miR-429对CRKL表达的靶向调控作用研究  

Target regulating of miR-429 on CRKL expression

作　　者：郭春梅[1] 刘淑清[2] 孙明忠[1] GUO Chunmei;LIU Shuqing;SUN Mingzhong(Department of Biotechnology,Dalian Medical University,Dalian 116044,China;Biochemistry,Dalian Medical University,Dalian 116044,China)

机构地区：[1]大连医科大学生物技术系,辽宁大连116044 [2]大连医科大学生物化学与分子生物学教研室,辽宁大连116044

出　　处：《大连医科大学学报》2018年第3期198-204,共7页Journal of Dalian Medical University

基　　金：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)

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psi CHECK-2-CRKL-3'-UTR-WT及突变型psi CHECK-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Hep G2-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Hep G2-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Hep G2-sh NC相比,CRKL下调使内源性miR-429表达上调了(102.8±20.0)%(P=0.0069),与Hep G2-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。Objective To explore the target regulating of miR-429 on CRKL expression. Methods We constructed the dual-luciferase expression vetors： psi CHECK-2-CRKL-3＇-UTR-WT（ wild-type） and psi CHECK-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 Hep G2 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 Hep G2-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 Hep G2-sh NC and CRKL overexpression significantly decreased endogenous miR-429 expression level（92. 4 ± 3. 2） %（ P = 0. 0009） compared to Hep G2-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.

关 键 词：miR-429 CRKL 负性调控 

分 类 号：Q291[生物学—细胞生物学]