Mechanism Research Of Pancreatic Stellate Cells Activation Induced By Extracellular Vesicles Derived From Pancreatic Cancer Cells

BackgroundThe interaction between pancreatic cancer cells and pancreatic stellate cells?PSCs?creates a tumor microenvironment that supports the growth,metastasis,drug resistance and immune tolerance of pancreatic cancer.In pancreatic cancer,quiescent PSCs are transformed into activated PSCs and activated PSCs play a vital regulative role in the progression of pancreatic cancer.Extracellular vesicles?EVS?are nanoscale vesicles,which are rich in microRNAs,proteins,lipids and other types of nucleic acids,play an important role in mediating communication between cells.Pancreatic cancer cells can induce PSCs activation by paracrine,however,the role and mechanism of extracellular vesicles derived from pancreatic cancer cells in PSCs activation remain unclear.ObjectiveIn this study,we aim to explore the role and mechanism of extracellular vesicles derived from pancreatic cancer cells in the activation of pancreatic stellate cells and to search for potential diagnostic markers and therapeutic targets of pancreatic cancer.MethodsTranswell was used for indirect co-culture experiment,which was divided into three groups.PSCs cultured alone were setted as control group,PSCs co-cultured with PANC-1were setted as PANC-1 group,and PSCs co-cultured with PANC-1 and EVs inhibitor GW4869were setted as PANC-1+GW4869 group.Total cell proteins were extracted after co-culture for48 hours and Western Blot was adopted to measure the expression of NF-?B in PSCs.EVs were extracted from the supernatants of HPDE6-C7,PANC-1 and SW1990 by ultracentrifugation.The isolated EVs were identified by transmission electron microscope,Western blot and nanoparticle tracking analysis.PSCs were treated with isolated EVs derived from pancreatic cancer cells,and the effects of extracellular vesicles on the proliferation and migration of pancreatic stellate cells were detected by cell counting kit-8?CCK-8?and transwell,respectively.qRT-PCR of microRNA was used to detect the expression of miR-301a-3p in HPDE6-C7,PANC-1,SW1990,H-EVs,SW-EVs and P-EVs.PSCs were transfected with mi R-301a-3p mimics.CCK-8 and transwell methods were respectively employed to measure the functions of miR301a-3p overexpression on the proliferation and migration of pancreatic stellate cells.Western Blot was used to detect the effect of miR-301a-3p overexpression on the expression of NF-?B in pancreatic stellate cells.ResultsThe results of indirect co culture showed that PANC-1 could induce up-regulation of?-SMA and NF-?B in PSCs compared with the control group?P<0.001?,while GW4869,an EVs inhibitor,could attenuate this induction?P<0.05?.Therefore,we speculated that PANC-1 could induce PSCs activation by secreting EVs.Evs of pancreatic cancer cells could promote the proliferation,migration and expression of NF-?B of PSCs.The morphology of pellets obtained by ultracentrifugation under transmission electron microscope was consistent with that of classical EVs;the results of Western blot showed that the pellets were CD63 and TSG101 positive,while GM130 negative,which was consistent with the expression of EVs marker protein;the tracking analysis of nanoparticles showed that the diameter of most pellets was about 130nm,belonging to the category of small EVs.The results showed that the isolated pelltes were classical small EVs.The results of EVs uptake experiment showed that PSCs could swallow pancreatic cancer cell-derived EVs.CCK-8 results showed that SW-EVs at concentration of 50?g/ml and 80?g/ml could promote proliferation of PSCs?P<0.001and P<0.01?,and P-EVs at concentration of 50?g/ml and 80?g/ml also could promote proliferation of PSCs?P<0.001?.Transwell results showed that both SW-EVs and P-EVs?50?g/ml?could promote the migration of PSCs?P<0.01 and P<0.001?.The results of Western blot showed that co culture of P-EVs and SW-EVs with PSCs could induce up regulation of?-SMA expression?P<0.001 and P<0.01?and up regulation of NF-?B expression?P<0.01 and P<0.05?.qRT-PCR showed that the expression of miR-301a-3p in PANC-1 and SW1990 was higher than that in HPDE6-C7?P<0.001 and P<0.01?,and the expression of miR-301a-3p in P-EVs and SW-EVs was higher than that in H-EVs?P<0.001?,and P-EVs and SW-EVs co cultured with PSCs could up regulate the expression of miR-301a-3p in PSCs?P<0.001?.The expression of mi R-301a-3p in PSCs was significantly up-regulated?P<0.001?after transfected with mi R-301a-3p mimics.Overexpression of miR-301a-3p can promote the proliferation(P_24h<0.05,P_48h<0.05,P_72h<0.01),migration?P<0.01?and up regulate the expression of NF-?B?P<0.05?in PSCs.ConclusionThe expression of?-SMA and NF-?B in PSCs can be induced by EVs derived from pancreatic cancer cells.The EVs derived from pancreatic cancer cells can promote the proliferation and migration of PSCs.The expression of miR-301a-3p in the extracellular vesicles of pancreatic cancer cells was increased and the EVs could transfer miR-301a-3p to the PSCs to promote the proliferation,migration and the expression of NF-?B of PSCs.