Construction of RNAi Targeting CLU Gene and Effect of CLU on Biological Function of Nasopharyngeal Cancer Cells
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摘要:目的
构建CLU基因干扰重组质粒及建立CLU基因干扰转染鼻咽癌细胞及动物模型，检测CLU对鼻咽癌细胞生物学功能的影响。
方法首先以real-time PCR和Western blot法验证CLU在鼻咽癌细胞系5-8F，6-10B中的表达，以高表达CLU的5-8F细胞作为干扰转染的目标细胞；设计及构建shRNA表达载体pSR-GFP/Neo-CLU-shRNA，将其转染至5-8F鼻咽癌细胞系中，通过Transwell小室迁移及侵袭实验来检测对其迁移和侵袭功能的影响。
结果与6-10B细胞比较，CLU基因在5-8F中的表达显著上调（P<0.05）；干扰鼻咽癌细胞5-8F中的CLU可使细胞迁移和侵袭功能明显减弱（P<0.05），使其体外成瘤能力显著降低（P<0.05）。
结论CLU基因在鼻咽癌细胞系5-8F中明显高表达；pSR-GFP/Neo-CLUshRNA可成功干扰5-8F中CLU的表达；CLU可抑制5-8F细胞的迁移、侵袭及体内成瘤能力。
Abstract:ObjectiveTo construct the RNAi targeting Clusterin (CLU) gene, to explore the effect of interference targeting CLU on the biological behavior of nasopharyngeal carcinoma (NPC) cells.
MethodsWe detected the expression of CLU in NPC cell lines 6-10B and 5-8F through the real-time PCR and Western blot; then we constructed pSR-GFP/Neo-CLU-shRNA expression vector targeting CLU. Based on interference targeting CLU on NPC, we tested the cell migration and motility by Transwell migration and motility assay.
ResultsThe expression of CLU was increased in NPC cell line 5-8F compared with NPC cell line 6-10B (P<0.05) . When the gene CLU of 5-8F was interfered, the cell migration, invasion and tumorigenicity were weakened.
ConclusionThe expression of CLU is up-regulated in NPC cell line 5-8F. pSR-GFP/Neo- CLU-shRNA could successfully inhibit the expression of CLU in NPC cell line 5-8F. CLU could inhibit the migration, invasion and tumorigenicity of 5-8F cells in vivo.
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Key words:
- Nasopharyngeal cancer(NPC) /
- CLU /
- Neoplasm metastasis
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图 1 QRT-PCR法分析CLU-mRNA在6-10B和5-8F细胞系中的表达
Figure 1 Expression of CLU-mRNA in NPC cell lines 6-10B and 5-8F determined by QRT-PCR
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图 2 Western blot法检测不同鼻咽癌细胞系中CLU蛋白表达水平
Figure 2 Protein expression of CLU in NPC cell lines detected by Western blot
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图 3 Western blot法检测干扰转染后CLU蛋白表达
Figure 3 Protein expression of CLU detected by Western blot after interference
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图 4 CLU-shRNA对5-8F细胞侵袭及迁移能力的影响
Figure 4 Effect of CLU-shRNA on invasion and migration of 5-8F cells
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