CAJ | 학술논문

目的探讨小RNA干扰技术(siRNA)沉默猫肉瘤相关蛋白(Fer)的表达对前列腺癌细胞周期及凋亡的影响及其机制.方法设计合成3组针对Fer特异的siRNA转染人前列腺癌PC-3细胞,应用实时定量反转录聚合酶链反应(RT-qPCR)和Western blot法检测转染前后PC-3细胞中Fer mRNA及蛋白表达水平,并筛选出干扰效率最高的小干扰RNA进行后续实验,用流式细胞仪检测各组细胞的细胞周期变化并分析细胞凋亡,观察Fer基因表达抑制后细胞周期及凋亡相关基因表达水平的变化.结果利用RT-qPCR检测siRNA-1、siRNA-2、siRNA-3组的Fer mRNA相对表达量分别为0.288、0.369、0.623,与阴性对照(NC)组比较差异均具有统计学意义(以NC组FermRNA表达量为l,P＜0.05),Western blot方法同样证实3组小RNA干扰组显著抑制PC-3细胞的Fer蛋白的表达,其中siRNA-1干扰效率最高(P＜0.01).流式细胞仪结果显示RNA干扰(Si)组细胞G0/G1期细胞比例为(52.340±2.049)％,显著高于NC组(40.230±2.065)％,而在S期细胞比例为(35.040 ±3.810)％,显著低于NC组(50.650±3.693)％,两者与NC组比较差异均具有统计学意义(P＜0.05);Si组细胞的凋亡率为(33.6±3.3)％,显著高于NC组的凋亡率(7.8±1.0)％,差异有统计学意义(P＜0.05);同时随着Fer表达的抑制,细胞周期素(Cyclin) D1,B细胞淋巴瘤/白血病-2 (bcl-2)的表达显著下调,p21和活化型半胱氨酸-3的表达显著上升,与NC组比较差异均有统计学意义(P＜0.05).结论 Fer-siRNA可抑制Fer在前列腺癌细胞中的表达,抑制前列腺癌细胞G1/S期转化,从而诱导肿瘤细胞凋亡,其机制可能与调控细胞周期及凋亡相关基因的表达有关.
목적 탐토소RNA간우기술(siRNA)침묵묘육류상관단백(Fer)적표체대전렬선암세포주기급조망적영향급기궤제.방법 설계합성3조침대Fer특이적siRNA전염인전렬선암PC-3세포,응용실시정량반전록취합매련반응(RT-qPCR)화Western blot법검측전염전후PC-3세포중Fer mRNA급단백표체수평,병사선출간우효솔최고적소간우RNA진행후속실험,용류식세포의검측각조세포적세포주기변화병분석세포조망,관찰Fer기인표체억제후세포주기급조망상관기인표체수평적변화.결과 이용RT-qPCR검측siRNA-1、siRNA-2、siRNA-3조적Fer mRNA상대표체량분별위0.288、0.369、0.623,여음성대조(NC)조비교차이균구유통계학의의(이NC조FermRNA표체량위l,P＜0.05),Western blot방법동양증실3조소RNA간우조현저억제PC-3세포적Fer단백적표체,기중siRNA-1간우효솔최고(P＜0.01).류식세포의결과현시RNA간우(Si)조세포G0/G1기세포비례위(52.340±2.049)％,현저고우NC조(40.230±2.065)％,이재S기세포비례위(35.040 ±3.810)％,현저저우NC조(50.650±3.693)％,량자여NC조비교차이균구유통계학의의(P＜0.05);Si조세포적조망솔위(33.6±3.3)％,현저고우NC조적조망솔(7.8±1.0)％,차이유통계학의의(P＜0.05);동시수착Fer표체적억제,세포주기소(Cyclin) D1,B세포림파류/백혈병-2 (bcl-2)적표체현저하조,p21화활화형반광안산-3적표체현저상승,여NC조비교차이균유통계학의의(P＜0.05).결론 Fer-siRNA가억제Fer재전렬선암세포중적표체,억제전렬선암세포G1/S기전화,종이유도종류세포조망,기궤제가능여조공세포주기급조망상관기인적표체유관.
Objective To investigate the influence of small interference RNA (siRNA) silencing feline sarcoma-related protein (Fer) on cell cycle and apoptosis of prostate cancer cells.Methods PC-3 cells were transfected with three siRNAs targeting human Fer by liposome transfection reagent.Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression of Fer mRNA and protein respectively,and the most efficient siRNA was screened out for the following experiments.The cell cycle and apoptosis rate were analyzed by flow cytometry.Furthermore,we observed the expression levels of the cell cycle-and apoptosis-related genes in the transfected cells with down-regulation of the Fer expression.Results RT-qPCR showed that Fer mRNA relative expression levels of three siRNAs was 0.288,0.369 and 0.623,respectively,which were significantly lower than those in the negative control group (l,P ＜ 0.05).Western blotting also demonstrated that the expression of Fer protein in PC-3 cells transfected with three siRNAs could significantly inhibited,and siRNA-1 was selected as the most efficient siRNA (P ＜ 0.01).Flow cytometry revealed the percentage of G0/G1 phase cells in RNAi (Si) group was (52.340 ± 2.049) ％,significantly higher than that in negative control group [(40.230 ± 2.065) ％] (P ＜ 0.05),and that of S phase cells was (35.040 ± 3.810) ％,significantly lower than that in the negative control group [(50.650 ± 3.693) ％] (P ＜ 0.05).The apoptosis rate in Si group was (33.6 ± 3.3) ％,significanfly higher than that in negative control group [(7.8 ± 1.0) ％] (P ＜0.05).Meanwhile,with the inhibition of Fer,the expression levels of Cyclin D1 and B cell lymphoma/leukemia-2 (bcl-2) were reduced obviously,and p21 and cleaved Caspase-3 upregulated obviously as compared with negative control group (P ＜ 0.05).Conclusion Fer-siRNA could significantly inhibit the expression of Fer and the progression of G1/S in human prostate cancer cells,and then induced apoptosis of PC-3 cells in vitro.The mechanism probably correlates with regulation of the cell cycle-and apoptosis-related genes expression.