| Object:The nasopharyngeal carcinoma cell lines CNE1,CNE2 and 6-10 B were cultured by exogenous addition of S100A8 and S100A9 protein to explore the molecular mechanism of nasopharyngeal carcinoma cell line proliferation,especially invasion and migration ability.Does the P38 MAPK pathway play an important role in the invasion and migration of nasopharyngeal carcinoma cells by S100A8 / S100A9?Method:1.The effects of different concentrations of protein on the proliferation ability of each cell line were detected by CCK8 method after adding 0,0.25,0.5,1,3,5μg / ml S100A8 / S100A9 protein for 24 h in nasopharyngeal carcinoma cell lines.1μg / ml S100A8 / S100A9 protein in culture medium was used to detect the clonal ability of CNE1 and CNE2 cell lines.2.The medium with 1μg/ml S100A8 and S100A9 protein compound is taken as the experiment group,and the medium without S100A8 and S100A9 protein compound is taken as the control group.The invasion and migration of CNE2 cells were detected by scratch and adhesion experiments.Transwell chamber migration and invasion experiments are conducted respectively to detect the invasion and migration situations of experimental group and the control group.3.CNE1,CNE2 and 6-10 B cells were treated by 1μg / ml S100A8 /S100A9 protein for 0,0.5,1,3,6 and 12 hours.Western blot was used to detect p38 MAPK,JNK MAPK,NF-ΚB and Wnt / β-catenin pathway protein accumulation.At the same time,detected Epithelial-mesenchymal transition(EMT)key markers E-cadherin and N-cadherin protein expression.4.Pretreated nasopharyngeal carcinoma cell lines with p38 MAPK pathway inhibitor SB230580 and medium with 1μg/ml S100A8/S100A9 protein is taken as the experiment group;and that pretreated nasopharyngeal carcinoma cell lines with SB230580 diluent DMSO is taken as the control group.Transwell chamber migration and invasion experiments are conducted respectively to detect the invasion and migration situations of the nasopharyngeal carcinoma.Western blot and qRT-PCR were used to detect the expression of E-cadherin and N-cadherin in the experimental group and control group.The expression of matrix metalloproteinases(MMPs)was detected by qRT-PCR.Result:1.CCK8 method was detected the proliferation of CNE1,CNE2 and 6-10 B cells treat by 0 ~ 5μg / ml S100A8 / S100A9 protein.The nasopharyngeal carcinoma cell lines showed the growth trend.The clone formation ability of 1μg / ml S100A8 / S100A9 protein group was higher than that of the untreatedcontrol group(p <0.05).2.The cells were treated with 1μg / ml S100A8 / S100A9 protein at 6h,24 h,36h and 48 h after scratches.The migration area of CNE2 cell lines was larger than the control group.S100A8/S100A9 promoted the ability of migration(p<0.05,p<0.01)and adhesion to matrix.Transwell migration and invasion experiments showed that 1μg / ml S100A8 / S100A9 protein induced invasion and migration of CNE1,CNE2 and 6-10 B cells(p <0.05).3.The expression of p38 MAPK,JNK MAPK,ERK MAPK,NF-ΚB and Wnt / β-catenin pathway in 1μg / ml S100A8 / S100A9 protein was detected by Western blot.The results showed that p38 MAPK and Wnt / β-catenin pathway were activated,P-p38 protein and β-catenin protein in the nasopharyngeal carcinoma cell lines accumulation increased.The p-p38 protein of CNE1 cell lines was up-regulated at 0.5 h and reached its peak at 6 h(p<0.01),and the up-regulation of β-catenin protein was most obvious at 1 h.The p-p38 protein in CNE2 and 6-10 B cells was the most significant(p<0.05)at 1h.Western blot was used to detect E-cadherin protein expression,and the three nasopharyngeal carcinoma cell lines were down-regulated E-cadherin expression(p<0.05),and N-cadherin protein expression were up-regulated(p<0.05).4.SB230580 inhibited the p38 MAPK pathway,Transwell migration and invasion experiments showed that S100A8 / S100A9 protein induced invasion and migration of nasopharyngeal carcinoma cell lines decreased,and the number of cells passing through the polycarbonate membrane decreased(p< 0.05).The expression of E-cadherin was up-regulated and the expression of N-cadherin was down-regulated in the experimental group(p<0.05).The expression of MMPs was detected by qRT-PCR.Result show that,the expression of MMP1,MMP2,MMP3,MMP7 and MMP9 in the CNE1 cell lines was significantlylower than that in the control group(p<0.05).The expression of MMP1,MMP2 and MMP9 was down-regulated in the SB230580 pretreatment CNE2 cell lines group(p<0.05).The expression of MMP1,MMP2,MMP7 and MMP9 was down-regulated in 6-10 B cells,the difference was statistically significant(p<0.05)Conclusion:1μg / ml S100A8 / S100A9 protein in the medium can promote the proliferation,migration and invasion of nasopharyngeal carcinoma cell lines and the epithelial-mesenchymal transition is occurrence,at the same time.The mechanism of S100A8 / S100A9 protein to promote the invasion and migration of nasopharyngeal carcinoma cell lines may be regulated by p38 MAPK pathway,and then promote the up-regulation of EMT and MMPs. |
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