CAJ | 학술논문

目的：探讨泛素连接酶RING2对苯并[a]芘(BaP)染毒人支气管上皮16HBE细胞周期和P53蛋白表达的影响。方法：以16HBE未处理组为阴性对照组，二甲基亚砜(DMSO)组为溶剂对照组，MOCK组为序列对照组。在使用RNA干扰技术降低16HBE细胞泛素连接酶RING2基因表达前后，分别采用不同浓度BaP(1、2、4、8、16、32μmol/L)染毒24 h；或16μmol/L BaP染毒不同时间(1、2、4、8、12、24 h)。用流式细胞术检测干扰前后两组细胞周期分布情况，用Western-blot法检测干扰前后两组细胞P53蛋白表达水平。结果：流式细胞术检测结果显示，与阴性对照组比较，16HBE细胞染毒后各浓度和各时点组S期细胞所占的比例均增加(P<0.05)，而16HBE(siRNA-RING2)各浓度和各时点组S期细胞所占的比例均下降(P<0.05)。协方差分析显示分组因素(是否进行RNAi)和染毒浓度都对S期细胞比例有影响(P均<0.01)，16HBE(siRNA-RING2)细胞组的修正均数(17.09%)比16HBE细胞组(31.55%)明显降低(P<0.01)。分组因素和染毒时间都对S期细胞比例有影响(P均<0.01)，16HBE(siRNA-RING2)细胞组的修正均数(13.07%)比16HBE细胞组(28.04%)明显下降(P<0.01)。Western-blot结果显示，与阴性对照组比较，16HBE细胞染毒后各浓度和各时点组P53的表达水平均增加(P<0.05)，而16HBE(siRNA-RING2)细胞除16μmol/L染毒8 h组外，其余各组P53的表达水平均降低(P<0.05)。协方差分析显示分组因素和染毒浓度都对P53的表达水平有影响， P值分别为0.026和0.028。16HBE(siRNA-RING2)细胞组的修正均数(0.989)比16HBE细胞组(1.375)明显下降(P<0.05)；分组因素和染毒时间都对P53的表达水平有影响， P值分别为0.007和0.035。16HBE(siRNA-RING2)细胞组的修正均数(0.857)比16HBE细胞组(1.541)明显下降(P<0.05)。结论：RING2参与的组蛋白泛素化修饰可能通过影响P53表达和细胞周期S期的变化来发挥对DNA损伤修复的调控。目的：探討汎素連接酶RING2對苯併[a]芘(BaP)染毒人支氣管上皮16HBE細胞週期和P53蛋白錶達的影響。方法：以16HBE未處理組為陰性對照組，二甲基亞砜(DMSO)組為溶劑對照組，MOCK組為序列對照組。在使用RNA榦擾技術降低16HBE細胞汎素連接酶RING2基因錶達前後，分彆採用不同濃度BaP(1、2、4、8、16、32μmol/L)染毒24 h；或16μmol/L BaP染毒不同時間(1、2、4、8、12、24 h)。用流式細胞術檢測榦擾前後兩組細胞週期分佈情況，用Western-blot法檢測榦擾前後兩組細胞P53蛋白錶達水平。結果：流式細胞術檢測結果顯示，與陰性對照組比較，16HBE細胞染毒後各濃度和各時點組S期細胞所佔的比例均增加(P<0.05)，而16HBE(siRNA-RING2)各濃度和各時點組S期細胞所佔的比例均下降(P<0.05)。協方差分析顯示分組因素(是否進行RNAi)和染毒濃度都對S期細胞比例有影響(P均<0.01)，16HBE(siRNA-RING2)細胞組的脩正均數(17.09%)比16HBE細胞組(31.55%)明顯降低(P<0.01)。分組因素和染毒時間都對S期細胞比例有影響(P均<0.01)，16HBE(siRNA-RING2)細胞組的脩正均數(13.07%)比16HBE細胞組(28.04%)明顯下降(P<0.01)。Western-blot結果顯示，與陰性對照組比較，16HBE細胞染毒後各濃度和各時點組P53的錶達水平均增加(P<0.05)，而16HBE(siRNA-RING2)細胞除16μmol/L染毒8 h組外，其餘各組P53的錶達水平均降低(P<0.05)。協方差分析顯示分組因素和染毒濃度都對P53的錶達水平有影響， P值分彆為0.026和0.028。16HBE(siRNA-RING2)細胞組的脩正均數(0.989)比16HBE細胞組(1.375)明顯下降(P<0.05)；分組因素和染毒時間都對P53的錶達水平有影響， P值分彆為0.007和0.035。16HBE(siRNA-RING2)細胞組的脩正均數(0.857)比16HBE細胞組(1.541)明顯下降(P<0.05)。結論：RING2參與的組蛋白汎素化脩飾可能通過影響P53錶達和細胞週期S期的變化來髮揮對DNA損傷脩複的調控。
목적：탐토범소련접매RING2대분병[a]비(BaP)염독인지기관상피16HBE세포주기화P53단백표체적영향。방법：이16HBE미처리조위음성대조조，이갑기아풍(DMSO)조위용제대조조，MOCK조위서렬대조조。재사용RNA간우기술강저16HBE세포범소련접매RING2기인표체전후，분별채용불동농도BaP(1、2、4、8、16、32μmol/L)염독24 h；혹16μmol/L BaP염독불동시간(1、2、4、8、12、24 h)。용류식세포술검측간우전후량조세포주기분포정황，용Western-blot법검측간우전후량조세포P53단백표체수평。결과：류식세포술검측결과현시，여음성대조조비교，16HBE세포염독후각농도화각시점조S기세포소점적비례균증가(P<0.05)，이16HBE(siRNA-RING2)각농도화각시점조S기세포소점적비례균하강(P<0.05)。협방차분석현시분조인소(시부진행RNAi)화염독농도도대S기세포비례유영향(P균<0.01)，16HBE(siRNA-RING2)세포조적수정균수(17.09%)비16HBE세포조(31.55%)명현강저(P<0.01)。분조인소화염독시간도대S기세포비례유영향(P균<0.01)，16HBE(siRNA-RING2)세포조적수정균수(13.07%)비16HBE세포조(28.04%)명현하강(P<0.01)。Western-blot결과현시，여음성대조조비교，16HBE세포염독후각농도화각시점조P53적표체수평균증가(P<0.05)，이16HBE(siRNA-RING2)세포제16μmol/L염독8 h조외，기여각조P53적표체수평균강저(P<0.05)。협방차분석현시분조인소화염독농도도대P53적표체수평유영향， P치분별위0.026화0.028。16HBE(siRNA-RING2)세포조적수정균수(0.989)비16HBE세포조(1.375)명현하강(P<0.05)；분조인소화염독시간도대P53적표체수평유영향， P치분별위0.007화0.035。16HBE(siRNA-RING2)세포조적수정균수(0.857)비16HBE세포조(1.541)명현하강(P<0.05)。결론：RING2삼여적조단백범소화수식가능통과영향P53표체화세포주기S기적변화래발휘대DNA손상수복적조공。
[ABSTRACT]OBJECTIVE:To investigate the role of ubiquitin protein ligase RING2 in cell cycle and expression of P53 in human bronchial epithelial cells exposed to benzo[a]pyrene.METHODS:The untreated 16HBE cellsgroups were used as negative control groups,the DMSOgroups were used assolvent control groups,the MOCK groups were used as sequence control groups. 16HBE cells and 16HBE(siRNA-RING2) cells were exposed to BaP at different concentrations (1,2,4,8,16,32μmol/L) for 24 h,and were exposed to BaP at 16μmol/L for different time points (1,2, 4,8,12,24 h). Flow cytometry was used to evaluate the cell cycle. The levels of P53 protein were detected by Western-blot.RESULTS:After BaPtreatment,compared with the negative control groups,ateach concentration and each time point the proportion of S phaseof 16 HBE cellswere significantly increased (P<0.01). However at each concentration and each time point 16HBE (siRNA-RING2) cells groups the proportion of S phase cells were significantly decreased (P<0.01). Covariance analysis shows grouping factors (with or withoutRNAi) andall concentrations had impact on the proportion of S phase,P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE (siRNA-RING2) cell group (17.09%) was significantly lower than that of 16HBE cell group (31.55%). Grouping factors (with or without RNAi) and the exposure time all have impacted on theproportion of S phase,P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE (siRNA-RING2) cell group (13.07%) was significantly lower than that of 16HBE cell group (28.04%) (P<0.05). After BaPtreatment,compared with the negative control group,ateach concentration and each time pointP53 protein levels at16 HBE cell groups were significantly increased (P<0.05). However,ateach concentration and each time point 16HBE (siRNA-RING2) cell groups (except16μmol/LBaP treated 8 h) the levels of P53 protein were significantly decreased (P<0.05). Covariance analysis shows grouping factors (with or without RNAi) and all concentration showed impact on the level of P53 protein,P values were 0.026 and 0.028,respectively. The estimate mean ofthe levels of P53 protein in 16HBE (siRNA-RING2) cell group (0.989) was significantly lower than thatof 16HBE cell group (1.375) (P<0.05). Grouping factors (with or without RNAi) and the exposure time both impacted on the level of P53 protein,P values were 0.007 and 0.035,respectively. The estimate mean of the levels of P53 protein in 16HBE (siRNA-RING2) cell group (0.857) was significantly lower than that of 16HBE cell group (1.541) (P<0.05).CONCLUSION:The histone ubiquitination modifications in which RING2wasinvolved may regulate DNA repair by affecting the expression of P53 and cell cycle S phase changes.