基底样乳腺癌中microRNA-205靶向调控KLF12及对细胞侵袭能力的影响

KLF12 targeted regulation of microRNA-205 in basal-like breast cancer and its effect on cell invasion

目的探讨基底样乳腺癌(basal-like breast carcinoma,BLBC)特异性miRNAs和靶基因及生物学功能。方法提取细胞总RNA和蛋白,荧光素酶实验验证靶基因;采用qRT-PCR、Western blot和免疫组化En Vision法分析miRNA和靶基因表达;CCK8和Transwell实验检测细胞增殖和侵袭能力。结果荧光素酶实验示miR-205靶向调控KLF12(P=0.001 6)。qRT-PCR检测结果示MAD-MB-468细胞中miR-205表达下调(P=0.007),KLF12表达升高(P=0.039);转染miR-205 mimics过表达miR-205,miR-205表达显著升高(P=0.000),KLF12表达减少(P=0.038)。Western blot检测结果示miR-205过表达,MDAMB-468细胞KLF12表达显著升高(P=0.007 9)。CCK8实验示miR-205未参与细胞增殖。Transwell实验示过表达miR-205显著抑制细胞侵袭能力(P=0.001)。结论 miR-205是BLBC特异性的miRNA,通过负性靶向调控原癌基因KLF12和抑制侵袭具有抑癌基因的功能。miR-205和KLF12为BLBC的诊断和治疗提供潜在的分子标志物和新思路。

Purpose To identify the specific miRNAs and target gene of basal-like breast cancer, and to explore its distinct biological function. Methods Total RNA and protein was extracted from cells. KLF12 3＇ -UTR construct and luciferase reporter assays was performed for target gene. Expression levels of miRNAs including its target were analyzed by qRT-PCR, Western blot and EnVision immunohistochemistry. Cell proliferation and invasion was detected by CCK8 and Transwell assay. Results Lueiferase assays revealed miR- 205 directly targeted KLF12 （ P = O. 001 6 ）. MiR-205 expression levels were exclusively lower in MDA-MB-468 cell lines （ P = 0. 007 ） , but KLF12 were highly expressed level in MDA-MB-468 cells （P = 0. 039 ）. Over-expression of miR-205 by transfection with its mimics in MDA-MB-468 cells substantially increased miR-205 expression level （P = 0. 000 ） but reduced KLFI2 expression level （P = 0.038 ）. Western blot revealed that over-expression of miR-205 expression by transfection with its mimics in MDA-MB-468 cells substantially reduced KLF12 expression level （P = O. 007 9）. Modulation of miR-205 expression by transfection with its mimics or in- hibitor was not involved in MDA-MB-468 cell proliferation, but involved in invasion （ P = 0. 001 ）. Conclusion miR-205 may be a specific miRNA in BLBC that exerts tumor suppression role through negatively regulating proto-oncogene KLF12 and inhibiting cell invasion. MiR-205 and KLF12 may provide potentially diagnosis molecular biomarker and possible new approach for the treatment for BLBC.