Effect Of GPR30 On The Proliferation,Apoptosis And Cycle Of Human Thyroid Carcinoma SW579 Cells And Its Mechanism

Objectives:To investigate the expression of estrogen receptor in SW579 cells of human thyroid squamous cell carcinoma,and to explore the effect of genistein on cell proliferation,apoptosis and cycle of SW579 cells before and after blocking the expression of estrogen receptor and its mechanism.To provide experimental basis for understanding the possible mechanism of genistein action in human thyroid carcinoma SW579 cells.Methods:Human thyroid cancer cells SW579 were divided into blank control group,genistein treated group(10,20,40,80,160,200,320 μmol/L,G15 untreated group)and estrogen-related receptor blocking group(G15 pretreatment combined with genistein treatment).1.Real-time Quantitate PCR was used to detect the expression of the estrogen nuclear receptors ERα,ERβ,and the estrogen membrane receptor GPR30 in human thyroid cancer cells SW579 and the effect of genistein on them;2.MTT assay was used to determine the effective concentration and time of G15,a specific blocker of GPR30,in human thyroid cancer SW579;3.MTT assay was used to detect the effect of genistein in the proliferation of human thyroid cancer SW579 cells before and after specific blocking of GPR30.4.Annexin V/FITC flow cytometry was used to detect the effect of genistein on cell cycle and apoptosis of human thyroid cancer SW579 before and after specific blockade of GPR30.5.Transcriptome sequencing was used to detect the differentially expressed genes and signaling pathways of genistein in human thyroid carcinoma SW579 cells and to verify the screening results.6.Statistical methods:SPSS 21.0 software was used for statistical analysis of the data,and P<0.05 was considered to be statistically significant.Results:1.PCR results showed that SW579 cells expressed ERβ and GPR30 receptor,but ERa was not expressed.Moreover,when the concentration of genistein(?)40 μmol/L for 24h and the concentration of genistein(?)80 μmol/L for 48h,the expression of ERβ in SW579 cells was significantly increased(P<0.05).After the concentration of genistein(?)40 μmol/L for 24 and 48h,the expression of GPR30 in SW579 cells was significantly enhanced(P<0.05).These results suggested that genistein could up-regulate the expression of ERβ and GPR30 genes in SW579 cells.2.According to the MTT method,the IC50 of G15 in SW579 cells was 15.17μmol/L,and the G15 of 5μmol/L acted on SW579 cells for 2 hours,with no significant effect on their proliferation.It was determined that the time of G15 treatment of SW579 cells was 2 hours and the concentration was 5 μmol/L.3.After the concentration of genistein(?)40 μmol/L for 24 and 48h,the proliferation of SW579 cells could be promoted.After G15 specifically blocked GPR30 and the concentration of genistein(?)80 μmol/L,genistein significantly inhibited the proliferation of SW579 cells(P<0.05).When the concentration of genistein increased(?)200μmol/L for 48h,the proliferation of SW579 cells was significantly inhibited regardless of whether GPR30 was blocked,and the inhibitory effect of G15-specific block of GPR30 was more significant(P<0.05).4.According to the results of flow cytometry technology to detect cell apoptosis,genistein in SW579 24 and 48h,cells had no significant effect on the apoptosis,but after GPR30 was blocked,when the concentration of genistein(?)80 μmol/L,SW579 apoptosis rate increased significantly,and increase the rate of apoptosis significantly increased over time.And according to the results of the cell cycle before GPR30 was blocked,SW579 main block the cell cycle in S phase,after blocking,the concentration of genistein(?)80 μmol/L,SW579 cell cycle block in G2/M phase.5.Transcriptome sequencing results showed that the effect of genistein in human thyroid cancer SW579 cells resulted in significant changes in 33 genes related to the cancer pathway,including up-regulation of 21 genes and down-regulation of 12 genes.The up-regulated CDKN1A,FOXO1,DAPK and FOS genes and down-regulated EGF and FTGS2 genes were randomly screened for real-time PCR verification,and the verification results were consistent with the results of transcriptome screening.6.According to the transcriptome sequencing results,after 160μmol/L of genistein was applied to SW579 cells for 48h,multiple genes in the PI3K-Akt,Ras,MAPK,Hippo and Wnt pathways were significantly up-regulated,while multiple genes in the PI3K-Akt,AGE-RAGE,MAPK and FoxO pathways were significantly down-regulated.Genes CDKN1A,FOXO1,FOS and PLCG2 were significantly up-regulated in multiple pathways,while EGF,PTGS2 and CXCL8 were significantly down-regulated in multiple pathways.Conclusions:1.The expression of ERa receptor in SW579 cells of human thyroid cancer is not expressed,but express ERβ and GPR30 receptors are expressed,and genistein can up-regulate the expression of ER and GPR30 genes in SW579 cells.2.Specific blocking of GPR30 and the concentration of genistein ≥80 μmol/L in human thyroid cancer SW579 cells can significantly promote the apoptosis of SW579 cells,and the cell cycle is blocked in G2/M phase.3.After the action of genistein in human thyroid cancer SW579 cells,33 genes related to the cancer pathway were significantly changed,including 21 genes up-regulated and 12 genes down-regulated.4.The proliferation promoting effect of genistein in SW579 cells is related to the regulation of PI3K-Akt,Ras,MAPK,Hippo,Wnt,AGE-RAGE,FoxO pathway and genes CDKN1A,FOXO1,FOS,PLCG2,EGF,PTGS2,CXCL8 may be its target gene.