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| miR-148-5p靶向H型血管调控因子Slit3的实验研究 |
| Study of targeting regulation of type H vessel regulator Slit3 by miR-148-5p |
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| DOI：10.3969/j.issn.1006-7108.2022.07.005 |
| 中文关键词: 绝经后骨质疏松症 H型血管 Slit3 miR-148-5p 双荧光素酶报告基因 |
| 英文关键词:postmenopausal osteoporosis type H vessels Slit3 miR-148-5p dual luciferase reporter assay |
| 基金项目:国家自然科学基金青年项目(81904228)；福建中医药大学校管科研项目(X2019015) |
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| 中文摘要: |
| 目的 观察绝经后骨质疏松症中H型血管调控因子Slit3表达差异，进一步探讨其上游靶向调控miRNAs。方法 雌性SD大鼠随机分假手术组和模型组，Real-time PCR和免疫组化检测骨组织Slit3 mRNA和蛋白表达水平；生物信息学预测可能与Slit3-3’UTR互作的miRNAs，构建野生型和突变型Slit3-3’UTR重组荧光素酶报告载体，和miRNAs表达载体共转染293T细胞，双荧光素酶报告基因测定荧光素酶活性验证miRNA对Slit3-3’UTR的靶向作用。结果 与假手术组相比，模型组Slit3 mRNA表达水平略上调和平均光密度显著上调，生物信息学预测Slit3-3’UTR与miR-148a-5p、miR-148b-5p、miR-410-3p、miR-129-5p和miR-374-5p存在碱基结合位点，野生型Slit3-3’UTR共转染miR-148b-5p荧光活性下调至空白对照组的68.91%，突变型Slit3-3’UTR共转染miR-148b-5p荧光活性未见下调。结论 Slit3可能是绝经后骨质疏松症H型血管的调控基因，miR-148b-5p与Slit3-3’UTR的靶向结合直接调控Slit3的表达水平。 |
| 英文摘要: |
| Objective To observe the expression of type H vessel regulator slit3 in patients with postmenopausal osteoporosis and to further explore its upstream targeted regulation miRNAs. Methods Female SD rats were randomly divided into sham operation group and model group. The expressions of slit3 mRNA and protein were detected with real-time PCR and immunohistochemistry. miRNAs that may interact with slit3-3’UTR were predicted using bioinformatics. Wild type and mutant slit3-3’UTR recombinant luciferase reporter vectors were constructed and co-transfected with miRNAs expression vector into 293T cells. Dual-luciferase reporter assay system was used to determine luciferase activity and to verify the targeted regulation of miRNA on slit3-3’UTR. Results The expression of slit3 mRNA was up-regulated and average optical density significantly increased in the model group comparing to those in the sham operation group. Bioinformatics predicted that slit-3'UTR had base binding sites with miR-148a-5p, miR-148b-5p, miR-410-3p, miR-129-5p, and miR-374-5p. The fluorescence activity of miR-148b-5p co-transfected with wild-type slit3-3'UTR was down regulated to 68.91% of that in the blank control group. The fluorescence activity of miR-148b-5p co-transfected with mutant slit3-3'UTR was not down regulated. Conclusion Slit3 may be an associated gene of type H vessels in postmenopausal osteoporosis. The targeted combination of miR-148b-5p and slit3-3'UTR directly and negatively regulates the expression level of slit3. |
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