中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2015年
8期
542-548
,共7页
廖婉玉%张斌%方诚%杨燕%张舒%丁希伟%邹晓平
廖婉玉%張斌%方誠%楊燕%張舒%丁希偉%鄒曉平
료완옥%장빈%방성%양연%장서%정희위%추효평
胆管上皮癌%沉默信息调节因子-1%RNA 干扰%细胞增殖%细胞凋亡%细胞运动
膽管上皮癌%沉默信息調節因子-1%RNA 榦擾%細胞增殖%細胞凋亡%細胞運動
담관상피암%침묵신식조절인자-1%RNA 간우%세포증식%세포조망%세포운동
Cholangiocarcinoma%SIRT1%RNA interference%Cell proliferation%Apoptosis%Cell movement
目的：了解沉默信息调节因子1（SIRT1）在胆管癌细胞中的表达水平，探讨其对胆管癌细胞增殖、凋亡和迁移能力的影响。方法采用实时定量 PCR 和 Western 印迹法比较人胆管癌细胞QBC939和 HuCCT1与正常胆管上皮细胞 HIBEpiC 中 SIRT1 mRNA 和蛋白的表达。将 QBC939和HuCCT1分成阴性对照组、转染序列1组和转染序列2组，分别转染阴性对照小干扰 RNA 、针对 SIRT1的小干扰 RNA 序列1和小干扰 RNA 序列2，采用实时定量 PCR 和 Western 印迹法鉴定干扰效果。转染后应用细胞计数试剂盒和平板克隆形成实验检测细胞增殖能力；采用流式细胞术检测细胞凋亡率；采用Transwell迁移实验检测细胞迁移能力。采用 Western 印迹法检测凋亡相关蛋白生存素、B 细胞淋巴瘤‐2基因相关 X 蛋白（Bax）基因、剪切型多聚腺苷二磷酸核糖聚合酶（PARP），以及上皮标志物上皮型钙黏蛋白和间质标志物神经型钙黏蛋白、波形蛋白的表达情况。统计学处理采用 t 检验。结果胆管癌细胞 QBC939和 HuCCT1中 SIRT1的 mRNA 表达水平分别比正常胆管上皮细胞 HIBEpiC 升高（3．30±0．43）和（4．55±0．50）倍，差异均有统计学意义（t ＝7．29、9．79，P 均＜0．05）；胆管癌细胞中SIRT1的蛋白表达水平升高与 mRNA 相似。转染 SIRT1小干扰 RNA 后，胆管癌细胞中 SIRT1 mRNA和蛋白表达水平显著降低。 HuCCT1细胞的转染序列1组和转染序列2组的细胞生存率在转染后72 h分别为（19．72±1．39）％和（27．48±2．67）％，均低于阴性对照组的（84．65±3．18）％，差异均有统计学意义（t＝37．42、27．54，P均＜0．05）；HuCCT1细胞中转染序列1组和转染序列2组早期凋亡细胞比例分别为（19．43±1．72）％和（19．80±0．86）％，均高于阴性对照组的（5．43±0．31）％，差异均有统计学意义（t＝11．30、22．23，P均＜0．05）；HuCCT1细胞中转染序列1组和转染序列2组24 h 穿过微孔膜的细胞数分别为（56．00±2．61）和（58．80±3．19）个，均少于阴性对照组的（76．60±5．08）个，差异均有统计学意义（t＝7．21、5．93，P 均＜0．05）。 QBC939细胞的细胞生存率、早期细胞凋亡比例和迁移能力与HuCCT1细胞相似。下调胆管癌细胞中 SIRT1的表达后，可显著下调生存素、神经型钙黏蛋白和波形蛋白的表达，上调 Bax 、剪切型 PARP 和上皮型钙黏蛋白的表达。结论 SIRT1在胆管癌细胞中高表达，下调胆管癌细胞中 SIRT1的表达可抑制细胞的增殖和迁移，促进其凋亡，机制可能与下调生存素，上调 Bax 蛋白的表达，抑制上皮间质转化的发生有关。
目的：瞭解沉默信息調節因子1（SIRT1）在膽管癌細胞中的錶達水平，探討其對膽管癌細胞增殖、凋亡和遷移能力的影響。方法採用實時定量 PCR 和 Western 印跡法比較人膽管癌細胞QBC939和 HuCCT1與正常膽管上皮細胞 HIBEpiC 中 SIRT1 mRNA 和蛋白的錶達。將 QBC939和HuCCT1分成陰性對照組、轉染序列1組和轉染序列2組，分彆轉染陰性對照小榦擾 RNA 、針對 SIRT1的小榦擾 RNA 序列1和小榦擾 RNA 序列2，採用實時定量 PCR 和 Western 印跡法鑒定榦擾效果。轉染後應用細胞計數試劑盒和平闆剋隆形成實驗檢測細胞增殖能力；採用流式細胞術檢測細胞凋亡率；採用Transwell遷移實驗檢測細胞遷移能力。採用 Western 印跡法檢測凋亡相關蛋白生存素、B 細胞淋巴瘤‐2基因相關 X 蛋白（Bax）基因、剪切型多聚腺苷二燐痠覈糖聚閤酶（PARP），以及上皮標誌物上皮型鈣黏蛋白和間質標誌物神經型鈣黏蛋白、波形蛋白的錶達情況。統計學處理採用 t 檢驗。結果膽管癌細胞 QBC939和 HuCCT1中 SIRT1的 mRNA 錶達水平分彆比正常膽管上皮細胞 HIBEpiC 升高（3．30±0．43）和（4．55±0．50）倍，差異均有統計學意義（t ＝7．29、9．79，P 均＜0．05）；膽管癌細胞中SIRT1的蛋白錶達水平升高與 mRNA 相似。轉染 SIRT1小榦擾 RNA 後，膽管癌細胞中 SIRT1 mRNA和蛋白錶達水平顯著降低。 HuCCT1細胞的轉染序列1組和轉染序列2組的細胞生存率在轉染後72 h分彆為（19．72±1．39）％和（27．48±2．67）％，均低于陰性對照組的（84．65±3．18）％，差異均有統計學意義（t＝37．42、27．54，P均＜0．05）；HuCCT1細胞中轉染序列1組和轉染序列2組早期凋亡細胞比例分彆為（19．43±1．72）％和（19．80±0．86）％，均高于陰性對照組的（5．43±0．31）％，差異均有統計學意義（t＝11．30、22．23，P均＜0．05）；HuCCT1細胞中轉染序列1組和轉染序列2組24 h 穿過微孔膜的細胞數分彆為（56．00±2．61）和（58．80±3．19）箇，均少于陰性對照組的（76．60±5．08）箇，差異均有統計學意義（t＝7．21、5．93，P 均＜0．05）。 QBC939細胞的細胞生存率、早期細胞凋亡比例和遷移能力與HuCCT1細胞相似。下調膽管癌細胞中 SIRT1的錶達後，可顯著下調生存素、神經型鈣黏蛋白和波形蛋白的錶達，上調 Bax 、剪切型 PARP 和上皮型鈣黏蛋白的錶達。結論 SIRT1在膽管癌細胞中高錶達，下調膽管癌細胞中 SIRT1的錶達可抑製細胞的增殖和遷移，促進其凋亡，機製可能與下調生存素，上調 Bax 蛋白的錶達，抑製上皮間質轉化的髮生有關。
목적：료해침묵신식조절인자1（SIRT1）재담관암세포중적표체수평，탐토기대담관암세포증식、조망화천이능력적영향。방법채용실시정량 PCR 화 Western 인적법비교인담관암세포QBC939화 HuCCT1여정상담관상피세포 HIBEpiC 중 SIRT1 mRNA 화단백적표체。장 QBC939화HuCCT1분성음성대조조、전염서렬1조화전염서렬2조，분별전염음성대조소간우 RNA 、침대 SIRT1적소간우 RNA 서렬1화소간우 RNA 서렬2，채용실시정량 PCR 화 Western 인적법감정간우효과。전염후응용세포계수시제합화평판극륭형성실험검측세포증식능력；채용류식세포술검측세포조망솔；채용Transwell천이실험검측세포천이능력。채용 Western 인적법검측조망상관단백생존소、B 세포림파류‐2기인상관 X 단백（Bax）기인、전절형다취선감이린산핵당취합매（PARP），이급상피표지물상피형개점단백화간질표지물신경형개점단백、파형단백적표체정황。통계학처리채용 t 검험。결과담관암세포 QBC939화 HuCCT1중 SIRT1적 mRNA 표체수평분별비정상담관상피세포 HIBEpiC 승고（3．30±0．43）화（4．55±0．50）배，차이균유통계학의의（t ＝7．29、9．79，P 균＜0．05）；담관암세포중SIRT1적단백표체수평승고여 mRNA 상사。전염 SIRT1소간우 RNA 후，담관암세포중 SIRT1 mRNA화단백표체수평현저강저。 HuCCT1세포적전염서렬1조화전염서렬2조적세포생존솔재전염후72 h분별위（19．72±1．39）％화（27．48±2．67）％，균저우음성대조조적（84．65±3．18）％，차이균유통계학의의（t＝37．42、27．54，P균＜0．05）；HuCCT1세포중전염서렬1조화전염서렬2조조기조망세포비례분별위（19．43±1．72）％화（19．80±0．86）％，균고우음성대조조적（5．43±0．31）％，차이균유통계학의의（t＝11．30、22．23，P균＜0．05）；HuCCT1세포중전염서렬1조화전염서렬2조24 h 천과미공막적세포수분별위（56．00±2．61）화（58．80±3．19）개，균소우음성대조조적（76．60±5．08）개，차이균유통계학의의（t＝7．21、5．93，P 균＜0．05）。 QBC939세포적세포생존솔、조기세포조망비례화천이능력여HuCCT1세포상사。하조담관암세포중 SIRT1적표체후，가현저하조생존소、신경형개점단백화파형단백적표체，상조 Bax 、전절형 PARP 화상피형개점단백적표체。결론 SIRT1재담관암세포중고표체，하조담관암세포중 SIRT1적표체가억제세포적증식화천이，촉진기조망，궤제가능여하조생존소，상조 Bax 단백적표체，억제상피간질전화적발생유관。
Objective To explore the expression of silent information regulator of transcription 1 (SIRT1 ) in cholangiocarcinoma cells , and to investigate its effects on proliferation , apoptosis and migration of cholangiocarcinoma cells .Methods The expression of SIRT1 in human cholangiocarcinoma cell lines QBC939 and HuCCT1 were compared with that in normal bile duct epithelial cell line HIBEpiC at mRNA and protein level by real‐time polymerase chain reaction (PCR) and Western blot .The QBC939 and HuCCT1 cells were divided into negative control group ,the first sequence transfected group and the second sequence transfected group , transfected with negative control small interfering RNA (siRNA ) , siRNA sequence 1 for SIRT1 and siRNA sequence 2 for SIRT1 ,respectively . The interference effects were identified by real‐time PCR and Western blot .After transfection ,cell proliferation was determined by cell count kit and clone forming test ,cell apoptosis was detected by flow cytometry ,and cell migration was assessed by Transwell migration assay . The expression of apoptosis related protein survivin , B‐cellymphoma‐2 associated X protein (Bax ) , cleaved poly adenosine diphosphate‐ribose polymerase (PARP) and epithelial cell marker E‐cadherin and mesenchymal cell marker N‐cadherin ,vimentin were determined by Western blot .The t test was performed for statistical analysis .Results The expressions of SIRT1 at mRNA level in cholangiocarcinoma cell lines QBC939 and HuCCT1 were 3 .30 ± 0 .43 and 4 .55 ± 0 .50 times higher than that in normal bile duct epithelial cell line HIBEpiC ,and the differences were statistically significant (t= 7 .29 and 9 .79 ;both P< 0 .05) .The increased expressions of SIRT1 in cholangiocarcinoma cell lines at protein level were similar to that at mRNA level .After transfected with SIRT1 siRNA ,the expression of SIRT1 in cholangiocarcinoma cell lines significantly reduced at mRNA and protein levels .At 72 hour after transfecting ,the cell viability rate of the first sequence transfected group and the second sequence transfected group in HuCCT 1 cells were (19 .72 ± 1 .39)% and (27 .48 ± 2 .67)% ,which were both lower than that of negative control group ((84 .65 ± 3 .18 )% ) , and the differences were statistically significant (t = 37 .42 and 27 .54 ;both P < 0 .05) .In HuCCT1 cells ,the portion of early apoptosis cells of the first sequence transfected group and the second sequence transfected group were (19 .43 ± 1 .72)% and (19 .80 ± 0 .86)% ,which were both significantly higher than that of negative control group ((5 .43 ± 0 .31)% ) ,and the differences were statistically significant (t= 11 .30 and 22 .23 ;both P< 0 .05) .In HuCCT1 cells ,the number of migrated cells through the micropore membranes of the first sequence transfected group and the second sequence transfected group were 56 .00 ± 2 .61 and 58 .80 ± 3 .19 ,which were both lower than that of negative control group (76 .60 ± 5 .08) , and the differences were statistically significant (t = 7 .21 and 5 .93 ;both P< 0 .05) .The cell viability rate ,the portion of early apoptosis cells and migration ability of QBC 939 cells were similar to those of HuCCT1 cells .After the expression of SIRT1 in cholangiocarcinoma cells downregulated ,the expressions at protein level of survivin ,N‐cadherin and vimentin remarkably reduced ,and the expressions of Bax ,cleaved PARP and E‐cadherin upregulated .Conclusions The expression of SIRT1 is higher in cholangiocarcinoma cells . As the expression of SIRT1 in cholangiocarcinoma cells downregulates ,cell proliferation and cell migration are inhibited ,and cell apoptosis is induced .The mechanism may be related with the downregulation of survivin ,upregulation of Bax and the suppression of epithelial‐to‐mesenchymal transition .
