Ursolic Acid Inhibits Proliferation,Migration And Invasion Of PTC Cells Through CAFs

Objective:1.The first part was to investigate the expression of CXCR4 and CXCR7 in PTC carcinoma and its adjacent tissues.Their correlation with pathological features was also discussed.2.The second part explored the effect of ursolic acid(UA)in Prunella vulgaris on NFs and CAFs cells,and explored the role of UA on the secretion of CXCL12 by CAFs;3.The third part confirmed that CXCL12 can promote their proliferation,migration and invasio;UA has direct inhibitory effect on TPC-1 and B-CPAP cells;4.The fourth part explored the difference of proliferation,migration and invasion ability of cancer cells cocultured with the cell medium(CM)of NFs and CAFs treated by UA.Methods:1.We collected 115 cases of PTC patients and collected their clinical data.The expression levels of CXCR4 and CXCR7 were detected by immunohistochemical method in 115 cases of PTC paraffin sections and corresponding paracancerous tissues.The correlation between the pathological characteristics of cancer tissues and the expression of CXCR4 and CXCR7 was analyzed by multivariate analysis.2.NFs and CAFs were observed under microscope;the contents of?-SMA,Vimentin and FAP-?in NFs and CAFs were detected by Western-blot and RT-PCR.The morphological changes of NFs and CAFs and the contents of?-SMA,Vimentin and FAP-?in NFs and CAFs were observed under different concentrations of UA in Prunella vulgaris.The CXCL12 content in the CM of two kinds of cells was detected by ELISA.3.Different concentrations of CXCL12 were used to induce TPC-1 and B-CPAP cells,and the optimal concentration was selected;Western-blot and RT-PCR were used to detect the expression of CXCR4 and CXCR7 in each group.Flow cytometry and CCK8 kit were used to detect the proliferation ability before and after stimulation.Scratch test and Transwell test were used to detect the changes of cell migration and invasion ability.TPC-1 and B-CPAP cells were treated with different concentrations of UA.The migration and invasion ability of TPC-1 and B-CPAP cells were detected by scratch test and Transwell test.Western blot and RT-PCR were used to detect the expression level of CXCR4 and CXCR7.4.The culture media of each group including NFs CM,CAFs CM,NFs(UA)CM and CAFs(UA)CM were collected and co-cultured with PTC cells respectively.The proliferation ability was detected by flow cytometry and CCK8 kit.The changes of cell migration and invasion ability were detected by scratch test and Transwell test.The expression changes of CXCR4 and CXCR7 were detected by Western blot and RT-PCR.Results:1.The high expression rates of CXCR4 and CXCR7 in cancer tissues were significantly higher than those in adjacent tissues,which were 53.91%(62/115)and 41.74%(48/115)respectively.In peritumoral nonmalignant tissues the high expression rate of CXCR4 and CXCR7 was 7.83%(9/115)and 23.48%(27/115),respectively.The expression of CXCR4 and CXCR7 were low in most adjacent tissues(106/115,88/115).The expression of CXCR4 and CXCR7 was correlated in cancer tissues and adjacent tissues,and the co-expression of CXCR4 and CXCR7 was significantly different(P<0.001).Strong positive staining of CXCR4 and CXCR7 was highly correlated with tumor invasion,including capsule invasion and regional lymph node metastasis(P<0.05).The expression of CXCR4 and CXCR7 had no correlation with tumor size,TNM stage and whether the adjacent tissues were thyroiditis or nodular goiter.2.RT-PCR and Western-blot results showed that?-SMA,Vimentin and FAP-?levels in CAFs were higher than those in NFs.UA in Prunella vulgaris at different concentrations could inhibit the proliferation of NFs and CAFs cells.The higher the concentration was,the greater the inhibition was in the range of 1-5 ng/ml.The IC₅₀(_24h)of NFs and CAFs were 4.572 ng/ml and 4.404 ng/ml,respectively.The expression of?-SMA,vimentin and FAP-?in CAFs cells treated with UA decreased(P<0.05).There was no significant difference in the content of?-SMA,vimentin and FAP-?between the two groups(P>0.05).The results showed that UA decreased the content of CXCL12 in the CM of NFs and CAFs,while the content of CXCL12 in CM of CAFs decreased significantly(P<0.01).3.Different concentrations of CXCL12(0,100,500,1000,2000ng/L)were used to induce TPC-1 and B-CPAP cells.Compared with the control group,the cell proliferation rate increased with the increase of CXCL12 concentration,and 1000 ng/L was the best stimulating concentration.Western-blot and RT-PCR showed that CXCL12 could increase the expression of CXCR4 and CXCR7 in TPC-1 and B-CPAP cells.The results of scratch test and Transwell test showed that the migration and invasion ability of cells increased gradually with the increase of CXCL12 concentration,and the migration and invasion ability was the strongest at the concentration of 1000ng/L.UA at different concentrations could inhibit the proliferation of TPC-1 and B-CPAP cells,and the higher the concentration was,the greater the inhibition ability was in the range of 1-5 ng/ml.The IC₅₀(_24h)of TPC-1 and B-CPAP were 5.991ng/ml and 3.925 ng/ml,respectively.Western-blot and RT-PCR showed that the expressions of CXCR4 and CXCR7 were decreased in TPC-1 and B-CPAP cells treated with UA(P<0.05).At the concentration gradient of 0ng/ml,2ng/ml and5ng/ml,the migration and invasion ability of TPC-1 and B-CPAP gradually decreased,and there were differences between groups(P<0.05).After UA treatment,the expression of CXCR4 and CXCR7 in TPC-1 and B-CPAP cells decreased,and the difference was statistically significant(P<0.05).4.The results of flow cytometry and CCK8-kit showed that the proliferation ability of TPC-1 and B-CPAP cells in CAFs(UA)CM group was the weakest.The results of wound healing test and Transwell test showed that scratch healing was the most obvious and the number of migration and invasion cells was the most in CAFs group.The results of Western-blot and RT-PCR showed that the expression of CXCR4 and CXCR7 in each group was different,the content in CAFs CM group was the highest,and the content in CAFs CM group was the lowest.The difference in each group was statistically significant(P<0.05).Conclusion:1.The expression rates of CXCR4 and CXCR7 in PTC tissues were higher than those in adjacent non tumor tissues,and they were related to lymph node metastasis and capsule invasion.2.UA in Prunella vulgaris directly inhibited the proliferation,migration and invasion of TPC-1 and B-CPAP cells.3.UA reduced the expression of?-SMA,Vimentin and FAP-?of CAFs,and reduced the secretion of CXCL12 by CAFs.4.CXCL12promoted the proliferation,migration and invasion of TPC-1 and B-CPAP cells.5.The proliferation,migration and invasion of PTC cells decreased after co-culture with CAFs medium and PTC cells with UA;the expression of CXCR7 and CXCR4 decreased.UA affected the proliferation,invasion and migration of PTC cells by affecting the secretion of CXCL12 in CAFs.