中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
Chinese Journal of Pathophysiology
2015年
9期
1572-1577
,共6页
邱瑜%黄建平%周勤仙%王欢%石回刚%何金花
邱瑜%黃建平%週勤仙%王歡%石迴剛%何金花
구유%황건평%주근선%왕환%석회강%하금화
人微小RNA-218%LIM和SH3蛋白1%宫颈癌%HeLa细胞
人微小RNA-218%LIM和SH3蛋白1%宮頸癌%HeLa細胞
인미소RNA-218%LIM화SH3단백1%궁경암%HeLa세포
Hsa-miR-218%LIM and SH3 protein 1%Cervical cancer%HeLa cells
目的：探讨人微小RNA－218（Homo sapiens microRNA－218， hsa－miR－218）对宫颈癌HeLa细胞生长的影响及分子机制。方法：构建hsa－miR－218的重组慢病毒表达载体pmiR－218，并将pmiR－218感染至HeLa细胞，台盼蓝拒染法检测细胞数量的变化，WST－8法检测细胞活力，构建萤光素酶报告基因载体验证miR－218与LIM和SH3蛋白1（LASP1）的相互结合作用，real－time PCR检测LASP1的mRNA相对表达水平，Western blot 检测LASP1蛋白的表达水平。结果：经DNA测序证实与设计完全一致，测序结果显示成功构建了重组慢病毒表达载体pmiR－218。 pmiR－218转染HeLa细胞72 h后HeLa细胞存活数量为176±9，对HeLa细胞活力的抑制率具有时间效应关系，较空白对照组比较，差异显著（P＜0．05）；萤光素酶活性结果显示，克隆LASP1－3’UTR的质粒与miR－218 mimics共转染293T细胞，引起萤光素酶活性的减低；real－time PCR 结果显示转染pmiR－218后，miR－218表达水平增加；过表达miR－128能下调LASP1 mRNA及蛋白的相对表达水平，与空白对照组及阴性对照组比较，差异显著（ P＜0．01）。结论：pmiR－218有效抑制HeLa细胞的生长，并具有时间－效应关系，其机制可能是miR－218通过靶向结合LASP1的3’UTR，从而下调其在HeLa细胞的表达有关。
目的：探討人微小RNA－218（Homo sapiens microRNA－218， hsa－miR－218）對宮頸癌HeLa細胞生長的影響及分子機製。方法：構建hsa－miR－218的重組慢病毒錶達載體pmiR－218，併將pmiR－218感染至HeLa細胞，檯盼藍拒染法檢測細胞數量的變化，WST－8法檢測細胞活力，構建螢光素酶報告基因載體驗證miR－218與LIM和SH3蛋白1（LASP1）的相互結閤作用，real－time PCR檢測LASP1的mRNA相對錶達水平，Western blot 檢測LASP1蛋白的錶達水平。結果：經DNA測序證實與設計完全一緻，測序結果顯示成功構建瞭重組慢病毒錶達載體pmiR－218。 pmiR－218轉染HeLa細胞72 h後HeLa細胞存活數量為176±9，對HeLa細胞活力的抑製率具有時間效應關繫，較空白對照組比較，差異顯著（P＜0．05）；螢光素酶活性結果顯示，剋隆LASP1－3’UTR的質粒與miR－218 mimics共轉染293T細胞，引起螢光素酶活性的減低；real－time PCR 結果顯示轉染pmiR－218後，miR－218錶達水平增加；過錶達miR－128能下調LASP1 mRNA及蛋白的相對錶達水平，與空白對照組及陰性對照組比較，差異顯著（ P＜0．01）。結論：pmiR－218有效抑製HeLa細胞的生長，併具有時間－效應關繫，其機製可能是miR－218通過靶嚮結閤LASP1的3’UTR，從而下調其在HeLa細胞的錶達有關。
목적：탐토인미소RNA－218（Homo sapiens microRNA－218， hsa－miR－218）대궁경암HeLa세포생장적영향급분자궤제。방법：구건hsa－miR－218적중조만병독표체재체pmiR－218，병장pmiR－218감염지HeLa세포，태반람거염법검측세포수량적변화，WST－8법검측세포활력，구건형광소매보고기인재체험증miR－218여LIM화SH3단백1（LASP1）적상호결합작용，real－time PCR검측LASP1적mRNA상대표체수평，Western blot 검측LASP1단백적표체수평。결과：경DNA측서증실여설계완전일치，측서결과현시성공구건료중조만병독표체재체pmiR－218。 pmiR－218전염HeLa세포72 h후HeLa세포존활수량위176±9，대HeLa세포활력적억제솔구유시간효응관계，교공백대조조비교，차이현저（P＜0．05）；형광소매활성결과현시，극륭LASP1－3’UTR적질립여miR－218 mimics공전염293T세포，인기형광소매활성적감저；real－time PCR 결과현시전염pmiR－218후，miR－218표체수평증가；과표체miR－128능하조LASP1 mRNA급단백적상대표체수평，여공백대조조급음성대조조비교，차이현저（ P＜0．01）。결론：pmiR－218유효억제HeLa세포적생장，병구유시간－효응관계，기궤제가능시miR－218통과파향결합LASP1적3’UTR，종이하조기재HeLa세포적표체유관。
AIM:To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism .METHODS:The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed . pmiR-218 was transfected into HeLa cells .The number of viable HeLa cells was counted by the method of Trypan blue ex-clusion.The inhibitory rate of cell activity was detected by WST-8 assay.The expression of LIM and SH3 protein 1 (LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot.The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay .RESULTS:The lentivirus expression vector pmiR-218 tar-geting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing .Over-expression of miR-218 inhibi-ted the activity of HeLa cells with the inhibitory rates of 15%, 26%and 65%at 24 h, 48 h and 72 h, respectively .The difference between transfection group and blank control /negative control group was statistically significant .The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3’ UTR plasmid.The relative expression of miR-218 was increased after transfection with pmiR-218.Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25%and 75%respectively.Compared with blank control group and negative control group , the difference was statistically significant (P<0.05).CONCLUSION:pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner.miR-218 targets to the 3’UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells .
