CAJ | 학술논문

目的探讨miR-16、核转录因子-κB1(NF-κB1)在人脑胶质瘤中的表达及其与胶质瘤细胞株SHG44、U87和U373侵袭生长的相关性. 方法 (1)收集苏州大学第一附属医院神经外科自2000年1月至2011年1月手术切除的29例胶质瘤组织和同期行减压术切除的6例正常脑组织标本,qRT-PCR检测标本miR-16、NF-κB1的表达.(2)将体外培养的U87、U373和SHG44细胞分为空白组、无义序列转染组,miR-16模拟物转染组,分别转染miR-16无义序列和miR-16模拟物,48 h后qRT-PCR检测细胞miR-16、NF-κB1的表达;Transwell实验检测细胞侵袭能力;72 h后Western blotting检测细胞NF-κB1、基质金属蛋白酶(MMP)-9和MMP-2蛋白的表达.(3)荧光素酶实验检测miR-16对NF-κB1基因的靶向调控作用.(4)以U87细胞作为阴性对照组,构建稳定表达miR-16的U87细胞株并建立颅内肿瘤、皮下肿瘤裸鼠模型(U87-miR-16组),免疫荧光、免疫组化分别检测2组裸鼠颅内肿瘤MMP9和Ki-67、NF-κB1、MMP9蛋白的表达;测量2组裸鼠皮下肿瘤的体积并绘制其生长曲线. 结果 (1)qRT-PCR显示miR-16在人脑胶质瘤中表达低于正常脑组织,且miR-16在Ⅰ级、Ⅱ和Ⅲ级、Ⅳ级胶质瘤中的表达量逐渐降低;NF-κB1在胶质瘤中的表达高于正常脑组织,且NF-κB1在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达量逐渐增高,差异有统计学意义((P＜0.05).(2)与空白组和无义序列转染组比较,miR-16模拟物转染组细胞miR-16表达增加,NF-κB1基因表达下降,侵袭能力下降,NF-κB1和MMP-9蛋白的表达下降,差异均有统计学意义(P＜0.05).(3)荧光素酶实验显示pMIR-NF-κB1组的荧光标准化比值明显高于pMIR-NF-κB1 +pre-miR-16组,差异有统计学意义(P＜0.05).(4)与U87阴性对照组比较,U87-miR-16组模型后第24～42天裸鼠皮下肿瘤的体积较小,差异有统计学意义(P＜0.05).与U87阴性对照组比较,U87-miR-16组裸鼠颅内肿瘤MMP-9的表达较低,NF-κB1、MMP-9和Ki-67的表达较低(Ki-67增殖指数分别为13.91％、32.98％). 结论 miR-16通过调控NF-κB1、MMP-9的表达从而抑制胶质瘤细胞侵袭和生长. 目的探討miR-16、覈轉錄因子-κB1(NF-κB1)在人腦膠質瘤中的錶達及其與膠質瘤細胞株SHG44、U87和U373侵襲生長的相關性. 方法 (1)收集囌州大學第一附屬醫院神經外科自2000年1月至2011年1月手術切除的29例膠質瘤組織和同期行減壓術切除的6例正常腦組織標本,qRT-PCR檢測標本miR-16、NF-κB1的錶達.(2)將體外培養的U87、U373和SHG44細胞分為空白組、無義序列轉染組,miR-16模擬物轉染組,分彆轉染miR-16無義序列和miR-16模擬物,48 h後qRT-PCR檢測細胞miR-16、NF-κB1的錶達;Transwell實驗檢測細胞侵襲能力;72 h後Western blotting檢測細胞NF-κB1、基質金屬蛋白酶(MMP)-9和MMP-2蛋白的錶達.(3)熒光素酶實驗檢測miR-16對NF-κB1基因的靶嚮調控作用.(4)以U87細胞作為陰性對照組,構建穩定錶達miR-16的U87細胞株併建立顱內腫瘤、皮下腫瘤裸鼠模型(U87-miR-16組),免疫熒光、免疫組化分彆檢測2組裸鼠顱內腫瘤MMP9和Ki-67、NF-κB1、MMP9蛋白的錶達;測量2組裸鼠皮下腫瘤的體積併繪製其生長麯線. 結果 (1)qRT-PCR顯示miR-16在人腦膠質瘤中錶達低于正常腦組織,且miR-16在Ⅰ級、Ⅱ和Ⅲ級、Ⅳ級膠質瘤中的錶達量逐漸降低;NF-κB1在膠質瘤中的錶達高于正常腦組織,且NF-κB1在Ⅰ、Ⅱ、Ⅲ、Ⅳ級膠質瘤中的錶達量逐漸增高,差異有統計學意義((P＜0.05).(2)與空白組和無義序列轉染組比較,miR-16模擬物轉染組細胞miR-16錶達增加,NF-κB1基因錶達下降,侵襲能力下降,NF-κB1和MMP-9蛋白的錶達下降,差異均有統計學意義(P＜0.05).(3)熒光素酶實驗顯示pMIR-NF-κB1組的熒光標準化比值明顯高于pMIR-NF-κB1 +pre-miR-16組,差異有統計學意義(P＜0.05).(4)與U87陰性對照組比較,U87-miR-16組模型後第24～42天裸鼠皮下腫瘤的體積較小,差異有統計學意義(P＜0.05).與U87陰性對照組比較,U87-miR-16組裸鼠顱內腫瘤MMP-9的錶達較低,NF-κB1、MMP-9和Ki-67的錶達較低(Ki-67增殖指數分彆為13.91％、32.98％). 結論 miR-16通過調控NF-κB1、MMP-9的錶達從而抑製膠質瘤細胞侵襲和生長.
목적 탐토miR-16、핵전록인자-κB1(NF-κB1)재인뇌효질류중적표체급기여효질류세포주SHG44、U87화U373침습생장적상관성. 방법 (1)수집소주대학제일부속의원신경외과자2000년1월지2011년1월수술절제적29례효질류조직화동기행감압술절제적6례정상뇌조직표본,qRT-PCR검측표본miR-16、NF-κB1적표체.(2)장체외배양적U87、U373화SHG44세포분위공백조、무의서렬전염조,miR-16모의물전염조,분별전염miR-16무의서렬화miR-16모의물,48 h후qRT-PCR검측세포miR-16、NF-κB1적표체;Transwell실험검측세포침습능력;72 h후Western blotting검측세포NF-κB1、기질금속단백매(MMP)-9화MMP-2단백적표체.(3)형광소매실험검측miR-16대NF-κB1기인적파향조공작용.(4)이U87세포작위음성대조조,구건은정표체miR-16적U87세포주병건립로내종류、피하종류라서모형(U87-miR-16조),면역형광、면역조화분별검측2조라서로내종류MMP9화Ki-67、NF-κB1、MMP9단백적표체;측량2조라서피하종류적체적병회제기생장곡선. 결과 (1)qRT-PCR현시miR-16재인뇌효질류중표체저우정상뇌조직,차miR-16재Ⅰ급、Ⅱ화Ⅲ급、Ⅳ급효질류중적표체량축점강저;NF-κB1재효질류중적표체고우정상뇌조직,차NF-κB1재Ⅰ、Ⅱ、Ⅲ、Ⅳ급효질류중적표체량축점증고,차이유통계학의의((P＜0.05).(2)여공백조화무의서렬전염조비교,miR-16모의물전염조세포miR-16표체증가,NF-κB1기인표체하강,침습능력하강,NF-κB1화MMP-9단백적표체하강,차이균유통계학의의(P＜0.05).(3)형광소매실험현시pMIR-NF-κB1조적형광표준화비치명현고우pMIR-NF-κB1 +pre-miR-16조,차이유통계학의의(P＜0.05).(4)여U87음성대조조비교,U87-miR-16조모형후제24～42천라서피하종류적체적교소,차이유통계학의의(P＜0.05).여U87음성대조조비교,U87-miR-16조라서로내종류MMP-9적표체교저,NF-κB1、MMP-9화Ki-67적표체교저(Ki-67증식지수분별위13.91％、32.98％). 결론 miR-16통과조공NF-κB1、MMP-9적표체종이억제효질류세포침습화생장.
Objective To explore the microRNA-16 (miR-16) and nuclear-transcription factor-κB1 (NF-κB1) expressions in human brain gliomas and their correlations with cell invasion and growth of malignant gliomas SHG44,U87 and U373.Methods (1) Twenty-nine cases of human glioma tissue samples and 6 normal brain tissues,collected in our hospital from January 2000 to January 2011,were chosen in our study; quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expressions ofmiR-16 and NF-κB1 in these tissues.(2) In vitro cultured U87,U373 and SHG44 cells were divided into blank-control group,nonsense sequence transfected group and miR-16 mimics transfected group; 48 h after the transfection,qRT-PCR was used to detect the expressions of miR-16 and NF-κB1; tmnswell assay was used to observe the cell invasion capability; 72 h after the transfection,Western blotting was employed to detect the protein expressions of NF-κB1,matrix metallopeptidase 9 (MMP-9) and MMP-2.(3) Luciferase reporter assay was used to detect the target regulating role of miR-16 in NF-κB1 gene.(4) U87 cells were used as negative control group,and U87 cells carried stably expressed miR-16 gene were implanted into intracranial and subcutaneous nude mice (U87-miR-16 group); immunofluorescence was used to detect the MMP-9 expression,and immunohistochemical staining was used to detect the protein expressions of Ki-67,NF-κ B1 and MMP-9; subcutaneous tumor volume was measured and the growth curve was drawn.Results (1) The qPCR results showed that the expression of miR-16 in human brain glioma tissues was significantly lower than that in normal brain tissues; and gradually decreased miR-16 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05); NF-κB1 expression in human brain glioma tissues was significantly higher than that in normal brain tissues; and gradually increased NF-κB1 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05).(2) As compared with those in the blank-control group and nonsense sequence transfected group,miR-16 mimics transfected group had significantly increased miR-16 expression,decreased NF-κB1 mRNA expression,decreased invasiveness,and decreased protein expressions of NF-κB1 and MMP-9 (P＜0.05).(3) Luciferase reporter assay showed that the fluorescence normalized ratio in the pMIR-NF-κB1 group was signfcaintly higher than that in the pMIR-NF-κB1+pre-miR-16 group (P＜0.05).(4) As compared with the negative control group,the U87-miR-16 group on the 24-42 d of implantation had significantly smaller volume of tumors (P＜0.05),and lower MMP9 expression,and NF-κB1,MMP-9 and Ki-67 expressions (the proliferation index of Ki-67:13.91％ and 32.98％).Conclusion MiR-16 inhibits glioma cell invasion and growth through down-mgulating NF-κB1 and MMP-9 expressions.