miR-423-3p通过下调CARNS1促进乳腺癌细胞恶性生物学行为

miR-423-3p Promotes Malignant Biological Behavior in Breast Cancer Cells by Downregulating CARNS1

摘要: 目的：探讨miR-423-3p对乳腺癌细胞恶性生物学行为的影响及作用机制。方法：体外培养人乳腺癌细胞MDA-MB-231、MDA-MB-468、MCF-7、BT-549及正常乳腺上皮细胞MCF-10A，采用实时荧光定量PCR (RT-qPCR)法和Western Blot方法，分别检测miR-423-3p及CARNS1 mRNA和蛋白相对表达量。选取MCF-7细胞，将细胞分为转染mimics NC组(A组)、转染miR-423-3p mimics组(B组)、转染inhibitors NC组(C组)、转染inhibitors组(D组)。在239T细胞中分别转染并分为CARNS1-3'-UTR-wt+miR-423-3p mimics (E组)、CARNS1-3'-UTR-wt-mimics NC (F组)、CARNS1-3'-UTR-mut+miR-423-3p mimics (G组)及CARNS1-3'-UTR-mut+mimics NC (H组)。采用RT-qPCR和Western Blot法，检测A~D组细胞miR-423-3p及CARNS1 mRNA和蛋白表达情况，采用CCK8法、细胞划痕实验、Transwell法和流式细胞术检测细胞增殖、迁移、侵袭和凋亡情况。在线数据库预测miR-423-3p与CARNS1基因3'非翻译区(3'-UTR)的互补结合位点，双荧光素酶报告基因实验验证预测结果。结果：RT-qPCR结果显示，MCF-7、MDA-MB-231、MDA-MB-468及BT-549细胞中miR-423-3p的相对表达量均明显高于正常乳腺上皮细胞MCF-10A (P < 0.05)，RT-qPCR和Western Blot检测结果显示，CARNS1 mRNA以及蛋白的相对表达量均明显低于正常乳腺上皮细胞MCF-10A (P < 0.05)。CCK8实验结果显示，在细胞培养的第24、48、72小时，B组细胞增殖活力明显高于A组，D组明显低于C组。细胞划痕实验显示，B组细胞愈合率明显高于A组(P < 0.05)。D组细胞愈合率明显低于C组(P < 0.05)。Transwell实验结果显示，过表达miR-423-3p后可促进乳腺癌细胞侵袭，而抑制miR-423-3p后会抑制乳腺癌细胞的侵袭，差异有统计学意义(P < 0.05)。流式细胞实验结果显示，B组较A组凋亡能力明显降低(P < 0.05)，D组较C组凋亡能力明显增强(P < 0.05)。starBase数据库预测miR-423-3p与CARNS1基因3'非翻译区(3'-UTR)存在互补结合位点，双荧光素酶报告基因实验结果显示，E组较F组细胞相对荧光素酶显著下调(P < 0.05)，G组与F组细胞间相对荧光素酶活性差异无统计学意义(P > 0.05)。RT-qPCR检测结果显示，B组细胞的miR-423-3p相对表达量明显高于A组(P < 0.05)，而CARNS1 mRNA相对表达量明显低于A组(P < 0.05)，D组细胞的miR-423-3p相对表达量明显低于C组(P < 0.05)，而CARNS1 mRNA相对表达量明显高于C组(P < 0.05)。结论：miR-423-3p可通过靶向调控CARNS1促进乳腺癌细胞的恶性生物学行为。

Abstract: Objective: To investigate the effect and mechanism of miR-423-3p on the malignant biological be-havior of breast cancer cells. Methods: Human breast cancer cells MDA-MB-231, MDA-MB-468, MCF-7, BT-549 and normal breast epithelial cells MCF-10A were cultured in vitro, and the relative mRNA and protein expressions of miR-423-3p and CARNS1 were detected by real-time quantitative PCR (RT-qPCR) and Western Blot, respectively. MCF-7 cells were divided into mimics NC transfection group (group A), miR-423-3p mimics group (group B), transfection inhibitors NC group (group C), and transfection inhibitors group (group D). 239T cells were transfected and divided into CARNS1-3'-UTR-wt miR-423-3p mimics (group E), CARNS1-3'-UTR-wt-mimics NC (group F), CARNS1-3'-UTR-mut miR-423-3p mimics (group G) and CARNS1-3'-UTR-mut mimics NC (group H). The mRNA and protein expressions of miR-423-3p and CARNS1 in group A~D cells were detected by RT-qPCR and Western Blot, and the proliferation, migration, invasion and apoptosis of cells were detected by CCK8 method, cell scratch assay, Transwell method and flow cytometry. The comple-mentary binding sites of miR-423-3p and the 3 untranslated region (3'-UTR) of CARNS1 gene were predicted by an online database, and the prediction results were verified by double luciferase re-porter gene assays. Results: RT-qPCR results showed that the relative expression levels of miR-423-3p in MCF-7, MDA-MB-231, MDA-MB-468 and BT-549 cells were significantly higher than those in normal mammary epithelial cells MCF-10A (P < 0.05), and the relative expression levels of mRNA and protein in CARNS1 were significantly lower than those in normal mammary epithelial cells (P < 0.05). The results of CCK8 experiments showed that at the 24th, 48th and 72nd hours of cell culture, the cell proliferation activity of group B was significantly higher than that of group A, and that of group D was significantly lower than that of group C. The cell scratch test showed that the cell healing rate of group B was significantly higher than that of group A (P < 0.05). The cell healing rate of group D was significantly lower than that of group C (P < 0.05). The results of Transwell assay showed that overexpression of miR-423-3p could promote the invasion of breast cancer cells, while inhibition of miR-423-3p would inhibit the invasion of breast cancer cells, and the difference was statistically significant (P < 0.05). The results of flow cytometry showed that the apoptosis ability of group B was significantly lower than that of group A (P < 0.05), and the apopto-sis ability of group D was significantly higher than that of group C (P < 0.05). The starBase database predicted that miR-423-3p had complementary binding sites in the 3' untranslated region (3'-UTR) of CARNS1 gene, and the results of double luciferase reporter gene assay showed that the relative luciferase of group E was significantly down-regulated compared with group F (P < 0.05), and there was no significant difference in relative luciferase activity between group G and group F (P > 0.05). The results of RT-qPCR showed that the relative expression of miR-423-3p in group B was signifi-cantly higher than that in group A (P < 0.05), while the relative expression of mRNA in CARNS1 was significantly lower than that in group A (P < 0.05), the relative expression of miR-423-3p in group D was significantly lower than that in group C (P < 0.05), and the relative expression of mRNA in CARNS1 was significantly higher than that in group C (P < 0.05). Conclusion: miR-423-3p can pro-mote the malignant biological behavior of breast cancer cells by targeting CARNS1.