Exosomal Transfer Of MiR-501 Confers Doxorubicin Resistance And Tumorigenesis Via Targeting Of BLID In Gastric Cancer

Research Backgroud:Gastric cancer is the fifth most common type of malignancy and the third leading cause of cancer-related death globally.Gastric cancer is commonly diagnosed at advanced stages due to a lack of novel molecular biomarkers and resulting in approximately 20-30%5-year survival rate.Currently,traditional chemotherapy still represents the primary option for advanced gastric cancer.Doxorubicin(adriamycin,ADR)has been used to treat numerous malignancies,including gastric cancer,in combination with fluorouracil,cisplatin,paclitaxel and mitomycin.However,resistance to doxorubicin remains an obstacle to gastric cancer treatment.Exosomes are a class of 30-100 nm sized extracelular vesicles(EVs)with lipid bilayer membranes.Exosomes carrying biomolecules including lipids,proteins and RNAs can be released into the extracelular environment and thereafter affect their biological activities.The cargo of RNAs in the exosomes has garnered a lot of attention from the researchers,including DNA,mRNA,long non-coding RNA(LnRNAs),microRNA(miRNAs)and circular RNA(circRNAs),especially miRNAs.The lipid membranes of exosomes protect the internal miRNAs from being digested by the RNases.Emerging evidence has demonstrated that tumor cels secreted exosomes play a crucial role in regulating recipient cell growth,invasion and metastasis,and chemoresistance through transmitting miRNAs.MicroRNA-501-5p(miR-501)is a recently identified novel oncomir.Several studies have reported that miR-501 is overexpressed in hepatocellular carcinoma(HCC),lung adenocarcinoma,cervical cancer and gastric cancer tissues.Elevated miR-501 promotes the progression of lung adenocarcinoma and cervical cancer,and predicts poor prognosis of gastric cancer.Our previous study has demonstrated that miR-501 enhances doxorubicin resistance and tumorigenesis in gastric cancer by targeting BH3-like motif containing,cell death inducer(BLID).However,whether miR-501 can be transmitted via exosomes and the roles of exosomal miR-501 in the chemoresistance and tumorigenesis of gastric cancer are not clear.Objectives:(1)To investigate the relationship between exosomal miR-501 and BLID.(2)To investigate the mechanism of exosomal miR-501 via targeting of BLID to enhance the resistance of gastric cancer to doxorubicin in vivo and in vitro.Methods:(1)SGC7901/ADR and SGC7901 cells were cultured in medium containing 10%exosome-free FBS,exosomes(ADR Exo and 7901 Exo)were isolated from conditioned medium using ultracentrifugation method(120000×g/min).Transmission electron microscope,Nanosight particle tracking analysis and Western blot analysis are used to confirm exosomes.(2)After co-culturing with ADR Exo,the expression level of miR-501 in SGC7901 cels was detected by real-time qRT-PCR,and the IC50 of SGC7901 cels was measured by CCK-8 assay.ADR Exo labeled with PKH26,which is a fluorescent tracer,was coincubated with SGC7901 recipient cels which were stained the nuclei with DAPI(blue).Confocal microscopy showing exosomes having been internalization by SGC7901.SGC7901/ADR cels were transfected with Cy3-miR-501 mimic and replated in the upper chamber of a coculture system with 0.4?m pores,which inhibit direct contact with the cels and alow the transmission of exosomes but not larger particles.After 24 h of coculture,strong red fluorescence was observed in SGC7901 cels seeded in the lower chamber.(3)Lentiviral vectors expressing a miR-501 inhibitor(7901 501KD),negative control miRNA inhibitor(7901 NCi)or BLID(7901 BLID)infected SGC7901 cels coculture with ADR Exo or PBS respectively.SGC7901 cels were cocultured with SGC7901/ADR cels prior treated with GW4869(ADR GW4869)to block exosome secretion.SGC7901cels were cocultured with SGC7901/ADR cels pre-treated with DMSO(ADR DMSO)as a negative control.Additionally,SGC7901/ADR cels transfected with miR-501 inhibitor(ADR 501i)to knock down miR-501 expression were cocultured with SGC7901 cels.The co-transfection of miR-501 inhibitor and siRNA specific for BLID(ADR 501i+siBLID)and negative control inhibitor(ADR NCi)were as the negative controls.The levels of miR-501 and BLID mRNA were detected by TaqMan miRNA probes and 2×TransStar Top Green pPCR SuperMix.The proteins of BLID and cleaved caspase-9/-3,phosphorylation of Akt(p-Akt)were examined by Western blot analysis.The CCK-8 assay was detected the IC50 of doxorubicin.The flow cytometry analysis,colony formation and Transwell assays were used to detect the effects of exosomal miR-501 on apoptosis,proliferation,migration and invasion.(4)Twenty male BALB/c nude mice randomly divided into four groups.Groups 1 and 2were injected with 7901 NCi,group 3 was injected with 7901 501KD,and group 4 was injected with 7901 BLID.Infected SGC7901 cels(1×10~6)in 100?l PBS and Matrigel were subcutaneously injected into each nude mouse.When the volume of the xenografts reached approximately 50 mm~3,the mice in groups 2,3 and 4 were intratumorally injected with ADR Exo(50?g)three times for a week.The negative control of group 1 was injected with the same volume of PBS.When the volume of the xenografts was approximately up to 100 mm~3,the mice of all groups were administered ADR(5 mg/kg)through the tail vein three times a week.After 2 weeks,the mice were sacrificed,and the subcutaneous tumors were excised and frozen in liquid nitrogen for extration of RNA to detect the levels of miR-501 and BLID mRNA and protein that was used to detect BLID and the downstream of cleaved caspase-9/-3,p-Akt.Results:(1)we first isolated exsomes from the conditioned medium of SGC7901 and SGC7901/ADR using ultracentrifugation method.The vesicles exhibited a round shaped with bilayered membranes,and the diameter ranged from 30 nm to 100 nm under the transmission electron microscope.The Nanosight particle tracking analysis(NTA)further identified the size distribution and concentration of exosomes extracted from the two cell lines.The predominant size was 100 nm,and the concentrations at the size of 100 nm were4.6×10~8 particles/ml and 2.8×10~9 particles/ml generated from SGC7901 and SGC7901/ADR cels,respectively,after multiplying by the dilution factors.The exosomal markers CD63,CD81 and HSP70,were detected in the exosomes.These results indicate that the vesicles isolated from SGC7901 and SGC7901/ADR display typical characteristics of exosomes.(2)The miR-501 in SGC7901/ADR secreted exosomes(ADR Exo)was 2.54±0.31-fold greater than that in SGC7901 exosomes(7901 Exo)(P<0.01).Confocal microscopy showing that PKH26 labeled ADR Exo are successfuly uptaken by SGC7901 recipient cels.SGC7901/ADR cels transfected with Cy3-miR-501 mimic(red fluorescence)were placed in the upper chamber and coincubated with SGC7901 cels seeded in the lower chamber in a coculture system with a 0.4?m pore membrane.The red fluorescence was observed in the SGC7901 recipient cels under a fluorescence microscope.The level of miR-501 was dramatically upregulated in SGC7901 cels after co-incubation with ADR Exo.The CCK-8 assay showed that ADR Exo induced doxorubicin resistance in SGC7901cels in a dose-dependent manner.(3)The 7901 NCi,7901 501KD and 7901 BLID cells are respectively incubated with ADR Exo.The 7901 NCi cels incubated with the equivalent amount of PBS(7901 NCi+PBS)were used as the negative control.The real-time qRT-PCR analysis showed that miR-501expression in 7901 NCi was dramatically increased in the presence of ADR Exo(P<0.001).On the contrary,the expression of BLID at both the mRNA and protein level was decreased in 7901 NCi+ADR Exo compared with that of 7901 NCi+PBS(P<0.001);however,miR-501 knockdown or BLID overexpression abolished the ADR Exo-induced BLID decrease(P>0.05).ADR Exo dramatically enhanced the doxorubicin resistance of7901 NCi cels(P<0.05),whereas no significant difference was seen in miR-501-silenced or BLID-overexpressing SGC7901 cels(P>0.05).After coculturing with GW4869blocked the exosome secretion of SGC7901/ADR cels(ADR GW4869),the expression of miR-501 of SGC7901 cels was notably decreased compared to that of DMSO-treated cels(P<0.05).BLID expression in SGC7901 cels cocultured with inhibited exosome secretion or knocked down the miR-501 of SGC7901/ADR at both the mRNA and protein levels was higher than that in the negative controls(P<0.01).The doxorubicin resistance of SGC7901 cels was reversed when coculturing with exosome block or miR-501knockdown SGC7901/ADR cels(P<0.05).(4)The 7901 NCi cels coculturing with ADR Exo showed a significant decline(35.57%±1.46%,P<0.01)in apoptosis compared to that of the negative control.But the apoptotic rates in 7901 501KD+ADR Exo and 7901 BLID+ADR Exo did not show any obvious difference to the 7901 NCi+PBS(P>0.05).The apoptotic rate of SGC7901 in the lower transwell chamber was increased about 31.20%±1.65%(P<0.05)after coculturing with GW4869-treated SGC7901/ADR than DMSO-treated.Meanwhile,SGC7901 cels exhibited higher apoptotic rate when coculturing with ADR 501i than those with ADR NCi or ADR 501i+siBLID respectively(P<0.05).The cleaved caspase-9 and caspase-3were inhibited in the 7901 NCi+ADR Exo group than those in the 7901 NCi+PBS group.No notable difference was observed in the 7901 501KD+ADR Exo and 7901 BLID+ADR Exo cels compared with the 7901 NCi+PBS cels.On the contrary,the cleaved caspase-9and caspase-3 were overexpressed in the 7901+ADR GW4869 group and 7901+ADR 501i group than corresponding negative control.(5)The 7901 NCi cels treated with ADR Exo exhibited increased colony formation,migration and invasion capacities compared to treated with PBS(P<0.05),however,there was no obvious alteration when 7901 501KD or 7901 BLID cels co-culturing with ADR Exo compared to 7901 NCi cels co-culturing with PBS(P>0.05).The proliferation,migration,and invasion abilities of SGC7901 cels after co-culturing with ADR GW4869cels were suppressed compared to co-culture with ADR DMSO cells(P<0.05).SGC7901cels cocultured with ADR 501i cels were attenuated in the capacities of proliferation,migration and invasion(P<0.05).SGC7901 cels cocultured with ADR NCi or ADR501i+siBLID cels respectively did not display dramatic difference in the numbers of colonies,cels passing through the membrane with or without Matrigel(P>0.05).The phosphorylation of Akt at Ser 473(p-Akt)was significantly activated in 7901 NCi cels after co-incubating with ADR Exo instead of PBS.The 7901 501KD or 7901 BLID cels after co-culturing with ADR Exo showed no dramatic difference compared to 7901NCi+PBS cels.Conversely,ADR GW4869 cels co-culturing with SGC7901 cels inactivated the phosphorylation of Akt compared to ADR DMSO cels.Consistently,p-Akt was depressed in SGC7901 cels co-culturing with ADR 501i cels compared with co-culturing with ADR NCi cels.No significant difference was seen in SGC7901 cels co-culturing with ADR NCi or ADR 501i+siBLID cels.(6)The tumor volumes of 7901 NCi+ADR Exo+ADR group were significantly larger than those of 7901 NCi+PBS group after doxorubicin treatment,and no obvious difference was seen among the three groups of 7901 NCi+PBS+ADR,7901 501KD+ADR Exo+ADR and7901 BLID+ADR Exo+ADR(P<0.01).The 7901 NCi group injected with ADR Exo and doxorubicin had higher expression of miR-501 compared to 7901 NCi group injected with PBS and doxorubicin(P<0.05).There was no marked alteration between 7901501KD+ADR Exo+ADR and 7901 NCi+PBS+ADR(P>0.05).On the contrary,the expressions of BLID at both mRNA and protein levels were decreased in the 7901NCi+ADR Exo+ADR group(P<0.05).No obvious difference was reached among three groups of 7901 NCi+PBS+ADR,7901 501KD+ADR Exo+ADR and 7901 BLID+ADR Exo+ADR(P>0.05).The cleavage of caspase-9 and caspase-3 were markedly decreased,and the protein of p-Akt was increased significantly in 7901 NCi group injecting with ADR Exo and ADR compared with 7901 NCi group injecting with PBS and ADR.No difference was found among 7901 NCi+PBS+ADR,7901 501KD+ADR Exo+ADR and7901 BLID+ADR Exo+ADR.Conclusions:(1)Exosomal miR-501 promotes gastric cancer proliferation,migration,invasion and doxorubicin resistance,whereas inhibits apoptosis in vitro and in vivo.(2)Exosomal miR-501 promotes doxorubicin resistance by inhibiting the expression of BLID,thereby inactivating the caspase-9/-3 pathway.Additionally,exosomal miR-501 can enhance gastric cancer cels proliferation,migration and invasion through phosphorylation of Akt.As a result,the exosomal miR-501 might serve as a potential diagnostic biomarker and a novel therapeutic target for gastric cancer.