微小RNA—10b沉默对人三阴性乳腺癌细胞生物学行为的影响及其机制

Effect and mechanism of silencing microRNA-lOb on biological behavior of human triple-negative breast cancer cell

目的观察微小RNA-10b（miR-10b）沉默对人三阴性乳腺癌MDA—MB-231细胞生物学行为的影响，并探讨其机制。方法用人工合成的miR-10binhibitor转染MDA—MB-231细胞，同时设空白细胞组和无义序列转染组为对照组，实时定量聚合酶链反应（Real—timePCR）检测转染后miR-10b的表达，以检验沉默效果。CCK-8法、流式细胞术、Transwell侵袭实验分析细胞增殖、凋亡、侵袭和迁移能力变化。Real—timePCR和Westernblot法分别检测T淋巴细胞侵袭转移诱导因子1（TIAM1）mRNA和蛋白的表达。结果与对照组比较，miR-10binhibitor转染组的2^-△△cr。值（0．26±0．01）明显下调（P〈0．01），表明成功沉默miR-10b的表达。miR-10b沉默后，48h和72h细胞增殖率分别为（409．10±8．38）％和（610．70±17．59）％，均明显上调（P〈0．01），增殖效应在48h之后出现，并一直维持到72h；早期和晚期细胞凋亡比例分别为1．77％和1．61％，均明显下调（P〈0．05）；穿过基质膜的侵袭和迁移细胞数分别为（212．17±13．57）个和（322．67±8．62）个，均明显上调（P〈0．01）。TIAM1基因在mRNA水平变化不大（P〉0．05），在蛋白水平表达上调（P〈0．05）。结论miR，10b沉默可以促进MDA—MB-231细胞的增殖、侵袭和迁移，并抑制凋亡，其机制可能与miR-10b沉默后在转录后水平上调TIAM1蛋白的表达有关。

Objective To investigate the effect of silencing microRNA-10b, on the biological behav- ior of human triple-negative breast cancer MDA-MB-231 ceils and the mechanism Methods MDA-MB-231 cells were transfected with artificially synthesized miR-10b inhibitor. Clank cell group and nonsence se- quence-transfected group were established as a control. Real-time quantitative polymerase chain reaction （ Realime PCR） was conducted to detect the expression of miR-10b after transfection. Cell counting kit-8 （CCK-8） assays, flow cytometry and Transwell assays were used to detect variations in cell proliferation, apoptosis, invasion and migration respectively. Real-time PCR and Western blotting were used to detect the expression of T lymphoma invasion and metastasis inducing factor 1 （ TIAM1 ） mRNA and protein respec- tively. Results As compared with control group, the value of 2^-△△cr（0. 26 ± 0. 01 ） in miR-10b inhibitortransfected group was significantly reduced （ P 〈 0.01 ） , suggesting the expression of miR-10b was suppressed by miR-10b inhibitor successfully. The proliferation rate at 48 h [ （409. 10 ± 8. 38）% ] and at 72 h [ （610. 70 ± 17.59） % ] was significantly increased （ P 〈 0. 01 ） after silencing miR-10b. Proliferation effect occured at 48 h and continued to 72 h. Early apoptosis rate （1.77%） and late apoptosis rate （ 1.61% ） were significantly reduced （ P 〈 0± 05 ）. The number of invasion （212. 17 ± 13.57 ） and migration （322. 67±8. 62） cells was significantly increased （P 〈0. 01 ）. TIAM1 gene had no significant changes at mRNA level （ P 〉 0. 05 ）, but the protein expression was up-regnrated （ P 〈 0. 05 ）± Conclusion Silencing miR-10b could promote proliferation, invasion and migration, and inhibit apoptosis of MDA-MB-231 cells, which is probably related to the positive regulation of TIAMI protein at the post-transcriptional level.