右美托咪定对口腔鳞癌细胞增殖、迁移和侵袭的影响

Effects of dexmedetomidine on abilities of cell proliferation,migration and invasion of oral squamous cell carcinoma cells

目的探讨右美托咪定对口腔鳞癌细胞增殖、迁移和侵袭的影响及机制。方法实验一:选用人口腔鳞癌细胞系HN6和CAL27,根据右美托咪定浓度不同随机均分为六组:阴性对照组(NC组)、右美托咪定1 nmol/L组(D1组)、右美托咪定10 nmol/L组(D2组)、右美托咪定100 nmol/L组(D3组)、右美托咪定1μmol/L组(D4组)和右美托咪定10μmol/L组(D5组)。培养24、48 h后分别采用CCK-8法、划痕实验和Transwell实验检测细胞增殖、迁移和侵袭能力。实验二:利用转录组测序技术筛选右美托咪定1μmol/L处理HN6细胞12 h后差异表达的基因。采用慢病毒转染细胞,建立稳定敲低细胞色素P4501B1(CYP1B1)的HN6细胞株。将细胞随机分为四组:shCtrl组(C组)、shCYP1B1组(CYP组)、shCtrl+右美托咪定组(CD组)和shCYP1B1+右美托咪定组(CYPD组)。采用划痕实验和Transwell实验检测HN6细胞迁移和侵袭能力。结果实验一:与NC组比较,D2组、D3组、D4组和D5组HN6和CAL27细胞迁移率均明显升高,基质胶内穿膜细胞数均明显增多;D1组HN6细胞迁移率明显升高(P<0.05)。与D1组比较,D2组、D3组、D4组和D5组HN6和CAL27细胞迁移率均明显升高,基质胶内穿膜细胞数均明显增多(P<0.05)。与D2组比较,D3组、D4组和D5组HN6和CAL27细胞迁移率均明显升高,基质胶内穿膜细胞数均明显增多(P<0.05)。与D3组比较,D4组HN6、CAL27细胞和D5组HN6细胞迁移率明显升高,基质胶内穿膜细胞数均明显增多(P<0.05)。与D4组比较,D5组HN6细胞迁移率明显升高,基质胶内穿膜细胞数明显增多;D5组CAL27细胞迁移率明显降低,基质胶内穿膜细胞数明显减少(P<0.05)。培养24、48 h后六组HN6和CAL27细胞存活率差异均无统计学意义。实验二:对右美托咪定处理后的HN6细胞进行转录组测序,筛选出54个差异表达基因,其中CYP1B1表达明显上调。慢病毒成功转染HN6细胞,构建稳定敲低CYP1B1的HN6细胞株。与C组比较,CYP组迁移率明显降低,基质胶内穿膜细胞数明显减少;CD组和CYPD组迁移率明显升高,基质胶内穿膜细胞数明显增多(P<0.05)。与CYP组比较,CD组和CYPD组迁移率明显升高,基质胶内穿膜细胞数增多(P<0.05)。与CD组比较,CYPD组迁移率明显降低,基质胶内穿膜细胞数明显减少(P<0.05)。结论右美托咪定能增强口腔鳞癌细胞迁移能力和侵袭能力,该作用可能与右美托咪定处理后CYP1B1表达相关。

Objective To investigate the effects of dexmedetomidine on the abilities of cell proliferation,migration,and invasion of oral squamous cell carcinoma(OSCC)cells and molecular mechanism.Methods Experiment 1:human OSCC HN6 and CAL27 cells were both randomly divided into 6 groups based on the concentrations of dexmedetomidine:negative control group(group NC),dexmedetomidine 1 nmol/L group(group D1),dexmedetomidine 10 nmol/L group(group D2),dexmedetomidine 100 nmol/L group(group D3),dexmedetomidine 1μmol/L group(group D4)and dexmedetomidine 10μmol/L group(group D5).The abilities of cell proliferation,migration,and invasion of HN6 and CAL27 cells after dexmedetomidine treatment for 24 to 48 hours were examined using cell counting kit-8,wound healing and Transwell assays,respectively.Experiment 2:the differentially expressed genes in HN6 cells under 1μmol/L dexmedetomidine treatment for 12 hours were screened by transcriptome sequencing.HN6 cells were transfected with lentivirus to knockdown Cytochrome P4501B1(CYP1B1)gene expression.HN6 cells were randomly divided into four groups:group shCtrl(group C),group shCYP1B1(group CYP),group shCtrl+DEX(group CD)and group shCYP1B1+DEX(group CYPD).The migration and invasion abilities of HN6 cells were detected.Results Experiment 1:compared with group NC,the migration rate and the number of cells penetrating the membrane in the matrix glue of HN6 and CAL27 cell were significantly increased in groups D2,D3,D4,and D5,the migration rate of HN6 cell in group D1 was significantly increased(P<0.05).Compared with group D1,the migration rate and the number of cells penetrating the membrane in the matrix glue of HN6 and CAL27 cell in groups D2,D3,D4,and D5 were significantly increased(P<0.05).Compared with group D2,the migration rate and the number of cells penetrating the membrane in the matrix glue of HN6 and CAL27 cell in groups D3,D4,and D5 were significantly increased(P<0.05).Compared with group D3,the migration rate and the number of cells penetrating the membrane in matrix glue of HN6,CAL27 cells in group D4,and HN6 cell in group D5 were significantly increased(P<0.05).Compared with group D4,the migration rate and the number of cells penetrating the membrane in the matrix glue of HN6 cellin group D5 were significantly increased,the migration rate of CAL27 cell and the number of cells penetrating the membrane in the matrix glue were significantly decreased(P<0.05).After 24 and 48 hours of cultivation,there were no significantly differences in the survival rates of HN6 and CAL27 cells between the six groups.Experiment 2:fifty-four differentially expressed genes were screened via transcriptome sequencing,among which CYP1B1 was upregulated significantly.The lentivirus transfection successfully constructed HN6 cell line with stable CYP1B1 down regulation.Compared with group C,the migration rate and the number of cells penetrating the membrane in the matrix glue were significantly decreased in group CYP,while the migration rate and the number of cells penetrating the membrane in the matrix glue were significantly increased in groups CD and CYPD(P<0.05).Compared with group CYP,the migration rate and the number of cells penetrating the membrane in the matrix glue were significantly increased in groups CD and CYPD(P<0.05).Compared with group CD,the migration rate and the number of cells penetrating the membrane in the matrix glue were significantly decreased in group CYPD(P<0.05).Conclusion Dexmedetomidine can enhance the abilities of migration and invasion of OSCC cells,which may be related to CYP1B1 expression following dexmedetomidine treatment.