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| hsa-miR-655靶向调控绝经后骨质疏松症肾阴虚证关联基因CLCF1的实验研究 |
| Experimental study on the hsa-miR-655 targeted regulation of the CLCF1 gene in postmenopausal osteoporosis associated with Kidney Yin deficiency |
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| DOI：10.3969/j.issn.1006.7108.2016.08.001 |
| 中文关键词: 中西医结合；肾阴虚证 绝经后骨质疏松症；CLCH基因；微小RNA；3'非编码区；荧光素酶报告系统 |
| 英文关键词:Integrative medicine Kidney Yin deficiency Postmenopausal osteoporosis CLCF1 gene microRNAs 3’ Non encoding area Dualluciferase assay |
| 基金项目:国家自然科学基金资助项目（81403420)；福建省卫生厅青年科研课题(2013-2-73) |
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| 中文摘要: |
| 目的 研究靶向调控绝经后骨质疏松症肾阴虚证关联基因CLCF1的microRNAs。方法 通过生物信息学预测可能与CLCF1基因3'非编码区（3'Non encoding area,3'UTR)作用的miRNAs；将人工合成的野生型和突变型CLCF1基因3'UTR区序列克隆至荧光素酶报告质粒；将重组荧光素酶报告载体和miRNAs表达载体共转染293T细胞，用双荧光素酶检测试剂盒测定荧光素酶活性，验证miRNA对CLCF1基因翻译水平的抑制作用。结果 生物信息学预测结果显示，CLCF1基因3'UTR与 hsa-miR-655、hsa-miR-300、hsa-miR-381、hsa-miR-374c、hsa-miR-515 等 5 个 miRNAs 存在互补结合位点；双荧光素酶实验结果表明，与对照组比较，hsa-miR-655组荧光活性下调至33.09%，差异有显著性意义（P = 0. 001)，当使用突变型CLCH基因3' UTR,荧光活性上调至67. 22%，说明hsa-miR-655对CLCF1 3'UTR的基因表达水平有显著抑制作用；与对照组比较，hsa-miR- 374c和hsa-miR-515组荧光活性分别下调27. 27% (P = 0. 000)和27. 74% (P = 0. 006)，二者对CLCF1 3'UTR的基因表达水平有一定抑制作用；hsa-miR-300和hsa-miR-381组与对照组相比，荧光活性分别下调6. 35% (P = 0. 213)和9. 89% (P = 0. 127), 二者对CLCH 3’UTR的基因表达无明显抑制作用。结论hsa-miR-655通过靶向结合CLCF1 mRNA的3'UTR区，在转录后水平负性调控绝经后骨质疏松症肾阴虚证关联基因CLCF1的表达。 |
| 英文摘要: |
| Objective To investigate the microRNA regulation on the gene expression of CLCF1 in postmenopausal osteoporosis associated with Kidney Yin deficiency. Methods Bioinformatics were used to predict the possible miRNAs that target bind to CLCF1 gene 3’ non coding region (3'UTR). Artificial synthesized wild type and mutation type of CLCF1 gene 3'UTR region sequences were cloned into the luciferase reporter plasmid； recombinant luciferase reporter vector and miRNAs expression vectors were transfected into 293 T cells and dualluciferase assay kit was used for the determination of luciferase activity, and to verify the miRNA inhibition of candidate target genes at the level of translation. Results Bioinformatics prediction results showed that the CLCF1 gene 3’UTR and the hsa-miR-655, hsa-miR-515, hsa-miR-300, hsa-miR-381, hsa-miR-374c et al. miRNAs binding sites are complementary. Compared with the control group, the fluorescence activity of the hsa-miR-655 group reduced to 33. 09%， which reached statistical significance (P = 0. 001). When using the mutant CLCF1 gene 3’UTR，fluorescence activity increased to 67. 22% , indicating that hsa-miR-655 has a significant inhibitory effect on the CLCF1 3'UTR gene. Compared with the control group, the fluorescence activity of the hsa-miR-374c and hsa-miR-515 groups reduced by 27. 27% (P = 0. 000) and 27. 74% (P = 0.006), respectively, confirming that these two microRNAs have inhibitory effect on the expression of CLCF1 gene. The fluorescence activity of hsa-miR-300 and hsa-miR-381 groups decreased by 6. 35% (P=0.213) and 9. 89% (P=0. 127)，respectively, compared with the control group, both had no obvious inhibitory effect on the gene expression of CLCF1 3fUTR. Conclusion hsa-miR-655 can target bind to the 3'UTR of CLCFlmRNA, and negatively regulates the gene expression of CLCF1 in postmenopausal osteoporosis associated with Kidney Yin deficiency at the post transcriptional level. |
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