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目的研究microRNA-21参与乳腺癌侵袭转移的调控机制。方法采用实时定量反转录-聚合酶反应(RT-PCR)方法检测MDA-435、MDA-231和MCF-7乳腺癌细胞株microRNA-21的表达;采用transwell方法测定上述三株乳腺癌细胞体外侵袭转移能力;人工合成microRNA-21干扰序列,转染MDA-231、MDA-435乳腺癌细胞,观察对细胞侵袭能力的影响;应用Western blot检测逆转诱导蛋白(RECK)的表达;采用荧光报告基因实验检测RECK与microRNA-21的结合情况。结果 MCF-7、MDA-231及MDA-435细胞株中均具有microRNA-21高表达,且表达量MDA-435>MDA-231>MCF-7,其中,具有高侵袭特性的MDA-435细胞具有最高水平microRNA-21的表达为(4.51±0.71),MDA-231细胞具有中等水平的microRNA-21表达为(2.67±0.27),MCF-7细胞表达microRNA-21水平相对较低为(1.23±0.11),比较差异有统计学意义(P<0.01)。三株细胞中,MDA-435细胞侵袭力最高为(425.4±35.0)细胞数/视野,MCF-7最低为(142.7±13.4)细胞数/视野,MDA-231居中为(263.7±24.4)细胞数/视野,三组细胞侵袭力比较差异有统计学意义(P<0.01)。将anti-microRNA-21分别转染入MDA-231和MDA-435细胞,与对照组比较,两种细胞株转染anti-microRNA-21后分别引起56%和67%的microRNA-21表达量降低,比较差异有统计学意义(P<0.05)。将anti-microRNA-21转染入MDA-231细胞,与阴性对照组比较,anti-microRNA-21引起了34%细胞侵袭数目的减少。将anti-microRNA-21转染入MDA-435细胞,结果表明anti-microRNA-21引起68%侵袭细胞数目的下降。Western blot检测显示,microRNA-21表达下调,MDA-435细胞中RECK表达明显增强;荧光素酶实验显示共转染RECK和anti-microRNA-21的MDA-435细胞荧光酶活性增加38%。结论本研究提示microRNA-21可促进乳腺癌侵袭转移,其作用可能与逆向调控RECK有关。
Objective To investigate the regulatory mechanism of microRNA-21 in invasion and metastasis of breast cancer. Methods The expression of microRNA-21 in MDA-435, MDA-231 and MCF-7 breast cancer cell lines was detected by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) Invasion and metastasis ability of human breast cancer cell line. The microRNA-21 interference sequence was synthesized and transfected into MDA-231 and MDA-435 breast cancer cells to observe the effect on cell invasiveness. Western blot was used to detect the expression of RECK. The combination of RECK and microRNA-21 was tested experimentally. Results The expression of microRNA-21 was highly expressed in MCF-7, MDA-231 and MDA-435 cells, and the expression levels of MDA-435> MDA-231> MCF- The highest level of microRNA-21 was (4.51 ± 0.71), the moderate level of MDA-231 cells was (2.67 ± 0.27), and the lower level of microRNA-21 in MCF-7 cells was (1.23 ± 0.11 ), The difference was statistically significant (P <0.01). Among the three cells, the highest invasiveness of MDA-435 cells was (425.4 ± 35.0) cells / field, the lowest of MCF-7 cells was (142.7 ± 13.4) cells / / Field of vision, the invasiveness of the three groups was significantly different (P <0.01). Transfection of anti-microRNA-21 into MDA-231 and MDA-435 cells resulted in a decrease of 56% and 67% of microRNA-21 expression levels after transfection with anti-microRNA-21 , The difference was statistically significant (P <0.05). Transfection of anti-microRNA-21 into MDA-231 cells resulted in a 34% reduction in cell invasion compared to the negative control. Transfection of anti-microRNA-21 into MDA-435 cells showed that anti-microRNA-21 caused a 68% reduction in the number of invasive cells. Western blot showed that the expression of microRNA-21 was down-regulated and the expression of RECK in MDA-435 cells was significantly increased. Luciferase assay showed that the luciferase activity of MDA-435 cells transfected with RECK and anti-microRNA-21 increased 38%. Conclusions This study suggests that microRNA-21 may promote the invasion and metastasis of breast cancer, which may be related to reverse regulation of RECK.