中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2012年
11期
846-850
,共5页
微 RNAs%卵巢肿瘤%细胞增殖%肿瘤浸润%细胞凋亡%原癌基因蛋白质c-bcl-2%体外研究
微 RNAs%卵巢腫瘤%細胞增殖%腫瘤浸潤%細胞凋亡%原癌基因蛋白質c-bcl-2%體外研究
미 RNAs%란소종류%세포증식%종류침윤%세포조망%원암기인단백질c-bcl-2%체외연구
MicroRNAs%Ovarian neoplasms%Cell proliferation%Neoplasm invasiveness%Apoptosis%Proto-oncogene proteins c-bcl-2%In vitro
目的 探讨微小RNA-16(miR-16)调控卵巢上皮性癌(卵巢癌)细胞增殖、侵袭及凋亡的作用及机制.方法 脂质体介导miR-16成熟体模拟物及相应的阴性对照片段转染人卵巢癌细胞SKOV-3,分别作为转染组、阴性对照组.实时荧光定量PCR技术检测各组细胞中miR-16的表达,蛋白印迹法检测血管内皮生长因子(VEGF)、基质金属蛋白酶2(MMP-2)、凋亡抑制蛋白bcl-2的表达,采用四甲基偶氮唑蓝法、5-乙炔基-2’脱氧尿苷(EdU)标记法测定细胞的增殖能力,transwell穿膜小室侵袭实验检测细胞的侵袭能力,流式细胞仪检测血清饥饿及低氧条件下细胞的凋亡情况.结果(1)转染组、阴性对照组SKOV-3细胞中miR-16的表达量分别为125.93±15.30、0.78±0.16,转染组明显高于阴性对照组,两组比较,差异有统计学意义(P＜0.01).(2)转染组、阴性对照组SKOV-3细胞中VEGF蛋白的相对表达量为0.58±0.05、1.22±0.03,MMP-2蛋白的相对表达量为0.63±0.03、1.16±0.03,bcl-2蛋白的相对表达量为0.52±0.03、1.19±0.05,转染组各蛋白的相对表达量均明显低于阴性对照组,分别比较,差异均有统计学意义(P＜0.01).(3)转染后第4天,转染组、阴性对照组细胞的增殖抑制率分别为(37.2±6.2)％、(3.6±3.2)％,两组比较,差异有统计学意义(P=0.001).(4)转染组、阴性对照组细胞的增殖率分别为(12.3±0.8)％、(23.4±1.8)％,转染组明显低于阴性对照组,两组比较,差异有统计学意义(P＜0.05).(5)转染组穿过聚碳酸酯膜的细胞数为(6±3)个,明显少于阴性对照组的(40±9)个,两组比较,差异有统计学意义(P＜0.01).(6)血清饥饿及低氧条件培养24 h后,转染组细胞的早期凋亡率[(16.9±2.1)％]、晚期凋亡率[(13.4±3.3)％]均明显高于阴性对照组[分别为(10.3±1.7)％、(3.2±1.8)％],分别比较,差异均有统计学意义(P＜0.01);培养48 h后,转染组细胞总凋亡率明显高于阴性对照组(P＜0.01).结论 miR-16可能通过下调卵巢癌细胞中VEGF、MMP-2及bcl-2蛋白的表达,抑制细胞的增殖、侵袭能力,增强其对凋亡刺激的敏感性.
目的 探討微小RNA-16(miR-16)調控卵巢上皮性癌(卵巢癌)細胞增殖、侵襲及凋亡的作用及機製.方法 脂質體介導miR-16成熟體模擬物及相應的陰性對照片段轉染人卵巢癌細胞SKOV-3,分彆作為轉染組、陰性對照組.實時熒光定量PCR技術檢測各組細胞中miR-16的錶達,蛋白印跡法檢測血管內皮生長因子(VEGF)、基質金屬蛋白酶2(MMP-2)、凋亡抑製蛋白bcl-2的錶達,採用四甲基偶氮唑藍法、5-乙炔基-2’脫氧尿苷(EdU)標記法測定細胞的增殖能力,transwell穿膜小室侵襲實驗檢測細胞的侵襲能力,流式細胞儀檢測血清饑餓及低氧條件下細胞的凋亡情況.結果(1)轉染組、陰性對照組SKOV-3細胞中miR-16的錶達量分彆為125.93±15.30、0.78±0.16,轉染組明顯高于陰性對照組,兩組比較,差異有統計學意義(P＜0.01).(2)轉染組、陰性對照組SKOV-3細胞中VEGF蛋白的相對錶達量為0.58±0.05、1.22±0.03,MMP-2蛋白的相對錶達量為0.63±0.03、1.16±0.03,bcl-2蛋白的相對錶達量為0.52±0.03、1.19±0.05,轉染組各蛋白的相對錶達量均明顯低于陰性對照組,分彆比較,差異均有統計學意義(P＜0.01).(3)轉染後第4天,轉染組、陰性對照組細胞的增殖抑製率分彆為(37.2±6.2)％、(3.6±3.2)％,兩組比較,差異有統計學意義(P=0.001).(4)轉染組、陰性對照組細胞的增殖率分彆為(12.3±0.8)％、(23.4±1.8)％,轉染組明顯低于陰性對照組,兩組比較,差異有統計學意義(P＜0.05).(5)轉染組穿過聚碳痠酯膜的細胞數為(6±3)箇,明顯少于陰性對照組的(40±9)箇,兩組比較,差異有統計學意義(P＜0.01).(6)血清饑餓及低氧條件培養24 h後,轉染組細胞的早期凋亡率[(16.9±2.1)％]、晚期凋亡率[(13.4±3.3)％]均明顯高于陰性對照組[分彆為(10.3±1.7)％、(3.2±1.8)％],分彆比較,差異均有統計學意義(P＜0.01);培養48 h後,轉染組細胞總凋亡率明顯高于陰性對照組(P＜0.01).結論 miR-16可能通過下調卵巢癌細胞中VEGF、MMP-2及bcl-2蛋白的錶達,抑製細胞的增殖、侵襲能力,增彊其對凋亡刺激的敏感性.
목적 탐토미소RNA-16(miR-16)조공란소상피성암(란소암)세포증식、침습급조망적작용급궤제.방법 지질체개도miR-16성숙체모의물급상응적음성대조편단전염인란소암세포SKOV-3,분별작위전염조、음성대조조.실시형광정량PCR기술검측각조세포중miR-16적표체,단백인적법검측혈관내피생장인자(VEGF)、기질금속단백매2(MMP-2)、조망억제단백bcl-2적표체,채용사갑기우담서람법、5-을결기-2’탈양뇨감(EdU)표기법측정세포적증식능력,transwell천막소실침습실험검측세포적침습능력,류식세포의검측혈청기아급저양조건하세포적조망정황.결과(1)전염조、음성대조조SKOV-3세포중miR-16적표체량분별위125.93±15.30、0.78±0.16,전염조명현고우음성대조조,량조비교,차이유통계학의의(P＜0.01).(2)전염조、음성대조조SKOV-3세포중VEGF단백적상대표체량위0.58±0.05、1.22±0.03,MMP-2단백적상대표체량위0.63±0.03、1.16±0.03,bcl-2단백적상대표체량위0.52±0.03、1.19±0.05,전염조각단백적상대표체량균명현저우음성대조조,분별비교,차이균유통계학의의(P＜0.01).(3)전염후제4천,전염조、음성대조조세포적증식억제솔분별위(37.2±6.2)％、(3.6±3.2)％,량조비교,차이유통계학의의(P=0.001).(4)전염조、음성대조조세포적증식솔분별위(12.3±0.8)％、(23.4±1.8)％,전염조명현저우음성대조조,량조비교,차이유통계학의의(P＜0.05).(5)전염조천과취탄산지막적세포수위(6±3)개,명현소우음성대조조적(40±9)개,량조비교,차이유통계학의의(P＜0.01).(6)혈청기아급저양조건배양24 h후,전염조세포적조기조망솔[(16.9±2.1)％]、만기조망솔[(13.4±3.3)％]균명현고우음성대조조[분별위(10.3±1.7)％、(3.2±1.8)％],분별비교,차이균유통계학의의(P＜0.01);배양48 h후,전염조세포총조망솔명현고우음성대조조(P＜0.01).결론 miR-16가능통과하조란소암세포중VEGF、MMP-2급bcl-2단백적표체,억제세포적증식、침습능력,증강기대조망자격적민감성.
Objective To study the role and mechanism of microRNA-16(miR-16)in the proliferation,invasion and apoptosis of ovarian epithelial carcinoma cells in vitro.Methods The SKOV-3 cells were transfected with miR-16 mimics or negative control RNA(NC)by lipofectamine 2000.The expression of miR-16 was detected by real-time reverse transcription(RT)-PCR in SKOV-3 cells,and western blot was used to detect the expression of vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2)and bcl-2 protein.Methyl thiazolyl tetrazolium(MTT),5-ethynyl-2'-deoxyuridine(EdU)and transwell assay were used to determine the proliferation and invasion abilities.And the rate of apoptotic cell was detected by flow cytometry method.Results(l)The expression level of miR16 in the transfection cells group was significantly higher than that in NC group(125.93 ± 15.30 versus 0.78 ± 0.16,P ＜ 0.01).(2)The rclative expression level of VEGF protein in transfection cells,NC and blank control group was 0.58 ± 0.05,1.22 ± 0.03,1.20 ± 0.03,MMP-2 protein was 0.63 ± 0.03,1.16 ±0.03,1.21 ± 0.03,and bel-2 protein 0.52 ± 0.03,1.19 ± 0.05,1.28 ± 0.06,respectively.The level of VEGF,MMP-2 and bcl-2 protein in the transfection group were lower than those in other control groups,and there were significantly differences among them(all P ＜0.01).(3)After transfected 4 days,the inhibition rate of cell proliferation in the transfection group was dramatically higher than that in NC group[(37.2 ±6.2)％ versus(3.6 ± 3.2)％,P =0.001].(4)The percentage rate of proliferative cells in the transfection,NC and blank control group was(12.3 ± 0.8)％,(23.4 ± 1.8)％,(31.1 ± 4.9)％.And it was lower in the transfection group(P ＜ 0.05).(5)Decreased cells via the transwell member in the transfection group(6 ± 3)were detected as compared with NC group(40 ± 9)and blank control group (48 ± 8,P ＜ 0.01).(6)Twenty-four hours after cultured in serum starvation and hypoxia,the rate of the viable and late apoptotic cells in the transfection group were significantly higher than those in NC group and blank control group[the rate of viable apoptotic cell was(16.9 ± 2.1)％,(10.3 ± 1.7)％ and(9.0 ±0.8)％ respectivcly,P＜0.01;the rate of late apoptotic cell was(13.4±3.3)％,(3.2 ±1.8)％ and (0.7 ±0.6)％ respectively,P ＜ 0.01].After cultured 48 hours,total apoptotic cells in the transfection group was significantly more than those in other groups(P ＜ 0.01).Conclusion miR-16 might inhibit the proliferation,invasion of ovarian epithelial carcinoma cells and enhance their sensitivity to apoptotic stimuli via downregulation of the expression of VEGF,MMP-2 and bcl-2 protein.
