Exosomal MicroRNA-4535 Of Melanoma Stem Cells Promotes Melanoma Metastasis By Inhibiting Autophagy Of Melanoma Cells

Part I Establishment,culture and identification of melanoma stem cell lineObjective: To isolate and verify the stemness of melanoma stem cells(MSC)from melanoma parental cells(MPC).Methods:(1)The cells suspended in MPCs culture medium were collected and cultured again in High Glucose DMEM culture medium containing 10% FBS.When the cells grew to about 50%,the suspended cells in the culture medium were collected for subculture again.The process of "collecting suspended cells in culture medium--continue to culture these cells and then collect suspended cells in culture medium for subculture" was carried out for a total of eight times.Then,the last suspension cells were inoculated into a96-well plate for single cell cloning formation assay to select the cells with strong spheroid formation ability.The experiment was carried out five times in a row.The last monoclonal pelletized cells were subcultured and amplified.(2)To detect the expression of stemness genes oct4,sox2,klf4,aldh1,CD133,bmi1,nanog in MSCs and MPCs.(3)The stemness of MSCs was detected by spheroid formation assay of 1000 cells in 6-well plate.(4)The stemness of MSCs was detected by spheroid formation assay of 1 cell in 96-well plate.(5)The tumorigenicity of MSCs in vivo was tested by subcutaneous tumorigenicity assay in nude mice.Results:(1)Melanoma stem cells(MSCs)from melanin parental cells(MPCs)were successfully isolated by eight suspension polymerization methods.(2)The expression levels of stemness genes oct4,sox2,aldh1,klf4,nanog in MSCs were significantly higher than those in MPCs.(3)The spheroid forming ability of 1000 MSCs in 6 well plate was significantly stronger than that of MPCs.(4)The spheroid forming ability of 1 MSC cell in 96 well plate was significantly stronger than that of MPC cell.(5)The tumorigenicity of MSCs was significantly stronger than that of MPCs in nude mice.Conclusion: Melanoma stem cells screened by eight suspension polymerizations have stemness and can maintain stable stemness continuous passage,which provides a good basis for the subsequent exploration of biological characteristics of melanoma stem cells and their effect on melanoma metastasis.Part II Melanoma stem cell exosomal micro RNA-4535 promotes melanoma metastasis Objective:(1)To investigate whether exosomes exist in the culture medium of MSCs and MPCs.(2)To explore whether the exosomes of MSCs have effects on the function of MPCs.(3)To explore whether micro RNAs in MSC exosomes and MPC exosomes are different.(4)To explore micro RNAs with high expression in MSC exosomes compared with MPC exosomes,and studying the effects of these micro RNAs on MPC functions.Methods:(1)Exosomes were extracted by ultracentrifugation,exosome markers were detected by WB,particle size range and particle concentration were detected by NTA,and exosome structure and particle size were detected by transmission electron microscopy.(2)By co-culturing MSC exosomes and MPC cells,the migration and invasion ability of MPC cells in vitro was detected to verify whether MSC exosomes could affect the metastasis of melanoma.(3)Construction of MSC-sh-RAB27 cell line by Lentiviral stable knockdown of RAB27 in MSC.(4)The secretion of exosomes of MSC-sh-RAB27 cells was detected by NTA.(5)After co-culture MSC-sh-RAB27 cell exosomes with MPC cells,the migration and invasion ability of MPC cells was detected.(6)Micro RNA sequencing and analysis were performed on MPC and MSC exosomes to find micro RNAs with high expression in MSC exosomes.In MPC and MSC cells and their exosomes,the differential micro RNAs obtained through sequencing analysis were screened again by QPCR to obtain micro RNAs with stable and high expression in MSC cells and their exosomes.(7)After micro RNA mimics were used to overexpress micro RNA-4535 in MPC cells,the effects of micro RNA-4535 on MPC cell functions were verified by migration and invasion assay.After inhibiting the expression of micro RNA-4535 in MSC cells with inhibitor,exosomes were collected and co-cultured with MPC cells to detect the migration and invasion ability of MPC cells.(8)Construction of MPC-OEmi R-4535 cell line with stable over-expressing micro RNA-4535 by lentivirus in MPC cells,and then MPC-OE-mi R-4535 cells were injected into NOD/SCID mice through tail vein to detect the ability of metastasize in vivo.(9)By labeling exosomes with PKH26 and micro RNA-4535 with cy5,the two are co-located in MPC cells to detect whether the exosomes of MSC cells carry micro RNA-4535 into MPCs.(10)By overexpressing micro RNA-4535 in MSC cells with mimics,exosomes were collected and co-culture with MPCs,and changes in micro RNA-4535 expression level and migration and invasion ability of MPCs were detected.(11)Micro RNA-4535 mimics were transfected into the exosomes of MSCs by electroporation transfection method.After co-culture these exosomes with MPCs,changes in micro RNA-4535 expression level in MPCs and changes in migration and invasion ability of MPCs were detected.Results:(1)There were exosomes in MPC and MSC cells.The exosome protein markers CD63,CD81,Alix were expressed and the results of NTA showed that the particle size range of exosomes was reasonable.Transmission electron microscopy(TEM)observed the structure and particle size of exosomes.(2)MSC exosomes can promote the migration and invasion ability of MPCs in vitro.(3)After stable knockdown of RAB27 in MSCs,the secretion of MSC exosomes was inhibited.When the exosomes secreted by the same number of 5*10^7 MSC-sh-NC cells and MSC-shRAB27 cells after cultured for 72 h were co-cultured with MPC cells,the ability of MSC-sh-RAB27 group exosomes to promote the migration and invasion of MPCs was weakened.(4)There are some micro RNAs with high expression in MSC exosomes compared with MPC exosomes,such as micro RNA-4535,micro RNA-1268 a,micro RNA-1246,micro RNA-592,micro RNA-4281,micro RNA-93-3p,micro RNA-3529-3p.(6)Highly expressed micro RNA-4535 in MSC exosomes can promote the migration and invasion ability of MPC cells in vitro.(7)MSC exosomal micro RNA-4535 can promote the ability of melanoma metastasis in NOD/SCID mice.(8)MSC exosomes and micro RNA-4535 can co-locate in MPC cells.(9)The expression of micro RNA-4535 in MPCs was increased after the exosomes secreted by MSCs overexpressing micro RNA-4535 were cocultured with MPCs,and the migration and invasion ability of MPCs was enhanced.(10)After inhibiting the expression of micro RNA-4535 in MSCs,micro RNA-4535 content in exosomes secreted by MSCs decreased,and the ability of promoting migration and invasion of MPCs weakened.Conclusions: Micro RNA-4535 from MSC exosomes can be transported into MPCs by exosomes to promote the metastasis ability of melanoma cells.Part III Micro RNA-4535 promotes melanoma metastasis by inhibiting autophagy of MPC cells Objective: To explore the mechanism of melanoma stem cell exosomal micro RNA-4535 promoting melanoma metastasis.Methods:(1)Target genes of micro RNA-4535 were enriched by KEGG signaling pathway to select signaling pathways that micro RNA-4535 mainly act on.(2)After micro RNA mimics was used to overexpress micro RNA-4535 in MPC cells,the expression of autophagy proteins was detected by Western Blot(WB),the changes of autophagy flux were detected by immunofluorescence,and the changes of autophagosomes were detected by transmission electron microscopy(TEM).(3)Micro RNA mimics was used to overexpress micro RNA-4535 in MPC cells and then Rapamycin(Ra Pa)was used to induce autophagy to detect changes in autophagy flux.(4)Micro RNA mimics was used to overexpress micro RNA-4535 in MPC cells,and then Ra Pa was used to induce autophagy,and then the migration and invasion ability of the cells was detected.(5)Screening possible target genes of micro RNA-4535 related to autophagy.(6)QPCR was used to verify whether the expression of ATG13 was affected by micro RNA-4535.(7)Whether micro RNA-4535 directly binds to ATG13 was verified by double luciferase assay.Results:(1)The target genes of micro RNA-4535 were most enriched in autophagy.(2)When micro RNA-4535 was overexpressed in MPC cells,the expression level of autophagic protein P62,LC3 I and LC3 II increased,and the accumulation of LC3 punta was also increased,TEM shows the accumulation of autophagosomes in cells as well.(3)MPC cells overexpressing micro RNA-4535 were treated with Ra Pa to induce autophagy,and the migration and invasion ability of the cells was enhanced.(4)ATG13 is a possible target gene of micro RNA-4535,and the expression level of ATG13 decreased after micro RNA-4535 was overexpressed in MPC cells.(5)The binding of ATG13 and micro RNA-4535 was detected by double luciferase assay,and no direct binding relationship was found between the two.Conclusions: MSC exosomal micro RNA-4535 promotes melanoma metastasis by inhibiting autophagy of MPC cells,but ATG13 is not the direct target gene of micro RNA-4535,and the regulation mechanism of micro RNA-4535 on autophagy needs to be further explored.