| 摘要: |
| 目的 探讨miR-429对CRKL的靶向调控作用。方法 构建双荧光素野生型psiCHECK-2-CRKL-3′-UTR-WT及突变型psiCHECK-2-CRKL-3′-UTR-MUT重组表达载体，双荧光素酶报告实验检测miR-429 与CRKL-3′-UTR的靶向结合；瞬时转染miR-429模拟物及其抑制剂，qRT-PCR和Western blot检测miR-429对内源性CRKL表达水平变化的影响；qRT-PCR检测CRKL上、下调对内源性miR-429表达水平变化的影响。结果 双荧光素报告实验结果表明miR-429直接靶向CRKL-3′-UTR第2个结合位点；同时与HepG2-miR-NC相比，miR-429上调使内源性CRKL 蛋白下调了(55.0±1.5) %（P=0.0089），与HepG2-LNA-miR-429相比，miR-429下调使内源性CRKL 蛋白上调了(44.3±1.0) %（P=0.0038），差异具有统计学意义，而miR-429上、下调对内源性CRKL mRNA 表达变化无影响；且与HepG2-shNC相比，CRKL下调使内源性miR-429表达上调了(102.8±20.0)%（P=0.0069），与HepG2-PCDH相比，CRKL上调使内源性miR-429表达下调了 (92.4±3.2) %（P=0.0009），差异具有统计学意义。结论 miR-429靶向作用于CRKL-3′-UTR在转录后蛋白翻译水平负性调控CRKL表达，同时CRKL负性调控内源性miR-429的表达。 |
| 关键词: miR-429 CRKL 负性调控 |
| DOI：10.11724/jdmu.2018.03.02 |
| 分类号:Q291 |
| 基金项目:基金项目：国家自然科学基金项目（81272186）；辽宁省自然科学基金项目（2015020266）；辽宁省教育厅基本科研项目（5061092） |
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| Target regulating of miR-429 on CRKL expression |
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GUO Chunmei1, LIU Shuqing2, SUN Mingzhong1
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1.Department of Biotechnology, Dalian Medical University, Dalian 116044, China;2.Department of Biochemistry, Dalian Medical University, Dalian 116044, China
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| Abstract: |
| Objective To explore the target regulating of miR-429 on CRKL expression. Methods We constructed the dual-luciferase expression vetors: psiCHECK-2-CRKL-3′-UTR-WT (wild-type) and psiCHECK-2-CRKL-3′-UTR-MUT (mutant). The dual luciferase reporter experiment was used to detecte the interaction between the miR-429 and CRKL-3′-UTR after miR-429 mimics and miR-429 inhibitors were transiently transfected into HepG2 cells. Real-time qRT-PCR and Western blot detected miR-429 and CRKL expression level. Results Luciferase reporter assay showed that miR-429 directly targeted to CRKL-3′-UTR at the second binding sites. MiR-429 overexpression and suppression significantly decreased and increased endogenous CRKL expression level (55.0±1.5)% (P=0.0089) and (44.3±1.0)% (P=0.0038) compared to HepG2-miR-NC. Interestingly, miR-429 expression increasing or decreasing showed no effect on the endogenous CRKL mRNA expression. Meanwhile, CRKL knockdown significantly increased endogenous miR-429 expression level (102.8±20.0)% (P=0.0069) compared to HepG2-shNC and CRKL overexpression significantly decreased endogenous miR-429 expression level (92.4±3.2)% (P=0.0009) compared to HepG2-PCDH. Conclusion MiR-429 may directly target CRKL-3′-UTR to suppress its expression at the post-transcriptional protein translation level. CRKL expression could regulate miR-429 expression negatively. |
| Key words: miR-429 CRKL negatively regulation |
