CAJ | 학술논문

目的研究泛素水解酶22(ubiquitin carboxyl-terminal hydrolase 22,USP22)基因在肝癌中的表达及沉默USP22基因对肝癌细胞的增殖活性、细胞凋亡和细胞周期的影响.方法采用免疫组化检测58例肝细胞癌组织及相应癌旁组织中USP22蛋白的表达情况,采用RT-PCR和Western blot检测肝癌细胞中USP22的表达水平,并合成针对USP22基因的siRNA转染肝癌细胞;采用RTPCR检测和Western blot检测转染效率;MTF法测定细胞生长抑制率;流式细胞仪测定细胞凋亡率和细胞周期;免疫印迹检测CyclinD1、CDK4和CDK6蛋白表达水平.结果发现USP22基因在肝癌组织及细胞中高表达;用针对USP22 siRNA干扰后,USP22表达水平明显降低;MTT法检测其细胞生长抑制率为42.24％,明显高于对照组(tsiUSP22组=22.47,P＜0.01);流式细胞仪检测其细胞凋亡率为33.08％ ±4.53％,明显高于对照组(tsiUSP22组=12.34,P＜0.01);流式细胞仪检测细胞周期,发现其处于G1期细胞的百分比为68.81％ ±2.71％,明显高于对照组(tsiUSP2组=33.91,P＜0.01);Western blot检测发现细胞周期蛋白CyclinD1、CDK4和CDK6在USP22 siRNA组中的表达水平明显低于对照组(tCyclinD1=10.85,tCDK4=10.51,tCDK6=10.59,P＜0.01). 结论 UPS22基因在肝癌组织及细胞中存在异常高表达,利用siRNA干扰UPS22表达,可抑制肝癌HepG3B细胞增殖活性,诱导其凋亡,并导致细胞周期被阻滞在G1期以内.
목적 연구범소수해매22(ubiquitin carboxyl-terminal hydrolase 22,USP22)기인재간암중적표체급침묵USP22기인대간암세포적증식활성、세포조망화세포주기적영향.방법 채용면역조화검측58례간세포암조직급상응암방조직중USP22단백적표체정황,채용RT-PCR화Western blot검측간암세포중USP22적표체수평,병합성침대USP22기인적siRNA전염간암세포;채용RTPCR검측화Western blot검측전염효솔;MTF법측정세포생장억제솔;류식세포의측정세포조망솔화세포주기;면역인적검측CyclinD1、CDK4화CDK6단백표체수평.결과 발현USP22기인재간암조직급세포중고표체;용침대USP22 siRNA간우후,USP22표체수평명현강저;MTT법검측기세포생장억제솔위42.24％,명현고우대조조(tsiUSP22조=22.47,P＜0.01);류식세포의검측기세포조망솔위33.08％ ±4.53％,명현고우대조조(tsiUSP22조=12.34,P＜0.01);류식세포의검측세포주기,발현기처우G1기세포적백분비위68.81％ ±2.71％,명현고우대조조(tsiUSP2조=33.91,P＜0.01);Western blot검측발현세포주기단백CyclinD1、CDK4화CDK6재USP22 siRNA조중적표체수평명현저우대조조(tCyclinD1=10.85,tCDK4=10.51,tCDK6=10.59,P＜0.01). 결론 UPS22기인재간암조직급세포중존재이상고표체,이용siRNA간우UPS22표체,가억제간암HepG3B세포증식활성,유도기조망,병도치세포주기피조체재G1기이내.
Objective To explore the expression of ubiquitin carboxyl-terminal hydrolase 22 (USP22) in hepatocellular carcinoma (HCC) and the impact of USP22 gene silence by siRNA on the proliferative activity,apoptosis and cell cycle of HCC cell line HepG3B.Methods Immunohistochemistry was used to detect the expression of USP22 in 58 HCC specimens and paraneoplastic liver tissues,RT-PCR and Western blot was used to detect the expression of USP22 in HCC cells,siRNA targeting USP22 mRNA was transfected into cell line HepG3B,RT-PCR and Western blot was used to detect the transfer efficiency,MTT assay was used to detect the cell proliferation inhibiting rate; Flow cytometry was used to analyze the apoptosis rate of cells and the cell cycle; Western blot was used to detect the expression of D1,CDK4 and CDK6 protein.Results USP22 was highly expressed in HCC; after transfected siRNA targeting USP22,the expression of USP22 decreased; The cell proliferation inhibiting rate was 42.24％,higher than the negative control group(tsiUSP2group =22.47,P ＜0.01) ; the apoptosis rate was 33.08％ ±4.53％,higher than negative control and blank control group(tsiUSP22 group =12.34,P ＜ 0.01) ; The rate of cells in the G1 phase was 68.81％ ± 2.71％,higher than negative control and blank control group (tsiUSP22group =33.91,P ＜0.01) ; D1,CDK4 and CDK6 protein expression was higher than negative control and blank control group(tCyclinD1 =10.85,tCDK4 =10.51,tCDK6 =10.59,P ＜ 0.01).Conclusions USP22 gene was highly expressed in HCC,specific siRNA targeting USP22 efficiently inhibits cell proliferation,promotes apoptosis and caused cell cycle arrest in G1 phase in HepG3B cells.