【摘要】 目的:探讨微小非编码RNA-516b-5p(miR-516b-5p)靶向RNA甲基转移酶家族成员2(NSUN2)对乳腺癌(BC)细胞增殖和侵袭的影响。方法:qRT-PCR实验检测BC癌组织、癌旁组织(n=45)及正常乳腺上皮细胞(MCF-10A)、BC细胞(MCF-7、MDA-MB-231、HCC1937)中miR-516b-5p、NSUN2 mRNA表达量。将MCF-7细胞分为NC组、miR-NC组、miR-516b-5p mimic组、sh-NC组、sh-NSUN2组、miR-516b-5p mimic+pcDNA-NC组、miR-516b-5p mimic+pcDNA-NSUN2组，通过克隆形成和Transwell侵袭实验分别检测各组MCF-7细胞增殖和侵袭能力；Western blot实验检测各组细胞中NSUN2、细胞周期蛋白1(CCND1)、细胞周期蛋白依赖性激酶1(CDK1)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)蛋白表达量；构建裸鼠BC移植瘤模型，并分为Model组、miR-516b-5p mimics组、miR-516b-5p mimic+pcDNA-NSUN2组，测量各组肿瘤质量和体积。结果:与癌旁组织相比，癌组织中miR-516b-5p表达量明显下降，NSUN2 mRNA表达量明显上升(P<0.05)。与MCF-10A细胞相比，MCF-7、MDA-MB-231、HCC1937细胞中miR-516b-5p表达量明显下降，NSUN2 mRNA表达量明显上升(P<0.05),其中MCF-7细胞以上指标变化趋势最为明显。与NC组相比，miR-NC组和sh-NC组细胞各指标无明显变化(P>0.05);与miR-NC组相比，miR-516b-5p mimic组细胞集落形成数量、侵袭细胞数减少，NSUN2 mRNA和NSUN2、CCND1、CDK1、MMP2、MMP9蛋白表达量均明显下降，miR-516b-5p表达量明显上升(P<0.05);与sh-NC组相比，sh-NSUN2组各指标变化趋势与miR-516b-5p mimic组一致(P<0.05);而在miR-516b-5p mimic+pcDNA-NSUN2组中各指标变化趋势与miR-516b-5p mimic组相反(P<0.05)。双荧光素酶实验证实miR-516b-5p与NSUN2存在靶向关系(P<0.05)。裸鼠成瘤实验中，与Model组相比，miR-516b-5p mimics组肿瘤组织质量和体积明显缩小(P<0.05);与miR-516b-5p mimics组相比，miR-516b-5p mimic+pcDNA-NSUN2组肿瘤组织质量和体积明显增大(P<0.05)。结论:miR-516b-5p可通过靶向下调NSUN2表达，抑制BC细胞增殖和侵袭。更多还原

【Abstract】 Objective: To discuss the impacts of micro non-coding RNA-516b-5p(miR-516b-5p) on the proliferation and invasion of breast cancer(BC) cells by targeting NOP2/Sun RNA methyltransferase 2(NSUN2). Methods: qRT-PCR experiments were performed to detect the miR-516b-5p and NSUN2 mRNA in BC cancer tissues, adjacent tissues, normal breast epithelial cells(MCF-10A), and BC cells(MCF-7, MDA-MB-231, HCC1937). MCF-7 cells were assigned into NC group, miR-NC group, miR-516b-5p mimic group, sh-NC group, sh-NSUN2 group, miR-516b-5p mimic+pcDNA-NC group, and miR-516b-5p mimic+pcDNA-NSUN2 group. Clone formation and Transwell invasion experiments were performed to detect the proliferation and invasion abilities of MCF-7 cells in each group. Western blot was performed to detect the NSUN2, cyclin 1(CCND1), cyclin dependent kinase 1(CDK1), matrix metalloproteinase 2(MMP2), and matrix metalloproteinase 9(MMP9) proteins. The nude mouse BC transplant tumor model was constructed and separated into Model group, miR-516b-5p mimic group, and miR-516b-5p mimic+pcDNA-NSUN2 group. The tumor mass and volume were measured in each group. Results: For adjacent tissues, the miR-516b-5p in cancer tissues was unusually declined, while the NSUN2 mRNA was clearly increased(P<0.05). Compared with MCF-10A cells, the miR-516b-5p in MCF-7, MDA-MB-231, and HCC1937 cells declined clearly, while the NSUN2 mRNA raised unusually(P<0.05), with the most unusual trend observed in MCF-7 cells. For the NC group, there were no unusual changes in various cellular indicators in the miR-NC group and sh-NC group(P>0.05). For the miR-NC group, the miR-516b-5p mimic group showed declined the number of colony forming cells and invasive cells, unusually decreased NSUN2 mRNA, and NSUN2, CCND1, CDK1, MMP2, and MMP9 proteins, and clearly raised miR-516b-5p(P<0.05). For the sh-NC group, the changes in various indicators in the sh-NSUN2 group were consistent with those in the miR-516b-5p mimic group(P<0.05). The trend of changes in various indicators in the miR-516b-5p mimic+pcDNA-NSUN2 group was opposite to that in the miR-516b-5p mimic group(P<0.05). The dual luciferase assay confirmed a targeted relationship between miR-516b-5p and NSUN2(P<0.05). In the nude mouse tumorigenesis experiment, the miR-516b-5p mimics group showed a clear reduction in tumor tissue mass and volume than the Model group(P<0.05). For the miR-516b-5p mimic group, the miR-516b-5p mimic+pcDNA-NSUN2 group showed a prominent increase in tumor tissue mass and volume(P<0.05). Conclusion: MiR-516b-5p can inhibit BC cell proliferation and invasion by targeting and downregulating NSUN2.更多还原