| Objective: To investigate the effect of skin squamous cell carcinoma-derived exosome miR-125 b on proliferation,migration and invasion of skin squamous cell carcinoma.Methods:(1)Human keratin-forming cells HACAT and skin squamous carcinoma cell lines A431 and SCL-1 were routinely cultured,and miR-125 b was detected in HACAT and A431 and SCL-1 cells by QPCR(Real-time quantitative polymerase chain reaction).expression in HACAT and A431 and SCL-1 cells.(2)Exosomes were extracted from the above three cell lines by differential centrifugation,and the above exosomes were identified by transmission electron microscopy,nanoparticle analysis and Western blot.(3)QPCR was used to detect the difference of miR-125 expression in exosomes.(4)A431 cells were transfected with mimic NC,miR-125 b mimic,and the supernatants were collected 48 h after transfection to isolate exosomes,as exo-NC,exo-miR-125 b,and identified by transmission electron microscopy.(5)The expression of miR-125 b in the composite exosomes was detected by QPCR.(6)After incubation with PKH67,the composite exosomes were incubated with A431 and SCL-1 cells stained with DAPI stain for 0h,6h and 12 h,respectively,followed by observation of exosome uptake in squamous carcinoma cell lines using laser confocal microscopy.(7)The expression of miR-125 b in the cells was detected by QPCR after incubation with A431 and SCL-1 cells for 48 h using miR-125 b overexpressed exosomes and NC exosomes,followed by MTT assay and Transwell assay to detect the effect of compound exosomes miR-125 b on the proliferation and migration of skin squamous carcinoma cell lines A431 and SCL-1,respectively,SCL-1proliferation,migration and invasion ability by MTT assay and Transwell assay,respectively.Results:(1)QPCR results showed that miR-125 b expression was down-regulated in A431 and SCL-1cells compared with HACAT cells(P<0.001).(2)After extracting the exosomes by differential centrifugation,the results from the electron microscopic detection showed that: the exosome structure was intact,the diameter was between 100-200 nm,and the number was high,which was the exosome structure and could be used for the subsequent experimental detection;the results from the particle size analysis graph showed that: the supernatant particle size of the isolated SCL-1 cells was 126.8±44.1,the supernatant particle size of HACAT1 cells was 134.0 ± 39.3,Most of the isolated exosomes were below 200 nm in size,indicating that the isolates were exosomes;the expression of CD9 and CD63 in the extracted exosome samples were positive,indicating that the exosome extraction was successful.(3)QPCR results could be obtained that miR-125 b expression was significantly down-regulated in A431 and SCL-1 exosomes compared with HACAT exosomes(P<0.001).(4)The results of exosome electron microscopy showed that the composite exosomes(exo-NC,exo-miR-125b)were structurally intact,with diameters between 100-200 nm and a high number of exosomes,and could be used for subsequent experiments.(5)QPCR results showed that miR-125 b expression was significantly upregulated in exo-miR-125 b compared with exo-NC group(P<0.001).(6)The graph of exosome uptake experiment showed that the uptake of exosomes by A431 and SCL1 cells increased with the extension of culture time.(7)QPCR results showed that miR-125 b expression was significantly up-regulated in exosomes compared with exo-NC group(P<0.001).(8)MTT assay results showed that the proliferation ability of cells treated with miR-125 b overexpressed exosomes was decreased compared with A431 and SCL-1 control groups(P<0.001).From the Transwell migration assay,it was obtained that the cell migration ability of A431 and SCL-1 cells decreased after incubation with miR-125 b overexpressing exosomes for 48h(P<0.001).As evidenced by Transwell invasion assay: A431 and SCL-1 cells showed decreased cell invasion ability after incubation with miR-125 b overexpressing exosomes for48h(P<0.001).Conclusion: Skin squamous cell carcinoma-derived exosome miR-125 b can inhibit the proliferation,migration,and invasion of CSCC cells.CSCC-derived exosome miR-125 b has potential as a novel diagnostic biomarker and clinical therapeutic target for CSCC in the future. |
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