circSLCO1B3在三阴乳腺癌脑转移中的作用及其调控机制

Role of circSLCO1B3 in brain metastasis of triple negative breast cancer and its regulatory mechanism

目的探讨circSLCO1B3在三阴乳腺癌(TNBC)脑转移中的作用及其可能的调控机制。方法采用qRT-PCR实验检测circSLCO1B3在乳腺癌细胞系中的表达。采用过表达质粒上调TNBC子代脑转移细胞231Br中circSLCO1B3的表达,设为circSLCOB3-OE组(转染对照质粒的为circCTR-OE组),采用Transwell侵袭实验检测circSLCO1B3对231Br细胞侵袭能力的影响。经小鼠左心室注射231Br细胞,构建体内脑转移模型,检测circSLCO1B3对TNBC脑转移能力的影响。通过CircInteractome及TargetScan在线软件分别预测与circSLCO1B3结合的miRNA及miRNA下游靶基因,并使用双荧光素酶报告基因和qRT-PCR、Western blotting实验验证其调控关系。结果相对正常乳腺上皮细胞,circSLCO1B3在TNBC细胞系中均显著下调,且在TNBC子代脑转移细胞231Br中下调最为显著(P<0.05)。与circCTR-OE组比较,circSLCOB3-OE组231Br细胞的侵袭能力、脑转移结节数目均显著降低(P均<0.05)。与细胞核比较,circSLCO1B3在细胞质中的相对含量高(P<0.05)。与野生型circSLCO1B3+miR-NC共转染组相比,野生型circSLCO1B3+miR-155组的相对荧光酶活性显著降低(P<0.05);与circCTR-OE组相比,circSLCO1B3-OE组miR-155的相对表达量显著降低(P<0.05)。与野生型PTEN+miR-NC共转染组相比,野生型PTEN+miR-155组的相对荧光酶活性显著降低(P<0.05)。与转染miR-NC相比,转染miR-155细胞PTEN mRNA的相对表达量显著降低(P<0.05)。与circCTR-OE组比较,circSLCOB3-OE组PTEN mRNA的相对表达量及PTEN蛋白表达显著增加,而与circSLCOB3-OE组比较,circSLCOB3-OE+miR-155组PTEN的表达则显著降低(P均<0.05)。结论circSLCO1B3通过吸附miR-155,上调PTEN蛋白的表达,进而抑制TNBC脑转移。

Objective To explore the role and possible regulatory mechanism of circSLCO1B3 in triple negative breast cancer(TNBC)brain metastasis.Methods The expression of circSLCO1B3 in breast cancer cell line was detect⁃ed by qRT-PCR.The overexpression plasmid was used to upregulate the expression of circSLCO1B3 in TNBC brain metas⁃tasis cell line 231Br,circSLCOB3-OE group(circCTR-OE group transfected with control plasmid),transwell invasion ex⁃periment was conducted to detect the effect of circSLCO1B3 on the invasion ability of 231Br cells.In vivo brain metastasis model was constructed by injecting 231Br cells into the left ventricle of mice,and detected the effects of circSLCO1B3 on TNBC brain metastasis ability;Extract cytoplasmic and nuclear RNA from 231Br cells for subcellular localization analysis of circSLCO1B3.The miRNA binding to circSLCO1B3 and downstream target genes of selected miRNA were predicted by CircInteractome and TargetScan online software,and their regulatory relationships were verified using dual luciferase re⁃porter assar,qRT PCR,and Western blotting experiments.Results circSLCO1B3 was significantly downregulated in TNBC cell lines compared to normal breast epithelial cells,and the downregulation was most significant in TNBC brain metastatic cells 231Br(P<0.05).Compared with the circCTR-OE group,the invasive ability and the number of brain met⁃astatic nodules of 231Br cells from circSLCOB3-OE group were significantly reduced(P<0.05).The relative content of circSLCO1B3 in the cytoplasm was higher than that in nucleus(P<0.05).Compared with the circCTR-OE group,when wild-type circSLCOB3 co-transfected with miR-155,luciferase activity was significantly reduced,and the expression level of miR-155 in the circSLCOB3-OE group was significantly reduced(P<0.05).Compared with the circCTR-OE group,wild-type PTEN mRNA co-transfected with miR-155 significantly reduced luciferase activity,while the relative expression of PTEN mRNA in the circSLCOB3-OE group significantly increased(P<0.05).Compared with the miR-NC transfection group,the relative expression of PTEN mRNA in the miR-155 transfection group was significantly reduced(P<0.05).Compared with the circCTR-OE group,the relative expression and protein expression of PTEN in the circSLCOB3-OE group significantly increased,and compared with the circSLCOB3-OE group,the expressions of PTEN in the circSLCOB3-OE+miR-155 group were significantly decreased(P<0.05).Conclusion circSLCO1B3 up-regulates the expression of PTEN protein by adsorbing miR-155,thereby inhibiting TNBC brain metastasis.