Effects Of Calprotectin (S100A8/A9) On The Proliferation,Migration And Invasion Of Oral Squamous Cell Carcinoma Cells

Objective: To observe the effects of exogenous calprotectin(S100A8/A9,Calprotectin)on the migration and invasion ability of oral squamous cell carcinoma(OSCC)cells,and to explore potential related mechanisms.Research on the mechanism of squamous cell carcinoma invasion and metastasis provides experimental basis.Methods:(1)CCK 8 method was used to detect the effects of different concentrations of S100A8/A9 protein on the proliferation of SCC-25 and CAL-27 cells,and determine the appropriate concentration.(2)The group added the exogenous 1ug/ml S100A8/A9 protein complex was used as the experimental group,and the group not containing the S100A8/A9 protein complex was used as the control group.The scratch test,Transwell cell migration experiment,and Transwell cell were performed Invasion test to detect the invasion and migration of SCC-25 and CAL-27 cells.(3)Add exogenous 1ug/ml S100A8/A9 protein to culture OSCC cells,collect the total protein of SCC-25 and CAL-27 cells at different time points,and use Western blot to detect the expression of p38 MAPK signaling pathway.(4)Immunofluorescence method was used to detect the expression of p38 MAPK signal pathway in SCC-25 and CAL-27 cells treated with 1ug/ml S100A8/A9 protein.(5)After adding p38 MAPK signaling pathway inhibitor to treat SCC-25 and CAL-27 cells,Transwell experiment was used to compare the effect of invasion and migration ability of the inhibitor group(p38MAPK pathway inhibitor SB230580)with the control group and the experimental group,and the invasion was detected by RT-q PCR The expression of MMP-2 and MMP-9 genes,the key markers of migration.Results:(1)The results of CCK 8 showed that the proliferation activity of SCC-25 and CAL-27 cells was concentration-dependent(P<0.05).(2)The scratch test results showed that the migration ability of SCC-25 and CAL-27 cells under the action of 1ug/ml S100A8/A9 protein was enhanced(P<0.05).the results of ranswell chamber experiments showed that compared with the control group,the SCC-25 and CAL-27 cells under the action of 1ug/ml S100A8/A9 protein had more SCC-25 and CAL-27 cells passing through the polycarbonate membrane and migrated and invaded.Increased ability with statistical significance(P<0.001).(3)Western blot detected the effect of 1ug/ml S100A8/A9 protein on the p38 MAPK pathway.The results showed that the p38 MAPK pathway was activated,and the amount of p-p38 MAPK protein increased cumulatively in SCC-25 and CAL-27 cells.The p-p38 MAPK protein in SCC-25 and CAL-27 cells was significantly up-regulated at 0.5h and started to be down-regulated after 12h(p<0.05).(4)The results of immunofluorescence showed that the expression of p-p38 MAPK in SCC-25 and CAL-27 cells was observed.The expression of p-p38 MAPK protein in SCC-25 and CAL-27 cells increased at 0.5h and decreased at 12h(p<0.05).(5)After 10?M SB230580 treatment of SCC-25 and CAL-27 cells,Transwell migration and invasion experiments showed that compared with the experimental group and the control group,the number of SCC-25 and CAL-27 cells passing through the polycarbonate membrane decreased,suggesting that the migration and invasion capabilities of SCC-25 and CAL-27 cells were weakened.The difference was statistically significant(P<0.001).RT-q PCR detection of MMP-2 and MMP-9 genes shows,under the effect of 1ug/ml S100A8/A9 protein,the expression of MMP-2 and MMP-9 genes in SCC-25 and CAL-27 cells in the experimental group was up-regulated,and the MMP-2 and MMP-9 genes in SCC-25 and CAL-27 cells in the inhibitor group Expression is down-regulated,the difference is statistically significant(P<0.001).Conclusion:The role of exogenous S100A8/A9 protein in SCC-25 and CAL-27 cells is concentration-dependent.Low concentrations of S100A8/A9 protein may promote the migration and invasion of OSCC cells by activating the p38 MAPK signaling pathway.