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微小RNA377靶向调控钙通道蛋白在心房颤动中的作用
李龙
2016-05-13
导师杨水祥
专业内科学
授予单位北京大学
授予地点北京大学第九临床医学院
学位硕士
关键词心房颤动 离子通道 小分子RNA L型钙通道 膜片钳技术
其他题名Effect of miR-377 in atrial fibrillation by regulating calcium channel proteins
分类号R541.7
摘要

目的:

采用过表达和敲低等手段,探讨微小RNA377(miR-377)与心房颤动相关钙离子通道蛋白的靶向调控关系。

方法:

本研究分为过表达组、敲低组、阴性组和空白组。将CACNA1C的重组荧光素酶报告质粒与miR-377 mimics及阴性对照共转染于人胚胎肾HEK293细胞,检测各组荧光素酶相对活性。并将miR-377 mimics、miR-377 inhibitor及阴性对照转染大鼠胚胎心肌H9c2细胞,观察其对靶基因的转录水平、蛋白表达水平和生物学功能的影响。

结果:

1 与对照组比较,miR-377过表达组CACNA1C荧光素酶相对活性降低31%(0.316±0.006 vs 0.455±0.008,P=0.009);miR-377敲低组CACNA1C荧光素酶相对活性无明显差异;

2与对照组比较,miR-377过表达组及敲低组CACNA1C、CACNB2基因转录水平无明显差异;

3 过表达miR-377抑制大鼠心肌H9c2细胞CACNA1C基因的蛋白表达(P<0.05);

4 过表达miR-377降低大鼠心肌H9c2细胞L型钙电流的电流密度,使峰值密度降低30%,从-3.23±0.64pA/pF降至-2.26±0.16pA/pF(P<0.05)。但没有改变峰值电位、翻转电位、I-V曲线形状及门控动力学特性。

结论:

L型钙通道α亚基编码基因CACNA1C可能是miR-377的直接靶基因; miR-377可能是通过抑制钙通道核心亚基CACNA1C的蛋白表达从而降低L型钙电流,参与房颤电重构,不是通过改变通道门控动力学特征来实现的。

英文摘要

Objective:

To study the relationship of miR-377 target-regulating atrial fibrillation (AF)-associated genes of calcium ion channel protein by means of overexpression and interference.

Methods:

The experiment was divided into overexpression, interference, negative control and blank control group. The recombinant luciferase reporter plass, miR-377 mimics, and negative control mimics were co-transfected into the HEK293 cell, the relative activity of luciferase was assayed. MiR-377 mimics, inhibitor and negative control mimics was transfected into H9C2 cells to investigate its role in regulation of target gene tranion,protein expression and function.

Results:

1 The relative activities of CACNA1C recombinant plass luciferase in miR-377 overexpression group were lower than those in negative control group (0.316±0.006 vs 0.455±0.008, P=0.009). The relative activities of CACNA1C recombinant plass luciferase in miR-377 interference group between negative control group shows no significant difference.

2 The mRNA level of CACNA1C and CACNB2 in overexpression group or interference group between negative control group shows no significant difference.

3 The CACNA1C protein level was inhibited by miR-377 mimics which was transfected into H9c2 cells.

4 L-type calcium current peak density decreased from -3.23±0.64pA/pF to -2.26±0.16pA/pF (P<0.05) when miR-377 mimics was transfected into H9c2 cells.

Conclusion:

Voltage-gated L-type calcium channel alpha subunit CACNA1C is probably the direct target gene of miR-377, indicating that miR-377 may become a new AF intervening target in future by influencing CACNA1C expression, L-type calcium current as well as AF electrical remolding.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/124809
专题北京大学第九临床医学院
作者单位北京大学第九临床医学院
推荐引用方式
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李龙. 微小RNA377靶向调控钙通道蛋白在心房颤动中的作用[D]. 北京大学第九临床医学院. 北京大学,2016.
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