| Objective:To investigate the expression ofβ1 adrenergic receptor (β1-AR)andβ2 adrenergic receptor(β2-AR)in CD133~+oral squamous cell carcinoma(OSCC)stem cells,OSCC lines and normal oral mucosa cells.To investigate the effects of blockingβ2-AR on proliferation,migration and invasion of CD133~+OSCC stem cells and OSCC cell lines.High throughput sequencing was used to screen differentially expressed genes of CD133~+OSCC stem cells with blockedβ2-AR and without blockedβ2-AR.The role ofβ2-AR and OSCC cancer stem cells in the genesis and development of OSCC was explored at the transcriptome level and gene expression direction.Method:The squamous epithelial cells of normal oral mucosa were cultured by trypsinase digestion.At the same time,6 cases of CD133~+OSCC stem cells,3 OSCC cell lines(Cal27,SAS and SCC9)and 6 cases of normal oral mucosal epithelial cells were examined for the expression ofβ1-AR andβ2-AR by RT-PCR and Western Blot,respectively.Secondly,the effects of different concentrations ofβ2-AR specific blocker ICI118,551 on the proliferation of 3 cases of CD133~+OSCC stem cells and 2 OSCC cell lines(SAS and SCC9)were assessed by Cell Counting Kit-8 assay(CCK-8).Colony formation assay was used to detect the effects ofβ2-AR specific blocker ICI118,551 on the single cell proliferative capacity of CD133~+OSCC stem cells.Then the effects of different concentrations ofβ2-AR specific blocker ICI118,551 on the migration of CD133~+OSCC stem cells and SCC9 were investigated by cell scratch test.Transwell assay was used to investigate the effects of different concentrations ofβ2-AR specific blocker ICI118,551 on the invasion of CD133~+OSCC stem cells and SCC9.Finally,the differentially expressed gene profiles of CD133~+OSCC stem cells with and without blockade ofβ2-AR were constructed by high throughput sequencing.Gene Ontology(Go)enrichment analysis and KEGG(kyoto encyclopedia of genes and genomes)pathway enrichment analysis were used to analyze the function of genes with distinct differences in expression.Result:Real-time fluorescence quantitative PCR and Western Blot showed that the expression ofβ1-AR andβ2-AR in CD133~+OSCC stem cells,OSCC cell lines(Cal27,SAS and SCC9)was higher than that in normal oral mucosal epithelial cells.CCK-8 results showed that the cell survival rate of CD133~+OSCC stem cells and OSCC cell lines decreased with the treatment of increasing concentrations of ICI118,551;The cell survival rate of SCC9 and CD133~+OSCC stem cells at drug concentrations of 0.1μM,0.5μM,1μM,2.5μM and 5μM was significantly different from that of 0μM(P<0.01).Colony formation experiment showed that the number of colonies in the drug group were 30,30 and 32 respectively,and the colonies formation rate(%)was61.33±2.31(n=3);The number of colonies in the control group were 34,35 and37 respectively,and the formation rate of colonies(%)was 70.67±3.06(n=3),the difference was statistically significant(P<0.05).Cell scratch test showed that the mobility of SCC9 and CD133~+OSCC stem cells decreased gradually with the increase of ICI118,551 concentration(P<0.05);the mobility of SCC9and CD133~+OSCC stem cells at drug concentrations of 0.1μM,0.5μM,1μM,2.5μM,5μM and 7.5μM was significantly lower than that at drug concentration of 0μM(P<0.05).Transwell assay showed that the invasion rate of SCC9 and CD133~+OSCC stem cells decreased with the increase of ICI118,551concentration(P<0.05);The invasive rate of SCC9 and CD133~+OSCC stem cells at drug concentrations of 0.1μM,0.5μM,1μM,2.5μM,5μM,7.5μM and 10μM was significantly lower than that at 0μM(P<0.05).High throughput sequencing screened 994 differentially expressed genes between the drug treatment group and the control group,of which 237 genes were up-regulated and 757 genes were down-regulated after drug treatment.According to GO analysis,there are 118 kinds of differentially expressed genes annotated to molecular function(MF),92 kinds annotated to cell component(CC)and 856kinds annotated to biological process(BP).KEGG analysis showed that differentially expressed genes were enriched in 19 signaling pathways,including cell cycle,MAPK signaling pathway,microRNAs in cancer,pathways in cancer,PI3K-Akt signaling pathway and Ras signaling pathway.Conclusion:The expression ofβ1-AR andβ2-AR in CD133~+OSCC stem cells and OSCC cell lines was significantly higher than that in normal oral mucosa cells.Blockingβ2-AR can inhibit the proliferation,migration and invasion of CD133~+OSCC stem cells and OSCC cell lines.After blockingβ2-AR,CD133~+OSCC stem cell genes with significant changes are mainly enriched in biological processes such as tissue development,cell proliferation,epithelial cell differentiation,cell migration and cell motility.And these differentially expressed genes maybe participate in the occurrence and development of OSCC through signaling pathways such as MAPK、PI3K/Akt and Ras to a certain extent. |
PDF Full Text Request