| Part 1 Inhibitory effect of salidroside on proliferation,invasion and migration of osteosarcoma cellsObjective:To investigate the effect of salidroside on the proliferation,invasion and migration of osteosarcoma 143B cells.Methods:Human osteosarcoma cell line 143B was cultured.After 24h,48,72h of treatment with 30 ? mol/L and 60 ? mol/L concentration salidroside,MTT assay was used to decteted the cell proliferation ability.After 24 hours of treatment with 30 mol/L and 60 ? mol/L concentration salidroside,cell invasion was detected by Transwell assay,cell migration was detected by scratch assay,mRNA and protein expression were detected wih RT-PCR and Western blot,respectively.Results:The results showed that after 24h treatment,absorbance value of 60 mol/L group(0.038 ± 0.002)and 30 ? mol/L group(0.054 ± 0.002)143B cell were lower than the control group(0.074 ± 0.004),t1=2.659,P=0.016;t2=1.993,P=0.039.After 48h treatment,absorbance value of 60 ? mol/L group(0.073 ± 0.001)and 30 ? mol/L group(0.087 ± 0.006)143B cell were lower than the control group(0.105± 0.007),t1=2.445,P=0.013;t2=1.659,P=0.041;After 72h treatment,absorbance value of 60 mol/L group(0.106 ± 0.043)and 30 ? mol/L group(0.144 ± 0.036)143B cells were lower than the control group 0.187 ± 0.023),tl=3.775,P=0.006;t2=2.036,P=0.01.Therefore,60 ? mol/L and 30 ? mol/L salidroside can inhibit the proliferation of osteosarcoma 143B cells,and with the increase of treatment time,the inhibitory effect is more obvious.Salidroside inhibits the proliferation ability of osteosarcoma 143B cells in a time dependent manner.The transmembrane number of 60 ? mol/L salidroside group(48.7 ± 8.2)and 30 ? mol/L salidroside group(76.8 ± 12.8)143B cells was lower than that of the control group(94.5 ± 11.9),t1=8.056,P<0.001,t2=2.472,P=0.038.The inhibitory effect of 60 ? mol/L salidroside on 143B cells was stronger than that of 30 ? mol/L salidroside,t=4.806,P=0.001.The scratch assay was used to determine the salidroside on migration ability of 143B osteosarcoma cells.The migration rate of control group,30 ? mol/L salidroside group,60 mol/L salidroside group 143B cells were 98.7.0 ± 0.3%,82.6 ± 3.7%,52.9 ± 6.8%,respectively.Compared to the control group,salidroside(30 and 60 ? mol/L))inhibited 143B cell migration,t1?12.576,P<0.001;t2= 9.794,P<0.001.At the same time,the inhibitory effect of 60 ? mol/L salidroside on the migration of 143B was stronger than that 30 ? mol/L salidroside group,t=7.654,P<0.001.RT-PCR results showed that the relative expression of MMP-2 mRNA in the 60 ?mol/L(0.935±0.15)and 30 ? mol/L(1.302 ± 0.019)salidroside treatment group was lower than that in the control group(1.622 ± 0.015),t1? 1.478,P<0.001,t2=6.784 and P=0.003.RT-PCR results showed that the relative expression of MMP-9 mRNA in the 60 ?mol/L(0.686 ± 0.007)and 30 ? mol/L(0.791 ± 0.016)salidroside treatment group was lower than that in the control group(1.142 ± 0.021),t1=7.556,P=0.002,t2=4.665 and P=0.007,respectively.The results showed that relative expression of MMP-2 protein of 60 ? mol/L(0.362 ± 0.071)and 30 ? mol/L(0.553 ± 0.112)were lower than that in control group(0.834:± 0,122),t1=9.698,P<0.001;t2=3.129,P=0.013.Relative expression of MMP-9 protein of 60 ? mol/L(0.135 ± 0.021)and 30 ? mol/L(0.31 ± 0.05)were lower than that in control group(0.42 ± 0.08),t1=6.447,P=0.006;t2=3.029,P=0.015.Conclusion:Salidroside can inhibit the proliferation and invasion of osteosarcoma,and its mechanism is closely related to the MMP family gene.Part 2 Mechanism of inhibition of salidroside on osteosarcoma cell invasion and migration is mediated by autophagyObjective:To investigate the role of autophagy in the inhibition of salidroside on invasion and metastasis of osteosarcoma.Methods:Human osteosarcoma cell 143B was cultured.After the 24 hours treatment of 60 ? mol/L salidroside on osteosarcoma 143B cells,the autophagy was detected by transmission electron microscope.After treatment with salidroside of 60,mol/L and 30 ? mol/L for 24 hours,the protein expression level of LC3 and Beclin-1 in each group was detected by Western-blotting.For the further study on the role of autophagy in inhibition of salidroside on proliferation and invasion of osteosarcoma cell,four group was set as follow:60 ? mol/L salidroside treatment group,60 ? mol/L salidroside+3-MA(autophagy inhibitor)treatment group,control group,3-MA treatment group.After treatment for 24,48 and 72 hours,cell proliferation was detected by MTT assay.After 24 hours treatment,the invasion ability of each group was detected by Transwell.The expression level of LC3? and MMP-2 protein in each group was detected by Western-blotting.Results:The number of autophagic bodies in the 60 ? mol/L salidroside group was(2.6 ± 0.5)more than that of the control group(1.3 ± 0.4),P<0.05.The LC3 and Beclin-1 protein level of 60 ? mol/L and 30 ? mol/L salidroside treatment group was higher than that of control group,and the differences were statistically significant.LC3 and Beclin-1 protein expression levels of 60 ? mol/L was higher than that of 30 ? mol/L salidroside treatment group,the difference was statistically significant.The LC3II/LC3I ratio is also one of the important indicators to observe the autophagic activity of the cells,and the LC3?/LC3I increases gradually,indicating that the autophagy is more active.The experimental results show that LC3II/LC3I ratio of 60 ? mol/L and 30 ? mol/L salidroside treatment group were higher than that of control group,the differences were statistically significant,and LC3II/LC3I ratio of 60 ? mol/L salidroside treatment group was higher than that of 30 ? mol/L salidroside treatment group,the difference was statistically significant.The cell proliferation ability of 60 ? mol/L salidroside +3-MA(autophagic inhibitor)group was higher than that of 60 ? mol/L salidroside group,and the cell proliferation ability of 3-MA treatment group was higher than that of control group at 24,48 and 72 hours,respectively.The invasiveness of 143B cells in the 60 ? mol/L salidroside+3-MA treatment group was higher than that of the 60 ? mol/L salidroside treatment group,and the cell invasiveness in the 3-MA treatment group was higher than that of the control group.The results of Western-blotting experiment showed that the expression level of MMP-2 protein in the 60 ? mol/L salidroside +3-MA treatment group was higher than that of the 60 ? mol/L salidroside treatment group,and the LC3II protein expression level in the 60 ? mol/L salidroside +3-MA treatment group was lower than that in the 60 ? mol/L salidroside treatment group.Conclusion:Salidroside can activate 143B osteosarcoma cells autophagy,and autophagy inhibitor 3-MA partially reversed the inhibitory effect of salidroside on the proliferation and invasion of osteosarcoma 143B cells.Salidroside may play a role on invasion and metastasis of osteosarcoma through activation of autophagy. |
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