the expression levels of p62 and Bcl-2 were significantly increased(P＜0.05),and the cellular globularity of cells was increased in the Exo group;the up-regulation of CD 133,OCT4,and SOX2 was found to be statistically significant(P＜0.05).The incubation of AG490 reversed the expression trend of the above indicators in the Exo group(P＜0.05).Conclusion Our findings suggest that AG490 may have the potential to induce autophagy-mediated apoptosis in cervical cancer cells exposed to M2-type TAM-derived exosomes,while also demonstrating the capacity to suppress the tumorigenic properties of cervical cancer.
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