miR-135a对卵巢上皮性癌细胞中HOXA10基因表达及细胞增殖和凋亡的影响
miR-135a대란소상피성암세포중HOXA10기인표체급세포증식화조망적영향
Effects of miR-135a on HOXA10 expression, proliferation and apoptosis of ovarian cancer cells
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2013年 5期 364-369 ,共6页

    汤伟伟%万贵平%万一聪%张林%程文俊

    탕위위%만귀평%만일총%장림%정문준

    微RNAs%卵巢肿瘤%细胞系,肿瘤%同源盒结构域蛋白质类%细胞增殖%细胞凋亡 微RNAs%卵巢腫瘤%細胞繫,腫瘤%同源盒結構域蛋白質類%細胞增殖%細胞凋亡
    미RNAs%란소종류%세포계,종류%동원합결구역단백질류%세포증식%세포조망
    Micro RNAs%Ovarian neoplasms%Cell line,tumor%Homeodomain proteins%Cell proliferation%Apoptosis
    目的 研究微小RNA-135a(miR-135a)对卵巢上皮性癌(卵巢癌)细胞系SKOV3细胞中HOXA1O基因表达及细胞增殖和凋亡的影响.方法 (1)应用生物信息学技术预测与miR-135a高度相关的靶基因(即HOXA10基因).(2)将miR-135a成熟体模拟物(mimics)、miR-135a成熟体抑制物(inhibitor)及阴性对照分别转染SKOV3细胞,采用逆转录(RT)-PCR技术和蛋白印迹法检测转染不同时间(分别为24、48和72 h)后SKOV3细胞中HOXA10 mRNA和蛋白的表达水平.(3)报告基因实验验证miR-135a对HOXA10基因表达的调节作用,采用含miR-135a的质粒和含HOXA10基因的重组质粒共同转染SKOV3细胞,以转染微小RNA (miRNA)慢病毒质粒者为对照.(4)采用四甲基偶氮唑蓝(MTT)比色法检测转染不同时间(分别为24、48和72 h)后SKOV3细胞的增殖能力[以吸光度(A)值表示],蛋白印迹法检测转染48 h后SKOV3细胞中凋亡相关蛋白[包括bcl-2、bax和半胱氨酸天冬氨酸蛋白酶3(caspase-3)]的表达.结果 (1)生物信息学技术预测,得出与miR-135a高度相关的靶基因为HOXA10基因.(2)RT-PCR技术检测显示,随着转染时间的延长(分别为24、48和72 h),miR-135a mimics转染后SKOV3细胞中HOXA10 mRNA的表达水平(分别为0.94±0.04、0.78 ±0.03、0.70±0.03)逐渐降低(P＜0.05)；miR-135a inhibitor转染后SKOV3细胞中HOXA10 mRNA的表达水平(分别为1.14 ±0.05、1.16 ±0.03、2.60±0.08)逐渐升高(P＜0.05)；且两者转染48及72 h后,分别与阴性对照(为1.00)比较,差异均有统计学意义(P＜0.01).蛋白印迹法检测显示,转染不同时间后SKOV3细胞中HOXA10蛋白的表达水平与HOXA10 mRNA的变化一致.(3)报告基因实验显示,含miR-135a的质粒和含HOXA10基因的重组质粒共同转染SKOV3细胞后,与对照比较,其荧光素酶活性下降了67.8％ (P ＜0.01).(4) MTT比色法检测显示,miR-135amimics转染48和72 h后SKOV3细胞的增殖能力(,4值分别为0.38±0.03、0.67 ±0.05),明显低于阴性对照(A值分别为0.52 ±0.05、0.75 ±0.06;P ＜0.05)；miR-135a inhibitor转染72 h后SKOV3细胞的增殖能力(A值为0.95±0.05),明显高于阴性对照(A值为0.75 ±0.06;P ＜0.01).蛋白印迹法检测显示,miR-135a mimics转染后SKOV3细胞中bcl-2蛋白表达水平(0.28±0.06)明显低于阴性对照(0.76±0.09；P ＜0.01);bax蛋白表达水平无明显变化(P=0.142)；caspase-3酶活性(115.0±2.4)明显高于阴性对照(95.4 ±2.1;P＜0.01).miR-135a inhibitor转染后SKOV3细胞中bcl-2蛋白表达水平(0.92±0.03)明显高于阴性对照(0.76 ±0.09;P =0.037)；bax蛋白表达水平无明显变化(P=0.066);caspase-3酶活性(59.5±4.1)明显低于阴性对照(95.4±2.1;P＜0.01).结论 miR-135a通过HOXA10基因及其下游通路中凋亡相关蛋白影响卵巢癌细胞的增殖和凋亡.
    목적 연구미소RNA-135a(miR-135a)대란소상피성암(란소암)세포계SKOV3세포중HOXA1O기인표체급세포증식화조망적영향.방법 (1)응용생물신식학기술예측여miR-135a고도상관적파기인(즉HOXA10기인).(2)장miR-135a성숙체모의물(mimics)、miR-135a성숙체억제물(inhibitor)급음성대조분별전염SKOV3세포,채용역전록(RT)-PCR기술화단백인적법검측전염불동시간(분별위24、48화72 h)후SKOV3세포중HOXA10 mRNA화단백적표체수평.(3)보고기인실험험증miR-135a대HOXA10기인표체적조절작용,채용함miR-135a적질립화함HOXA10기인적중조질립공동전염SKOV3세포,이전염미소RNA (miRNA)만병독질립자위대조.(4)채용사갑기우담서람(MTT)비색법검측전염불동시간(분별위24、48화72 h)후SKOV3세포적증식능력[이흡광도(A)치표시],단백인적법검측전염48 h후SKOV3세포중조망상관단백[포괄bcl-2、bax화반광안산천동안산단백매3(caspase-3)]적표체.결과 (1)생물신식학기술예측,득출여miR-135a고도상관적파기인위HOXA10기인.(2)RT-PCR기술검측현시,수착전염시간적연장(분별위24、48화72 h),miR-135a mimics전염후SKOV3세포중HOXA10 mRNA적표체수평(분별위0.94±0.04、0.78 ±0.03、0.70±0.03)축점강저(P＜0.05)；miR-135a inhibitor전염후SKOV3세포중HOXA10 mRNA적표체수평(분별위1.14 ±0.05、1.16 ±0.03、2.60±0.08)축점승고(P＜0.05)；차량자전염48급72 h후,분별여음성대조(위1.00)비교,차이균유통계학의의(P＜0.01).단백인적법검측현시,전염불동시간후SKOV3세포중HOXA10단백적표체수평여HOXA10 mRNA적변화일치.(3)보고기인실험현시,함miR-135a적질립화함HOXA10기인적중조질립공동전염SKOV3세포후,여대조비교,기형광소매활성하강료67.8％ (P ＜0.01).(4) MTT비색법검측현시,miR-135amimics전염48화72 h후SKOV3세포적증식능력(,4치분별위0.38±0.03、0.67 ±0.05),명현저우음성대조(A치분별위0.52 ±0.05、0.75 ±0.06;P ＜0.05)；miR-135a inhibitor전염72 h후SKOV3세포적증식능력(A치위0.95±0.05),명현고우음성대조(A치위0.75 ±0.06;P ＜0.01).단백인적법검측현시,miR-135a mimics전염후SKOV3세포중bcl-2단백표체수평(0.28±0.06)명현저우음성대조(0.76±0.09；P ＜0.01);bax단백표체수평무명현변화(P=0.142)；caspase-3매활성(115.0±2.4)명현고우음성대조(95.4 ±2.1;P＜0.01).miR-135a inhibitor전염후SKOV3세포중bcl-2단백표체수평(0.92±0.03)명현고우음성대조(0.76 ±0.09;P =0.037)；bax단백표체수평무명현변화(P=0.066);caspase-3매활성(59.5±4.1)명현저우음성대조(95.4±2.1;P＜0.01).결론 miR-135a통과HOXA10기인급기하유통로중조망상관단백영향란소암세포적증식화조망.
    Objective To investigate the effects of miR-135a on HOXA10 expression,proliferation and apoptosis of SKOV3 cells.Methods (1) Through computer-aided algorithms,the predicted target gene of miR-135a (HOXA10)were determined.(2) miR-135a mimics,miR-135a inhibitor and negative control were transfected into SKOV3 cells,respectively.Reverse transcription (RT)-PCR,western blot analysis were used to examine the expression levels of HOXA10 at different times (24,48 and 72 hours).(3) A luciferase reporter assay was used to confirm the direct regulation between miR-135a and HOXA10.(4) SKOV3 cells proliferation at different times (24,48 and 72 hours) was detected by methyl thiazolyl tetrazolium(MTT) assay [quantified by absorbance(A)].Western blot was used to examine the expression of apoptosis-associated protein bcl-2,bax and caspase-3 in SKOV3 cells after 48 hours transfection.Results (1) HOXA10 was predicted to be the target gene of miR-135a by computer-aided algorithms.(2) RT-PCR shown that HOXA10 mRNA levels were decreased over time (24,48 and 72 hours) after miR-135a mimics transfectionin SKOV3 cells (0.94 ±0.04 vs 0.78 ±0.03 vs 0.70 ±0.03,P ＜0.05).While,the expression of HOXA10 mRNA was increased over time after miR-135a inhibitor transfection (1.14 ± 0.05 vs 1.16 ±0.03 vs 2.60 ±0.08,P ＜0.05).After transfected with miR-135a mimics or miR-135a inhibitor over 48 and 72 hours,the HOXA10 expression levels in SKOV3 cells were significantly lower or higher than each control group,respectively (all P ＜ 0.01).Western blot analysis of HOXA10 expression in SKOV3 cells confirmed the results of RT-PCR detected.(3) After cotransfection of miR-135a plasmid and pMIR-REPORT luciferase plasmid containing HOXA10,luciferase reporter assays showed that the luciferase activity reduced by 67.8％ (P ＜0.01).(4) MTT showed that SKOV3 cells growth after miR-135a mimics transfection for 48 and 72 hours were significantly lower than those in control group (0.38 ± 0.03 vs 0.52 ± 0.05,0.67 ±0.05 vs 0.75 ± 0.06 ; respectively,all P ＜ 0.05).While,SKOV3 cells transfected with miR-135a inhibitor for 72 hours grew significantly faster than that in control group (0.95 ± 0.05 vs 0.75 ± 0.06,P ＜ 0.01).After miR-135a mimics transfection,the level of bcl-2 protein was significantly lower than that in control group (0.28 ±0.06 vs 0.76 ±0.09,P ＜0.01).The activity of caspase-3 was significantly higher than that in control group (115.0 ± 2.4 vs 95.4 ± 2.1,P ＜ 0.01).While,there was no statistical difference of bax expression (P =0.142).However,after miR-135a inhibitor transfection,the expression level of bcl-2 protein was significantly higher than that in control group (0.92 ± 0.03 vs 0.76 ± 0.09,P =0.037) and the activity of caspase-3 was significantly lower than that in control group (59.5 ± 4.1 vs 95.4 ± 2.1,P ＜ 0.01).There was also no statistical difference of bax expression (P =0.066).Conclusion miR-135a may play an important role in cell proliferation and apoptosis of ovarian cancer cells by regulating HOXA10 and its downstream pathways.
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