| Objective:Study of the effects of exo-miR-31 on the biological behavior of cutaneous squamous cell carcinoma cells in vitro.Methods:(1)The expression of miR-31 in Ha Ca T cell,A431 cell and SCL-1 cell were detected by q PCR.(2)Isolate and identify the exosomes from the above three cells.(3)The expression of miR-31 in three kinds of exosomes was found by q PCR.(4)A431 cells were transfected with mimic-NC and miR-31 mimic respectively,and then the supernatant was collected and exosomes were isolated to obtain exo-NC and exo-miR-31.(5)The observation of exosomes’ morphology and the expression of miR-31 in the two kinds of exosomes have been observed and detected.(6)The uptake of PKH67-labeled exosomes in A431 cells and SCL-1 cells was observed by laser confocal microscopy at 0h,6h and 12 h after co-incubation with DAPI tinting.(7)The expression of miR-31 in A431 and SCL-1 cells after incubation with exo-NC and exo-miR-31 has been tested by q PCR.(8)MTT and Transwell were used to discover the proliferation,migration and invasion of A431 and SCL-1 cells after incubation with exo-NC and exo-miR-31.Results:(1)The expression of miR-31 in A431 cells was significantly higher than that in Ha Ca T cells(P < 0.001).The expression of miR-31 in SCL-1 cells was significantly higher than that in Ha Ca T cells(P < 0.05).(2)The exosomes were round vesicles with intact structure,and the diameter was between 100 nm and 200 nm.(3)The particle sizes of Ha Ca T-exo,A431-exo and SCL-1-exo were 134.0±39.3nm,138.6±35.2nm and 126.8±44.1nm,respectively.CD63 and CD9 were expressed in Hacat-Exo,A431-Exo and SCL-1-Exo.(4)Compared with Ha Ca T-exo,miR-31 was highly expressed in A431-exo and SCL-1-exo,with statistically significant differences(P < 0.001).(5)Compared with exo-NC group,miR-31 was highly expressed in exo-miR-31 group,and the difference was statistically significant(P< 0.001).(6)Exosome uptake: A431 cells and SCL-1 cells took up a small amount of exosomes at 6 hours,and the uptake of exosomes was increased at 12 hours.(7)Compared with A431 group and A431+exo-NC group,the level of miR-31 in A431+exo-miR-31 group was significantly higher(P < 0.001).Compared with SCL-1 group and SCL-1+exo-NC group,the level of miR-31 in SCL-1+exo-miR-31 group was higher,and the difference was statistically significant(P < 0.001).(8)Compared with A431 group and A431+exo-NC group,the proliferation,migration and invasion ability of A431+exo-miR-31 group were enhanced.The difference was statistically significant(P < 0.001).Compared with SCL-1 group and SCL-1+exo-NC group,the proliferation,migration and invasion ability of SCL-1+exo-miR-31 group were enhanced.The difference was statistically significant(P <0.001).Conclusion:Exo-miR-31 enhanced the proliferation,migration and invasion of CSCC cells in vitro,which may play a role in promoting cutaneous squamous cell carcinoma. |
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