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目的探讨microRNA-143(miR-143)阻遏乳腺癌细胞免疫抑制的作用及其分子机制。方法采用MTT法检测乳腺细胞HBL-100和乳腺癌细胞MCF-7、MDA-MB-231、BT549补体依赖的细胞毒性(complement-dependent cytotoxicity,CDC);在脂质体介导下,将miR-143、miR-143对照物(scramble)和miR-143抑制剂(inhibitor)分别转染MCF-7细胞,将miR-143 scramble和miR-143 inhibitor分别转染HBL-100细胞,采用MTT法检测CDC,RT-PCR法检测细胞中补体调节蛋白CD46基因mRNA的转录水平,Western blot法检测CD46蛋白的表达水平。结果乳腺癌细胞MCF-7、MDA-MB-231、BT-549的A570值与正常乳腺细胞HBL-100相比,均明显升高(P<0.01);与miR-143 scramble转染的MCF-7细胞的A570值相比,miR-143转染组的A570值明显下降(P<0.001),与miR-143 scramble转染的HBL-100细胞的A570值相比,miR-143 inhibitor转染的HBL-100细胞的A570值明显上升(P<0.001);与HBL-100细胞相比,CD46蛋白在MCF-7、MDA-MB-231和BT-549细胞中表达均上调,且与抑制CDC的作用呈正相关;在HBL-100细胞中过表达miR-143 inhibitor能上调CD46蛋白的表达水平,而不影响CD46基因mRNA的转录水平;在MCF-7细胞中过表达miR-143能明显抑制CD46蛋白的表达水平。结论 CD46在乳腺癌细胞中的上调导致了癌细胞对CDC的抵抗作用,是产生免疫抑制的机制之一;miR-143通过抑制CD46蛋白的表达水平,使细胞对CDC的作用更为敏感,提高了补体系统对乳腺癌细胞的杀伤作用,降低了其免疫抑制能力。
Objective To investigate the role of microRNA-143 (miR-143) in suppressing the immunosuppression of breast cancer cells and its molecular mechanism. Methods The complement-dependent cytotoxicity (CDC) of breast cancer cells HBL-100 and breast cancer cells MCF-7, MDA-MB-231 and BT549 were detected by MTT assay. 143, miR-143 scramble and miR-143 inhibitor were transfected into MCF-7 cells, and miR-143 scramble and miR-143 inhibitor were transfected into HBL-100 cells respectively. MTT was used to detect CDC The transcription level of CD46 mRNA was detected by RT-PCR and the level of CD46 protein was detected by Western blot. Results The A570 values of breast cancer cells MCF-7, MDA-MB-231 and BT-549 were significantly higher than those of normal breast cells (P <0.01) Compared with the A570 value of miR-143 scramble-transfected HBL-100 cells, the A570 value of miR-143 transfection group was significantly decreased (P <0.001) as compared with the A570 value of miR-143 transfected Compared with HBL-100 cells, the expression of CD46 protein was up-regulated in MCF-7, MDA-MB-231 and BT-549 cells, 143 inhibitor could up-regulate the expression of CD46 protein in HBL-100 cells without affecting the transcription level of CD46 mRNA. Overexpression of miR-143 in MCF-7 cells significantly inhibited the expression of CD46 protein The level of expression. Conclusion Upregulation of CD46 in breast cancer cells leads to the resistance of cancer cells to CDC and is one of the mechanisms of immunosuppression. MiR-143 can make cells more sensitive to CDC by inhibiting the expression of CD46 protein The complement system on the killing of breast cancer cells, reducing its immunosuppressive capacity.