| Objective(s):To explore the effect of genistein on the proliferation of human thyroid cancer SW579 cells and the possible mechanism of action in this process.In order to provide reference for the possible application of genistein in the related clinical treatment of thyroid cancer.Methods:1.MTT assay was used to detect the effect of estrogen on the proliferation of human thyroid carcinoma SW579 cells and the effect of estrogen on the proliferation of human thyroid carcinoma SW579 cells before and after estrogen receptor blocking;2.MTT assay was used to detect the effects of genistein on the proliferation of human thyroid cancer SW579 cells and the effects of genistein on the proliferation of human thyroid cancer SW579 cells before and after estrogen receptor blocking;3.The effects of genistein on the cell cycle and apoptosis of human thyroid carcinoma SW579 before and after estrogen receptor blocking were examined by flow cytometry;4.qRT-PCR was used to detect the effects of genistein on the m RNA expression levels of CDKN1 A,Bcl-2 and Bax related to cell cycle and apoptosis of human thyroid cancer SW579 before and after estrogen receptor blocking;5.The effects of genstein on the expression levels of CDKN1 A,Bcl-2 and Bax proteins related to cell cycle and apoptosis of human thyroid cancer SW579 before and after estrogen receptor blocking were detected by Western Blot;6.Statistical analysis: Graph Pad Prism8.0 was used for plotting,SPSS 21.0 was used for analysis,inter-group comparison was conducted by one-way ANOVA method,ppair comparison was conducted by LSD method,P<0.05 showed significant difference.Results:1.MTT results showed that different concentrations of estrogen could significantly promote the proliferation of SW579 cells at 24 h and 48h(P<0.05).When GPR30 and ER-βwere blocked separately or simultaneously,estrogen at different concentrations could still significantly promote the proliferation of SW579 cells compared with the control group,and the proliferation capacity of SW579 cells was higher after the block than before the block(P<0.05).2.MTT results showed that the proliferation of SW579 cells was significantly inhibited by different concentrations of genistein at 24 h and 48h(P<0.05)in a dose-dependent manner.Separately and simultaneously blocking GPR30 and ER-β,compared with the control group,the proliferation of SW579 cells was still significantly inhibited by all concentrations of genistein.Compared with before blocking,when the concentration of genistein was less than 40μmol/L,the proliferation capacity of the cells after blocking was greater than that before blocking.When the concentration of genistein was more than 80μmol/L,there was no significant difference before and after blocking(P<0.05).3.The cell cycle results showed that SW579 cells were treated with 40μmol/L genistein for24h:the number of cells in G0/G1 phase increased,the number of cells in S phase decreased,there was no significant difference in G2/M phase,and the cell cycle was blocked in G0/G1 phase(P<0.05).When GPR30 or ER-βwas blocked,there was no significant difference in the proportion of cells in different phases,but when GPR30 and ER-βwere blocked simultaneously,the number of cells in G0/G1 phase was decreased,and the number of cells in S phase and G2/M phase was increased(P<0.05).After 48 h of treatment,the number of cells in G0/G1 phase increased,the number of cells in S phase decreased,and there was no significant difference in G2/M phase.The cell cycle was blocked in G0/G1 phase(P<0.05).Compared with before blocking,when GPR30 or ER-βwas blocked,the proportion of cells in each phase had no significant difference,while when GPR30 and ER-βwere blocked at the same time,the cells in G0/G1 phase decreased,the cells in S phase had no significant difference,and the cells in G2/M phase increased(P<0.05).The results showed that the total apoptosis rate of SW579 cells was significantly increased by 40μmol/L genistein treatment for 24 h and 48h(P<0.05),and GPR30 and ER-βwere blocked separately or simultaneously.Compared with the control group,Genistein could still significantly increase the total apoptosis rate of SW579 cells,and the total apoptosis rate was higher before and after blocking than after blocking(P<0.05).4.qRT-PCR results showed that the CDKN1 A m RNA expression level of SW579 cells could be significantly increased after treatment with 40μmol/L genistein for 24 h and 48h(P<0.05).After24 h treatment,there was no significant difference in the expression level of CDKN1 A m RNA after blocking GPR30 compared with before blocking.Blocking ER-βcould significantly improve the expression level of CDKN1 A m RNA(P<0.05),and blocking GPR30 and ER-βat the same time.CDKN1 A m RNA expression level was significantly decreased(P<0.05).After 48 h treatment,there was no significant difference in the expression level of CDKN1 A m RNA after blocking GPR30 or ER-βcompared with before blocking,while blocking GPR30 and ER-βat the same time could significantly reduce the expression level of CDKN1 A m RNA(P<0.05).The ratio of Bcl-2/Bax m RNA was decreased by treating SW579 cells with 40μmol/L genistein for 24 h and 48 h.After blocking the two receptors separately or simultaneously,genistein still reduced the ratio of Bcl-2/Bax m RNA compared with the control group.Compared with before blocking,the ratio of Bcl-2/Bax m RNA was higher after blocking than before blocking(P<0.05).5.Western Blot results showed that the protein expression of CDKN1 A was significantly increased in SW579 cells treated with 40μmol/L genistein for 24 h and 48h(P<0.05).After blocking GPR30 and ER-β,CDKN1 A protein expression was increased compared with the control group,and there was no significant difference before and after blocking;while GPR30 and ER-βwere blocked,there was no significant difference compared with the control group.Compared with before blocking,CDKN1 A protein expression was higher than that after blocking(P<0.05).The protein expression of Bcl-2/Bax was decreased in SW579 cells treated with 40μmol/L genistein for 24 h and 48h(P<0.05),and the two receptors were blocked separately or simultaneously.Compared with the control group,genistein still decreased the ratio of Bcl-2/Bax protein.The ratio of Bcl-2/Bax protein after blocking was higher than that before blocking(P<0.05).Conclusion(s):1.Estrogen can promote the proliferation of SW579 cells,and the combination of estrogen with GPR30 and ER-βwill weaken the pro-proliferation effect of estrogen.2.Genistein inhibited the proliferation of SW579 cells in a dose-dependent manner.The inhibitory effect of genistein may inhibit cell proliferation by up-regulating CDKN1 A expression,blocking cells in G0/G1 phase,and down-regulating Bcl-2/Bax ratio,inducing cell apoptosis.At the same time,GPR30 and ER-βmay play an auxiliary role in the process of genistein inhibiting the proliferation of SW579 cells. |
PDF Full Text Request