Exosomes from Echinococcus granulosus cyst fluid regulate the proliferation and apoptosis of Hepa1-6 mouse hepatocellular carcinoma cells through JAK2/STAT3 signaling pathway
Objective To investigate the existence of exosomes in the hydatid cyst fluid and whether they regulate the proliferation and apoptosis of mouse hepatocellular carcinoma cells Hepa1-6 through JAK2/STAT3 signaling pathway.Methods Hepa1-6 cells were di-rectly interfered with Echinococcus granulosus cyst fluid.CCK8 and EdU were used to detect the effect of Echinococcus granulosus cyst fluid on the proliferation of Hepa1-6 cells.Ultrafast centrifugation method was used to extract exosomes from the hydatid cyst fluid,and the exosomes were verified by transmission electron microscopy,Western-Blotting and particle size analysis.The entry of exosomes into cells was verified by exosom-PKH67 phagocytosis test;The effect of exosomes in the hydatid cyst fluid on the proliferation of He-pa1-6 cells was detected by CCK8 and EDU.The effect of exosomes on apoptosis of Hepa1-6 cells was detected by Western-Blotting.The expression of JAK2/STAT3 signaling pathway related proteins was detected by Western-Blotting,and the relationship between this pathway and the proliferation of hepatoma cells was explored through correlation studies.Results 1.The hydatid fluid promoted the proliferation of Hepa1-6;2.Western-Blotting results showed positive expressions of CD63,CD9,TSG101 in the exosome surface mark-ers of echinococcus granulosus cyst fluid.3.Transmission electron microscopy showed that the exosomes were disk-like vesicles with double-layer membrane structure,and particle size analysis showed that the vesicles were distributed in a diameter range of 159.2±81.1 nm;4.Exosomes can enter cells within 24 hours;5.CCK8 and EdU results showed that the exosomes of echinococcus granulosus cyst fluid could promote the proliferation of Hepa1-6 cells at both 24h and 48h(P＜0.05).6.Western-Blotting results showed that the exosomes of fine echinococcus hydatid fluid promoted the expression of Bcl-2 at 24h and 48h after Hepa1-6 intervention,and the promoting effect increased with the increase of concentration(P＜0.05).The expressions of Bax,caspase-3 and caspase-9 de-creased,and the inhibitory effect increased with the increase of concentration.7.Western-Blotting results showed that the expression levels of t-JAK2 and t-STAT3 did not change significantly after the intervention of Hepa1-6 by exosomes in the fine hydatid cyst fluid for 24 h,and the protein expressions of p-JAK2 and p-Stat3 increased with the increase of exosomes concentration,and they were posi-tively correlated with the proliferation of liver cancer cells.Conclusion Echinococcus granulosus cyst fluid and its exosomes can pro-mote the proliferation of Hepa1-6 and inhibit its apoptosis through JAK2/STAT3 signaling pathway.
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