Effects of exosomes targeted delivery of integrin αv/β3 small interfering RNA on proliferation,migration,and invasion of anaplastic thyroid carcinoma cells
Objective To explore the effects of exosomes targeted delivery of integrin αv/β3 small interfering RNA(si-αv/β3)on the proliferation,migration,and invasion of anaplastic thyroid carcinoma(ATC)cells.Methods The expression levels of integrin αv/β3 in ATC cell lines(8505C,Hth7)and human normal thyroid cells(Shanghai Institutes of Cell Biology for Biological Sciences)were detected by Western blotting.We transfected internalized arginine-glycine-aspartic acid peptide(iRGD)into HEK-293T cells and then obtained iRGD targeted exosomes by ultracentrifugation.The targeted ability of exosomes was validated by PKH26 staining.iRGD targeted exosomes delivering si-αv/β3(denoted as iR-GD-exos-si-av/β3 group)were obtained by transfection of si-αv/β3 into iRGD-293T cells.The mRNA and protein expression levels of integrin αv/β3 in 8505 C cells were detected by quantitative polymerase chain reaction(qPCR)and Western blotting.The proliferative,migratory,and invasive abilities were analyzed by cell counting kit-8(CCK-8)assay,scratch test,and Transwell assay in 8505C cells.The measurement data between groups were compared using a t-test and one-way analysis of variance was used for comparison between multiple groups.Results The expression level of integrin αv/β3 in 8505C and Hth7 cells was significantly higher than that in human normal thyroid cells(αv:1.04±0.12,0.90±0.11 vs.0.31± 0.01,F=50.11,P＜0.01;β3:0.93±0.23,0.89±0.16 vs.0.33±0.27,F=6.81,P＜0.05).The fluorescence signal in the 8505C cells of iRGD-targeted exosomes group was significantly stronger than that of non-targeted exosomes(47.18±10.39 vs.5.64±1.43,t=6.86,P＜0.05).After transfection with si-αv/β3 into 8505C cells,the mRNA expression level was significantly lower than that of the control group(αv:0.20±0.01 vs.1.21±0.14,t=12.49,P＜0.05;β3:0.21±0.02 vs.1.01±0.18,t=7.90,P＜0.05),and the protein expression was also significantly lower than that of the control group(αv:0.45±0.03vs.0.88±0.06,t=10.33,P＜0.05;β3:0.48±0.09vs.1.10±0.11,t=9.02,P＜0.05).The mRNA level of iRGD-exos-si-av/β3 group was significantly lower than that of the empty vector group after co-culture with 8505C cells(αv:0.30±0.08 vs.1.05±0.02,t=16.75,P＜0.05;β3:0.27±0.05 vs.1.29±0.20,t=8.71,P＜0.05),and the protein expression level was also significantly lower than that of the latter group(αv:0.60±0.30 vs.1.20±0.23,t=5.72,P＜0.05;β3:0.24± 0.11 vs.1.26±0.14,t=16.23,P＜0.05).The migratory rate of iRGD-exos-si-av/β3 group was signif-icantly lower than that of the empty vector group at 24 h and 48 h after scratch,respectively[si-αv at 24 h:(13.00±1.10)％vs.(36.00±4.00)％,t=9.60,P＜0.01 and si-β3 at24 h:(7.00±1.60)％vs.(36.00±4.00)％,t=11.34,P＜0.01;si-αv at 48 h:(17.00±1.20)％vs.(65.00±2.50)％,t=28.89,P＜0.01 and si-β3 at 48 h:(22.00±1.80)％vs.(65.00±2.50)％,t=24.01,P＜0.01].The number of migrating cells in the iRGD-exos-si-av/β3 group was significantly fewer than that in the empty vector group[si-αv:(3 361.67±860.52)cells vs.(7 620.67±1 008.46)cells,t=5.56,P＜0.05;si-β3:(2 868.33±1 499.92)cells vs.(7 620.67±1 008.46)cells,t=4.55,P＜0.01].Conclusion iRGD targeted exosomes could deliver si-αv/β3 to ATC cells,thus signifiicantly weakened their proliferative,migratory and invasive abilities by downregulating the expression levels of integrin αv/β3.
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