A Preliminary Investigation Of Autophagy In Angiosarcoma Cells Mediated By Exosomal HSP90α Via MTOR Signaling Pathway

Objective: We aim to explore the role of exosomal HSP90α derived from angiosarcoma stem-like cells(Sphere cells)on tumor progression by inhibiting autophagy in angiosarcoma cells(ISOHAS cells),and clarify the key molecular events of HSP90α in ISOHAS cell autophagy and its effect on ISOHAS cell proliferation.Methods:(1)Exosomes were isolated from cell culture supernatant by ultracentrifugation,and their morphology,size and colloidal gold labeling were observed by transmission electron microscopy to determine the localization of HSP90α on exosomes.The expression of exosomes markers CD63,CD81 and the level of HSP90α carried were detected by Western blot.(2)PKH67 labelled exosomes derived from ISOHAS cells and Sphere cells respectively.After incubation with ISOHAS cells,Cell Mask was used to labele the cell membrane and DAPI was used to labeled the nucleus.The uptake of exosomes by ISOHAS cells was observed under fluorescence inverted microscope.Exosomes on the regulation of HSP90αexpression in ISOHAS cells and their effects on autophagy-related molecules Beclin 1,P62 and LC3 were detected by Western blot.The formation of autophagic vesicles was observed by MDC staining and AO staining.(3)Western blot was used to verify the expression of HSP90α in ISOHAS cells and Sphere cells is consistent with the effect of exosomes on ISOHAS cells.Western blot verified whether the expression of autophagy-related molecules Beclin 1,P62,LC3 in ISOHAS cells and Sphere cells is consistent with the effect of exosomes on ISOHAS cells.The formation of autophagic vesicles was observed by MDC staining and AO staining.(4)HSP90α overexpression plasmid and si RNA were used to overexpress and silence HSP90α in ISOHAS cells respectively.The effect of intervention of HSP90α on the expression of autophagy-related molecules Beclin 1,P62 and LC3 was further observed by Western blot.The formation of autophagic vesicles was observed by MDC staining and AO staining.LC3 B was labeled by immunofluorescence staining to observe the effect of intervening HSP90α expression on autophagic flow after blocking autophagic flow.(5)Using the String database to screen the interacting proteins with high confidence with HSP90α,and co-immunoprecipitation to verify whether HSP90α binds to the interacting proteins at the protein level.The activation state of PI3K-Akt-mTOR signaling pathway after intervention of HSP90α expression was verified by Western blot.(6)The effect of HSP90α on the proliferation activity of ISOHAS cells was detected by CCK8.The autophagy inducer RAPA and autophagy inhibitor 3-MA were further used to evaluate the effect of autophagy on the proliferation viability of ISOHAS cells.Results:(1)Exosome-derived HSP90α is located on the surface of exosomes.ISOHAS cells and Sphere cell-derived exosomes can be taken up by ISOHAS cells after incubation with ISOHAS cells.Then the expression of HSP90α,Beclin 1 and P62 in ISOHAS were increased(P<0.05),the ratio of LC3II/LC3 I was decreased(P<0.001).Compared with ISOHAS cell-derived exosomes,Sphere cell-derived exosomes had more significant up-regulation effects on HSP90α,Beclin 1,P62 and down-regulation of LC3II/LC3 I ratio(P<0.001).The results of MDC and AO fluorescence staining indicated that the number of autophagosomes decreased after exosome treatment.(2)Compared to ISOHAS cells,HSP90α,Beclin 1,and P62 were highly expressed in Sphere cells(P<0.001),and the ratio of LC3II/LC3 I was lower than that of ISOHAS cells(P<0.01).(3)Overexpression of HSP90α can promote the expression of Beclin 1 and P62(P<0.001),and reduce the ratio of LC3II/LC3I(P<0.01).After expressing HSP90α,the number of autophagosomes decreased,and the autophagic flux was attenuated.(4)Inhibiting the expression of HSP90α can inhibit the expression of Beclin 1 and P62(P<0.001),increase the ratio of LC3II/LC3I(P<0.01).The results of MDC,AO fluorescence staining and LC3 B immunofluorescence staining showed that the number of autophagosomes increased and the autophagic flux was enhanced after silencing HSP90α.(5)The String database screened out the interacting proteins with high confidence(Score value =0.999)with HSP90α,and determined that AKT1 may interact with HSP90α to participate in the mTOR signaling pathway and affect the autophagy process.Co-immunoprecipitation verifies that HSP90α has a direct binding effect with AKT.After overexpression of HSP90α,the expressions of PI3 K,Akt,p-Akt,mTOR,and p-mTOR were up-regulated(P<0.001).After HSP90α was silenced,the expressions of PI3 K,Akt,p-Akt,mTOR,and p-mTOR were down-regulated(P<0.001),suggesting that HSP90α may inhibit autophagy by binding to Akt to activate the mTOR signaling pathway.(6)Overexpression of HSP90αsignificantly promoted the proliferation of ISOHAS cells(P<0.01),whereas silencing HSP90α significantly inhibited the proliferation of ISOHAS cells(P<0.01).RAPA inhibited the proliferation of ISOHAS cells after inducing autophagy within a certain concentration range(10n M～100n M)(P<0.05).3-MA inhibited autophagy in a certain concentration range(10μM～50μM)and promoted the proliferation of ISOHAS cells(P<0.05),while excessive 3-MA(100μM)inhibited the proliferation of ISOHAS cells(P<0.05).Conclusions:(1)Angiosarcoma stem cell-like cells-derived exosomes inhibit autophagy and promote sarcoma cell proliferation by up-regulating the expression of HSP90α in angiosarcoma cells.(2)HSP90αmay inhibit autophagy and promote the proliferation of angiosarcoma cells by combining with Akt to activate the mTOR signaling pathway.