【摘要】 目的 筛选人口腔癌细胞系显著差异表达circRNA,探讨hsa＿circ＿0002141对人口腔癌细胞增殖、迁移、侵袭、凋亡及对口腔癌裸鼠肿瘤生长的影响。方法 取人口腔癌细胞系HSC3、Sa3、SAS及人正常口腔角质形成细胞系HNOKs,采用高通量测序法检测circRNA表达，筛选显著差异表达的circRNA,取表达上调倍数最高的hsa＿circ＿0002141和高表达hsa＿circ＿0002141的SAS细胞进行后续实验。取SAS细胞分为对照组(正常培养)、空白转染组(转染sh-circNC慢病毒)、转染组(转染sh-circ＿0002141慢病毒),转染2周，采用实时荧光定量PCR法检测3组细胞hsa＿circ＿0002141相对表达量，采用CCK-8法检测细胞增殖，采用Transwell小室实验检测细胞迁移及侵袭，采用流式细胞术测定细胞凋亡率。将6只裸鼠随机分为空白转染模型组和转染模型组各3只，分别接种转染sh-circNC慢病毒、sh-circ＿0002141慢病毒的SAS细胞，于建模第3、6、9、12、15、18、21天测量肿瘤长径和短径，计算肿瘤体积；饲养21 d处死2组裸鼠，测定肿瘤组织质量。结果 HSC3、Sa3、SAS细胞与HNOKs细胞比较共发现152个显著差异表达circRNA,其中9个表达上调，143个表达下调，hsa＿circ＿0002141表达上调倍数最高。转染2周，转染组SAS细胞hsa＿circ＿0002141相对表达量(0.32±0.03)低于对照组(1.00±0.03)和空白转染组(1.02±0.07)(P<0.05)。转染后培养24、48、72、96 h,转染组细胞增殖率[(87.24±8.49)%、(106.33±9.82)%、(130.29±13.06)%、(148.52±14.61)%]均低于对照组[(100.00±9.52)%、(136.17±13.23)%、(170.54±15.92)%、(190.75±18.99)%]和空白转染组[(100.29±9.56)%、(138.58±12.78)%、(171.48±16.51)%、(189.76±18.34)%](P<0.05)。转染后培养24 h,转染组迁移细胞数[(120.05±11.54)个]、侵袭细胞数[(93.95±8.43)个]均少于对照组[(249.80±21.73)、(180.54±16.79)个]和空白转染组[(246.73±23.16)、(176.89±15.81)个](P<0.05)。转染2周，转染组细胞凋亡率[(22.68±2.15)%]高于对照组[(5.30±0.51)%]和空白转染组[(5.26±0.51)%](P<0.05)。对照组细胞hsa＿circ＿0002141相对表达量及不同时间点细胞增殖率、迁移细胞数、侵袭细胞数、细胞凋亡率与空白转染组比较差异均无统计学意义(P>0.05)。转染模型组裸鼠建模第3、6、9、12、15、18、21天肿瘤体积均大于空白转染模型组(P<0.05),建模第21天肿瘤组织质量[(0.42±0.03)g]低于空白转染模型组[(1.58±0.12)g](t=12.816,P=0.001)。结论 hsa＿circ＿0002141在人口腔癌细胞中呈高表达，下调其表达可抑制SAS细胞增殖，减少SAS细胞、迁移及侵袭，缩小口腔癌裸鼠肿瘤体积，hsa＿circRNA＿0002141可能成为口腔癌治疗的新靶点。更多还原

【Abstract】 Objective To screen the significantly differentially expressed circRNAs in human oral cancer cell lines, and to investigate the influences of hsa＿circ＿0002141 on the proliferation, migration, invasion and apoptosis of human oral cancer cells and the tumor growth in oral cancer nude mice. Methods The expressions of circRNA in human oral cancer cell lines HSC3, Sa3 and SAS as well as human normal oral keratinocyte cell lines HNOKs were detected by high-throughput sequencing, the significantly differentially expressed circRNA was screened, and hsa＿circ＿0002141 with the highest up-regulated expression and SAS cells with highly expressed hsa＿circ＿0002141 were selected for the subsequent experiment. The SAS cells were divided into control group(normal culture for 2 weeks), blank transfection group(transfected with sh-circNC lentivirus for 2 weeks) and transfection group(transfected with sh-circ＿0002141 lentivirus for 2 weeks). The relative expressions of hsa＿circ＿0002141 were detected by real-time fluorescence quantitative PCR in three groups, the cell proliferation rates were determined by CCK-8 assay, the numbers of migrated cells and invasive cells were determined by Transwell assay,and the apoptotic rates were determined by flow cytometry.Six nude mice were randomly and equally divided into blank transfection model group and transfection model group,inoculated with sh-circNC lentivirus and sh-circ＿0002141lentivirus transfected SAS cells,respectively.The tumor volume was calculated after the long diameter(a)and short diameter(b)were measured by day 3,6,9,12,15,18and 21of modeling.After21days of feeding,all nude mice were sacrificed and the tumor masses were measured.Results Comparing HSC3,Sa3and SAS with HNOKs,152differentially expressed circRNAs were found,among which 9were up-regulated and 143were down-regulated.With the highest up-regulated expression fold,hsa＿circ＿0002141was selected for the subsequent experiment.After 2-week transfection,the relative expression of hsa＿circ＿0002141of SAS cells was lower in transfection group(0.32±0.03)than that in control group(1.00±0.03)and blank transfection group(1.02±0.07)(P<0.05).The cell proliferation rates after 24-,48-,72-and 96-h transfection were lower in transfection group[(87.24±8.49)%,(106.33±9.82)%,(130.29±13.06)%,(148.52±14.61)%]than those in control group [(100.00±9.52)%,(136.17±13.23)%,(170.54±15.92)%,(190.75±18.99)%]and blank transfection group [(100.29±9.56)%,(138.58±12.78)%,(171.48±16.51)%,(189.76±18.34)%](P<0.05).After 24-h culture,the numbers of migrated cells and invasive cells were less in transfection group(120.05±11.54,93.95±8.43)than those in control group(249.80±21.73,180.54±16.79)and blank transfection group(246.73±23.16,176.89±15.81)(P<0.05).The apoptotic rate was higher in transfection group[(22.68±2.15)%]than that in control group[(5.30±0.51)%]and blank transfection group[(5.26±0.51)%](P<0.05).There were no significant differences in the relative expression of hsa＿circ＿0002141,proliferation rate at different time points,number of migrated cells,number of invasive cells,and apoptotic rate between control group and blank transfection group(P>0.05).The tumor volumes of nude mice by day 3,6,9,12,15,18and 21of modeling were larger in transfection model group than those in blank transfection model group(P<0.05).By day 21of modeling,the tumor mass was lower in transfection model group[(0.42±0.03)g]than that in blank transfection model group [(1.58±0.12)g](t=12.816,P=0.001).Conclusions hsa＿circ＿0002141is highly expressed in oral cancer cells.To down-regulate its expression can inhibit the proliferation of SAS cells,reduce the numbers of migrated and invasive SAS cells,and shrink the tumor volume of oral cancer nude mice,and it may become a new target for oral cancer treatment.更多还原