miR-212-3p Targeted Regulation of NAP1L1 Inhibits Glioma Cell Proliferation，Migration and EMT
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摘要:
目的 探究miR-212-3p在胶质瘤细胞增殖、侵袭和上皮-间充质转化（EMT）中的分子机制。 方法 RT-qPCR用于衡量胶质瘤细胞中miR-212-3p和NAP1L1表达，构建NC mimic、miR-212-3p mimic、oe-NC和oe-NAP1L1转染至细胞中，利用CCK-8 、Transwell和伤口实验评估细胞生物学行为。通过Western blot分析EMT相关标志物的蛋白表达。MiR-212-3p与NAP1L1的关系通过双荧光素酶报告基因和AgO2-RIP实验进行证实。 结果 miR-212-3p的表达水平在胶质瘤细胞中显著下调（P < 0.0001 ），过表达miR-212-3p 可以显著降低胶质瘤细胞增殖（P <0.0001 ）、侵袭（P =0.0011 ）和迁移能力（P <0.0001 ），并且抑制了EMT标志物N-钙黏蛋白（N-cadherin）（P =0.000861 ）和波形蛋白（Vimentin）（P =0.007430 ）的表达，而上调了E-钙黏蛋白（E-cadherin）（P <0.0001 ）的表达。MiR-212-3p靶向负调控NAP1L1的表达。过表达NAP1L1逆转了miR-212-3p对胶质瘤细胞增殖（P <0.0001 ）、迁移（P <0.0001 ）和EMT（P <0.0001 ）的抑制作用。结论 MiR-212-3p通过靶向负调节NAP1L1的表达抑制胶质瘤细胞增殖、迁移和EMT。 -
关键词:
- 胶质瘤 /
- miR-212-3p /
- NAP1L1 /
- 上皮-间充质转化 /
- 细胞增殖
Abstract:Objective To explore the molecular mechanism of miR-212-3p in glioma cell proliferation, invasion and epithelial-mesenchymal transition （EMT）. Methods The expression of miR-212-3p and NAP1L1 were detected by RT-qPCR in glioma cells. NC mimic, miR-212-3p mimic, oe-NC and oe-NAP1L1 were built and transfected in cells. CCK-8, Transwell and Wound healing assay were used to evaluate the cell biological behaviour. Western blot was used to detect the expression of EMT-related biomarkers. The relationship between miR-212-3p and NAP1L1 was confirmed by the dual-luciferase reporter gene and AgO2-RIP assay. Results MiR-212-3p was lowly expressed in glioma cells （P < 0.0001 ）. miR-212-3p mimic significantly inhibited the glioma cell proliferation （P <0.0001 ）, invasion （P =0.0011 ） and migration （P <0.0001 ）, and reduced the expression of EMT-related biomarkers N-cadherin （P =0.000861 ） and Vimentin （P =0.007430 ）, while upregulating the expression of E-cadherin （P <0.0001 ）. MiR-212-3p targeted and negatively regulated the NAP1L1 expression. Overexpression of NAP1L1 reversed the inhibitory effects of miR-212-3p on glioma cell proliferation （P <0.0001 ）, migration （P <0.0001 ）, and EMT （P <0.0001 ）.Conclusions MiR-212-3p inhibits the glioma cell proliferation, migration and EMT by targeting the negative regulation of NAP1L1 expression. -
Key words:
- Glioma /
- MiR-212-3p /
- NAP1L1 /
- Epithelial-mesenchymal transition /
- Cell proliferation
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图 1 miR-212-3p在胶质瘤细胞中低表达
与NHA组比较，***P < 0.001。
Figure 1. miR-212-3p is lowly expressed in glioma cells
图 2 miR-212-3p抑制胶质瘤细胞增殖、迁移和EMT
A：RT- qPCR检测miR-212-3p转染效率；B：CCK-8分析；C：Transwell评估细胞侵袭；D：伤口愈合实验评估细胞迁移；E：Western blot分析EMT相关标志物的表达（N-cadherin、E-cadherin和Vimentin）的蛋白水平。与NC组比较，**P < 0.01，***P < 0.001。
Figure 2. miR-212-3p inhibits glioma cell proliferation，migration and EMT
图 3 miR-212-3p靶向调控NAP1L1的表达
A：miR-212-3p与NAP1L1的结合序列；B：双荧光素酶基因报告验证miR-212-3p与NAP1L1的靶向关系；C：AgO2-RIP检测miR-212-3p与NAP1L1的关系；D：生存率预测，high-risk样本33例，low-risk样本41例，P-value=0.023500；E：RT-qPCR检测细胞中NAP1L1的表达；与NC-mimic组比较，***P < 0.001。
Figure 3. miR-212-3p targets and regulates NAP1L1 expression
图 4 miR-212-3p通过下调NAP1L1抑制胶质瘤细胞增殖、迁移和EMT
A：RT- qPCR检测NAP1L1转染效率；B：RT- qPCR检测NAP1L1的表达；C：CCK-8分析；D：Transwell评估细胞侵袭；E：伤口愈合实验评估细胞迁移；F：Western blot分析N-cadherin、E-cadherin和Vimentin的蛋白水平。与NC组比较，**P < 0.01，***P < 0.001；与miR-212-3p mimic组比较，##P < 0.05，##P < 0.01，###P < 0.001。
Figure 4. miR-212-3p inhibits glioma cell proliferation，migration and EMT through down-regulation of NAP1L1
表 1 PCR引物序列
Table 1. PCR primer sequences
基因 引物 序列（5’-3’） miR-212-3p forward 5’-GCGCGTAACAGTCTCCAGTC -3’ reverse 5’-AGTGCAGGGTCCGAGGTATT -3’ U6 forward 5’-CAAATTCGTGAAGCGTTCCA-3’ reverse 5’-AGTGCAGGGTCCGAGGTATT-3’ NAP1L1 forward 5’-GGCAGACATTGACAACAA-3’ reverse 5’-TTCCTCCGAAATCTCATC-3’ GAPDH forward 5’-TGACCACAGTCCATGCCATCAC-3’ reverse 5’-CGCCTGCTTCACCACCTTCTT-3’ 
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