中国肿瘤临床
中國腫瘤臨床
중국종류림상
CHINESE JOURNAL OF CLINICAL ONCOLOGY
2014年
23期
1512-1516
,共5页
Hsa-miR202%IL-10%肺癌%A549细胞%增殖
Hsa-miR202%IL-10%肺癌%A549細胞%增殖
Hsa-miR202%IL-10%폐암%A549세포%증식
hsa-mir-202%IL-10%lung cancer%A549 cells%proliferation
目的：探讨Hsa-miR202（人微小非编码RNA-202）对肺癌A549细胞增殖的影响及分子机制。方法：将Hsa-miR-202序列与ppG/miR/eGFP/Blasitidin质粒定向连接，构建靶向Hsa-miR-202基因的真核表达载体pmiR-202，运用脂质体将pmiR-202转染至A549细胞，WST-8法检测细胞增殖率，RT-PCR检测IL-10、miR-202的相对表达水平，Western blot与ELISA检测IL-10蛋白的表达水平，构建荧光素酶报告基因载体验证miR-202与IL-10的相互结合作用。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miR-202的真核表达载体（pmiR-202），pmiR-202对A549细胞的增殖抑制率（24、48、72 h）分别为12%、38%、52%，并具有时间效应关系，较空白对照组及阴性对照组比较，差异有统计学意义（P<0.05）；RT-PCR结果显示，转染pmiR-202后，miR-202的表达水平增加；过表达miR-202能下调IL-10基因及蛋白的相对表达水平，其相对水平分别为25%、0.75，IL-10活性的相对表达量为3.26±0.43 pg/mL，较空白对照组及阴性对照组比较，差异有统计学意义（P<0.05）：荧光素酶活性结果显示，克隆IL-10-3'UTR的质粒与miR-202 mimics共转染293T细胞，引起荧光素活性的减低。结论：pmiR-202有效抑制A549细胞的增殖，并具有时间-效应关系，其机制可能是miR-202通过靶向结合IL-10的3'UTR从而下调其在A549细胞的表达有关。
目的：探討Hsa-miR202（人微小非編碼RNA-202）對肺癌A549細胞增殖的影響及分子機製。方法：將Hsa-miR-202序列與ppG/miR/eGFP/Blasitidin質粒定嚮連接，構建靶嚮Hsa-miR-202基因的真覈錶達載體pmiR-202，運用脂質體將pmiR-202轉染至A549細胞，WST-8法檢測細胞增殖率，RT-PCR檢測IL-10、miR-202的相對錶達水平，Western blot與ELISA檢測IL-10蛋白的錶達水平，構建熒光素酶報告基因載體驗證miR-202與IL-10的相互結閤作用。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miR-202的真覈錶達載體（pmiR-202），pmiR-202對A549細胞的增殖抑製率（24、48、72 h）分彆為12%、38%、52%，併具有時間效應關繫，較空白對照組及陰性對照組比較，差異有統計學意義（P<0.05）；RT-PCR結果顯示，轉染pmiR-202後，miR-202的錶達水平增加；過錶達miR-202能下調IL-10基因及蛋白的相對錶達水平，其相對水平分彆為25%、0.75，IL-10活性的相對錶達量為3.26±0.43 pg/mL，較空白對照組及陰性對照組比較，差異有統計學意義（P<0.05）：熒光素酶活性結果顯示，剋隆IL-10-3'UTR的質粒與miR-202 mimics共轉染293T細胞，引起熒光素活性的減低。結論：pmiR-202有效抑製A549細胞的增殖，併具有時間-效應關繫，其機製可能是miR-202通過靶嚮結閤IL-10的3'UTR從而下調其在A549細胞的錶達有關。
목적：탐토Hsa-miR202（인미소비편마RNA-202）대폐암A549세포증식적영향급분자궤제。방법：장Hsa-miR-202서렬여ppG/miR/eGFP/Blasitidin질립정향련접，구건파향Hsa-miR-202기인적진핵표체재체pmiR-202，운용지질체장pmiR-202전염지A549세포，WST-8법검측세포증식솔，RT-PCR검측IL-10、miR-202적상대표체수평，Western blot여ELISA검측IL-10단백적표체수평，구건형광소매보고기인재체험증miR-202여IL-10적상호결합작용。결과：경DNA측서증실여설계완전일치，측서결과현시성공구건료miR-202적진핵표체재체（pmiR-202），pmiR-202대A549세포적증식억제솔（24、48、72 h）분별위12%、38%、52%，병구유시간효응관계，교공백대조조급음성대조조비교，차이유통계학의의（P<0.05）；RT-PCR결과현시，전염pmiR-202후，miR-202적표체수평증가；과표체miR-202능하조IL-10기인급단백적상대표체수평，기상대수평분별위25%、0.75，IL-10활성적상대표체량위3.26±0.43 pg/mL，교공백대조조급음성대조조비교，차이유통계학의의（P<0.05）：형광소매활성결과현시，극륭IL-10-3'UTR적질립여miR-202 mimics공전염293T세포，인기형광소활성적감저。결론：pmiR-202유효억제A549세포적증식，병구유시간-효응관계，기궤제가능시miR-202통과파향결합IL-10적3'UTR종이하조기재A549세포적표체유관。
Objective:To study the effects of the overexpression of hsa-miR-202 on the proliferation and molecular mechanism of lung cancer A549 cells. Methods:A sequence of hsa-miR-202 with ppG/miR/eGFP/Blasitidin pasmid was directionally connected and a eukaryotic expression vector pmiR-202 of the target hsa-miR-202 gene was constructed. pmiR-202 was transtected to A549 cell with Lipofectamine 2000. The WST assay was used to detect the cell proliferation rate, and RT-PCR was used to detect the relative gene expression levels. Western blot analysis was used to detect the IL-10 protein expression levels. The interaction between miR-202 and IL-10 was examined using a luciferase reporter assay. Results:The design from the DNA sequencing results shows that a eukaryot-ic expression vector of miR-202 was successfully constructed. The proliferation inhibition rates of A549 cells by Pmir-202 were 12%, 38%, and 52%. The differences in the treatment group compared with the blank control and negative control groups were statistically significant. The RT-PCR results showed that the relative expression levels of miR-202 increased after transfection with pmiR-202. Over-expression of miR-202 can downregulate the relative gene and protein expression levels of IL-10, and the relative levels were 25%and 0.75, respectively. Compared with the blank control and the negative control groups, the difference was statistically significant (P<0.05). The fluorescent activity was reduced when transfection was performed with miR-202 mimics, and IL-10-3'UTR plasmid was cloned. Conclusion: pmiR-202 effectively inhibited the proliferation of A549 cells and exhibited a time-effect relationship with miR-202 by targeted combination with IL-10 3'UTR to downregulate IL-10 expression in A549 cells.
