微RNA-29b对胃癌腹膜高转移潜能细胞系侵袭和增殖能力的影响
미RNA-29b대위암복막고전이잠능세포계침습화증식능력적영향
Effects of microRNA-29b on invasion and proliferation of human high peritoneal metastatic gastric cancer cells
  • 万方数据
    中华消化杂志 중화소화잡지
    Chinese Journal of Digestion
    2015年 4期 241-246 ,共6页

    辛瑞娟%谭斌%酒梦娜%冯雅宁%白飞虎

    신서연%담빈%주몽나%풍아저%백비호

    胃肿瘤%腹膜转移%细胞侵袭%细胞增殖%微RNA-29b 胃腫瘤%腹膜轉移%細胞侵襲%細胞增殖%微RNA-29b
    위종류%복막전이%세포침습%세포증식%미RNA-29b
    Stomach neoplasms%Peritoneal metastasis%Cell invasion%Cell proliferation%miRNA-29b
    目的 研究miRNA 29b在胃癌细胞株GC9811及胃癌腹膜高转移潜能细胞株GC9811 P中的表达及其对细胞侵袭、增殖及凋亡能力的影响.方法 通过实时荧光定量PCR测定miRNA 29b在GC9811和GC9811 P细胞中的相对表达量.将GC9811细胞分3组,miRNA-29b下调组转染慢病毒LV miRNA 29b抑制子,阴性对照组转染空载体,未转染细胞作为空白对照组.将GC9811P细胞分3组培养,miRNA-29b上调组转染慢病毒LV-miRNA 29b,阴性对照组转染空载体,未转染细胞作为空白对照组.Transwell小室实验检测细胞的侵袭能力,MTT实验检测细胞的增殖能力,平板克隆实验检测细胞的克隆形成能力,流式细胞术检测细胞的凋亡情况.实时荧光定量PCR检测胃癌细胞(GC9811-P、GC 9811、MKN28-M、MKN28-NM)中miRNA-29b与富含半胱氨酸的酸性分泌蛋白(SPARC)基因较正常胃黏膜细胞GES的相对表达水平并进行相关性分析.两样本均数之间的比较采用两独立样本的t检验和SNK-q检验,3组样本均数之间的比较采用单因素方差分析.结果 GC9811P细胞中miRNA-29b的相对表达量(0.21±0.04)明显低于GC9811细胞(1.00±0.03,t=28.140,P＜0.01).GC9811细胞转染慢病毒LV-miRNA-29b抑制子后,细胞中miRNA-29b的表达(0.21±0.04)较阴性对照组(0.89±0.07)或空白对照组(1.00±0.04)明显降低(q=12.76、14.73,P均＜0.01),跨膜细胞数(274.33±9.03)较阴性对照组显著增多(110.67±13.69,t=9.981,P＜0.01),克隆形成能力明显增强(131.33±4.91比69.67±2.33,t=11.340,P＜0.01),MTT实验亦显示其增殖能力显著增强.GC9811-P细胞转染慢病毒LV miRNA-29b后,细胞中miRNA-29b的表达(4.08±0.20)较阴性对照组(1.15±0.05)或空白对照组(1.00±0.10)明显升高(q=21.73、22.81,P均＜0.01);跨膜细胞数(51.33±5.55)较阴性对照组(104.00±6.24)明显减少(t=6.305,P＜0.01),MTT实验亦显示其增殖能力明显减弱.细胞凋亡实验证实miRNA-29b具有促进细胞凋亡的趋势,但差异无统计学意义(P＞0.05);胃癌细胞中miRNA-29b和SPARC mRNA的表达呈负相关(r=-0.97,P=0.03).结论 miRNA-29b在胃癌腹膜高转移潜能细胞系中低表达,对胃癌细胞的侵袭和增殖具有抑制作用,miRNA-29b可能成为抑制胃癌腹膜转移的一个新的靶点.
    목적 연구miRNA 29b재위암세포주GC9811급위암복막고전이잠능세포주GC9811 P중적표체급기대세포침습、증식급조망능력적영향.방법 통과실시형광정량PCR측정miRNA 29b재GC9811화GC9811 P세포중적상대표체량.장GC9811세포분3조,miRNA-29b하조조전염만병독LV miRNA 29b억제자,음성대조조전염공재체,미전염세포작위공백대조조.장GC9811P세포분3조배양,miRNA-29b상조조전염만병독LV-miRNA 29b,음성대조조전염공재체,미전염세포작위공백대조조.Transwell소실실험검측세포적침습능력,MTT실험검측세포적증식능력,평판극륭실험검측세포적극륭형성능력,류식세포술검측세포적조망정황.실시형광정량PCR검측위암세포(GC9811-P、GC 9811、MKN28-M、MKN28-NM)중miRNA-29b여부함반광안산적산성분비단백(SPARC)기인교정상위점막세포GES적상대표체수평병진행상관성분석.량양본균수지간적비교채용량독립양본적t검험화SNK-q검험,3조양본균수지간적비교채용단인소방차분석.결과 GC9811P세포중miRNA-29b적상대표체량(0.21±0.04)명현저우GC9811세포(1.00±0.03,t=28.140,P＜0.01).GC9811세포전염만병독LV-miRNA-29b억제자후,세포중miRNA-29b적표체(0.21±0.04)교음성대조조(0.89±0.07)혹공백대조조(1.00±0.04)명현강저(q=12.76、14.73,P균＜0.01),과막세포수(274.33±9.03)교음성대조조현저증다(110.67±13.69,t=9.981,P＜0.01),극륭형성능력명현증강(131.33±4.91비69.67±2.33,t=11.340,P＜0.01),MTT실험역현시기증식능력현저증강.GC9811-P세포전염만병독LV miRNA-29b후,세포중miRNA-29b적표체(4.08±0.20)교음성대조조(1.15±0.05)혹공백대조조(1.00±0.10)명현승고(q=21.73、22.81,P균＜0.01);과막세포수(51.33±5.55)교음성대조조(104.00±6.24)명현감소(t=6.305,P＜0.01),MTT실험역현시기증식능력명현감약.세포조망실험증실miRNA-29b구유촉진세포조망적추세,단차이무통계학의의(P＞0.05);위암세포중miRNA-29b화SPARC mRNA적표체정부상관(r=-0.97,P=0.03).결론 miRNA-29b재위암복막고전이잠능세포계중저표체,대위암세포적침습화증식구유억제작용,miRNA-29b가능성위억제위암복막전이적일개신적파점.
    Objective To investigate the expression of microRNA-29b (miR-29b) in gastric cancer cell line GC9811 and high peritoneal metastatic gastric cancer cell line GC9811-P and its effect on invasion,proliferation and apoptosis.Methods The relative quantitative expression of miR-29b was detected by quantitative realtime polymerase chain reaction(qRT PCR).GC9811 cells were divided into three groups,miRNA down-regulated and transfected with lentiviruses LV-miR-29b inhibitor group,negative control group with negative transfection,and untransfected blank control group.GC9811-P cells were divided into three groups,miR-29b up regulated and transfected with lentiviruses LV miR 29b group,negative control with negative transfection group,and untransfected blank control group.The cell invasion ability was detected with Transwell assay,the cell proliferation ability was measured by methyl-thiazolyl tetrazolium (MTT) test,the colony forming ability was determined by plate colony formation assay,and the apoptosis was tested by flow cytometry.The expressions of miR 29b and secreted protein,acidic and rich in cystenie (SPARC) in gastric cell line GC9811-P,GC9811,MKN28M,MKN28NM and normal gastric cell line GES were determined by qRT-PCR,and the correlation was analyzed.Two independent samples t test or SNK-q test was performed for mean comparison between two groups,and one way analysis of variance was used for mean comparison among three groups.Results The relative quantitative expression of miR-29b inGC9811-P (0.21±-0.04) was significantly lower than that of GC9811 (1.00±0.03,t 28.140,P＜ 0.01).After GC9811 cells transfected with lentiviruses LV-miR-29b inhibitor,the expression of miR-29b (0.21±0.04) was significantly lower than that of control group (0.89±0.07) and blank control group (1.00±0.04,q 12.76,14.73,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up-regulated group raised(274.33± 9.03 vs 110.67 ± 13.69,t=9.981,P＜0.01;131.33±4.91 vs69.67±2.33,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was increased.After GC9811-P cells transfected with LV-miR-29b,the expression of miR 29b (4.08±0.20) was significantly higher than that of negative control group (1.15±0.05) and blank control group (1.00±0.10,q=21.73,22.81,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up regulated group reduced (51.33±5.55 vs 104.00±6.24,t 6.305,P＜0.01; 48.00±5.51 vs 113.33±5.17,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was weakened.Apoptosis assays demonstrated miR-29b promoted apoptosis; however,the difference was not statistically significant.The expression of miR-29b was negatively correlated with SPARC mRNA in gastric cancer cells (r=-0.97,P=0.03).Conclusions The low expression of miR-29b in high peritoneal metastatic gastric cancer cell inhibited the ability of invasion and proliferation.MiR-29b might be a new target of inhibiting peritoneal metastasis in gastric cancer.
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