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| hsa-miR-655靶向CLCF1基因对人成骨肉瘤MG63 细胞OPG/RANKL系统表达的影响 |
| Effect of hsa-miR-655-targeted CLCF1 gene on OPG/RANK by MG63 cells |
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| DOI：10.3969/j.issn.1006.7108.2017.11.003 |
| 中文关键词: hsa-miR455 心肌营养素样细胞因子1 MG63细胞 OPG/RANKL 基因表达 |
| 英文关键词:hsa-miR-655 CLCF1 MG63 cell line OPG/RANKL Gene expression |
| 基金项目:国家自然科学基金（81403420);福建省公益类科研院所基本科研专项项目（2015R1035-13) |
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| 中文摘要: |
| 目的 探讨hsa-miR-655通过靶向调控心肌营养素样细胞因子1 ( cardiotrophin-like cytokine factor 1，CLCF1)基因表达，对人成骨肉MG63细胞系增殖、护骨素（osteoprotegerin，OPG)和核因子κB受体活化因子配体（receptor activator of NF-κB ligand，RANKL)表达的影响。方法通过hsa-miR-655慢病毒转染MG63细胞进行功能获得实验。实验分阴性对照组（NC组）和hsa-miR-655过表达组（OE组），荧光显微镜和流式细胞术（flow cytometry analysis，FCM)评估转染效率，Cell Counting Kit-8(CCK-8)法检测细胞增殖能力，FCM检测细胞周期分布，实时荧光定量PCR(qRT-PCR)检测hsa-miR-655、CLCF1、OPG和 RANKLmRNA的水平，蛋白质印迹法（Western blot)检测CLCF1、OPG及RANKL蛋白的变化。结果 荧光显微镜和FCM显示对照组和miR-655过表达组感染效率为80%以上;qRT-PCR结果显示，相对于对照组，过表达组MG63细胞hsa-miR-655表达上调，差异有统计学意义（P = 0. 0004) ； CLCF1 mRNA水平未见明显变化（P = 0. 0986)；过表达组OPG( P = 0. 0384)、RANKL( P = 0.0007) mRNA均有所上调，差异具有统计学意义（P <0. 05)；Western blot结果显示，过表达组CLCF1蛋白水平表达下调 (P = 0. 0053);与对照组比较，过表达组OPG(P =0. 0021)、RANKL(P =0. 0002)蛋白均上调，而OPG/RANKL比值却降低（P = 0. 0078)，差异具有统计学意义(P <0. 05) ;CCK-8细胞增殖实验结果显示，miR-655过表达后抑制MG63细胞增殖;FCM检测细胞周期结果显示，miR-655过表达后MG63细胞G0/G1期细胞比例增加，S期减少，差异具有统计学意义（P <0. 05)。结论 hsa-miR-655通过负调控CLCF1基因的表达，抑制MG63细胞的增殖，下调OPG/RANKL的比值。 |
| 英文摘要: |
| Objective To investigate the regulation of hsa-miR-655 by targeting CLCF1 gene on OPG/RANKL expression in MG63 cells. Methods Lentivirus mediated hsa-miR-655 overexpression stable cell line MG63 was constructed. Cells were divided into control group (NC) and hsa-miR-655 overexpression group (OE). Fluorescence microscopy and FCM were used to evaluated infection efficiency. CCK-8 assay was used to evaluate the cell proliferation. The cell cycle was detected by flow cytometry. Real-time PCR was used to detect the mRNA expressions of miR-655，CLCF1，OPG，and RANKL after lentivirus transfection. Western blotting was used to detect the protein expressions of CLCF1，OPG，and RANKL. Results Fluorescence microscopy and FCM showed that the infection efficiency in both empty vector group and miR-655 overexpression group was over 80%. Compared to that in NC group，the expression of hsa-miR655 in OE cells was up-regulated (P =0. 0004). CLCF1 mRNA level did not change significantly (P =0. 0986). OPG (P =0. 0384) and RANKL (P =0. 0007) increased in OE group，and the difference was statistically significant. Western blotting result showed that the expression of CLCF1 protein in OE group was down- regulated (P =0. 0053)，but OPG (P =0. 002) and RANKL (P =0. 0002) were up-regulated and OPG/RANKL ratio decreased (P =0. 003) compared with those in NC group. The result of CCK-8 cell proliferation test showed that miR-655 overexpression inhibited the proliferation of MG63 cells. FCM result showed that the overexpression of miR-655 increased proportion of cells in G0/G1 phase, and decreased cells in S phase, and the difference was statistically significant (P <0.05). Conclusion hsa-miR-655 inhibits proliferation of MG63 cells and down-regulates OPG/RANKL ratio through negative regulation of CLCF1 gene expression. |
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