hsa-miR-626对Hep G2细胞中载脂蛋白（a）表达的影响

发布时间：2018-10-13 16:36

【摘要】：目的：脂蛋白（a）[lipoprotein(a),Lp(a)]由载脂蛋白（a）[apolipoprotein(a),apo(a)]和载脂蛋白B-100（apolipoprotein B-100,apoB100）通过二硫键连接而成，主要在肝细胞中合成。许多临床研究表明高Lp(a)血浆水平是动脉粥样硬化(Atherosclerosis,As)相关疾病如早发性心血管疾病中风等的独立危险因子。Lp(a)血浆水平对许多药物和非药物的治疗方法不敏感，血液透析是唯一能将Lp(a)水平降低50%以上的方法。在机体内调节apo(a)表达机制方面，除发现apo(a)基因LPA的一些单核苷酸多态性（single nucleotide polymorphism，SNP)能影响Lp(a)血浆水平外，未见其它报道。目前已经发现多个miRNAs和动脉粥样硬化（Atherosclerosis,As）以及脂质代谢相关，本研究拟通过生物信息学的方法筛选调控LPA基因的microRNA，然后在肝细胞中验证其降apo(a)效用。方法：通过生物信息学分析LPA3’端非翻译区（3’-Untranslated Regions,3’-UTR）序列中的二级结构，并通过在线预测工具预测可能作用于LPA基因的miRNAs。用western blot筛选apo(a)高表达细胞株；通过荧光检测miRNA mimics转染效率。分别通过RT-PCR检测转染阴性对照miRNA mimic和转染预测所得miRNA mimics处理组6h、12h和24h Hep G2LPA mRNA表达水平；通过westernblot检测转染阴性对照miRNA mimic和转染预测所得miRNA mimics处理组24h、36h和48h Hep G2apo(a)表达水平。通过western blot检测转染阴性对照miRNA、miR-626mimic、miR-626mimic+miR-626inhibitor和miR-626inhibitor处理48hHep G2apo(a)表达水平。使用荧光素酶报告系统进行miR-626靶标验证实验。统计学分析：实验所得数据采用均数±标准差(±SD)表示，用GraphpadPrism5.0.1对数据进行分析和作图，选取95%可信区间，P0.05为差异有显著性意义。结果：生物信息学预测可能作用于LPA的miRNAs为miR-655、miR-590-3p、miR-519a、miR-519b-3p、miR-338-3p、miR-519c-3p、miR-590-5p、miR-425、miR-626。 Hep G2细胞apo(a)表达水平较高，可用来做后续实验。miR-655、miR-590-3p、miR-519a、miR-519b-3p、miR-338-3p、miR-519c-3p、miR-590-5p、miR-425和miR-626mimics处理组与对照组相比LPA mRNA表达水平均差异无统计学意义；miR-655mimic和miR-590-5p mimic能轻微下调Hep G2表达apo(a)，而miR-626mimic能显著下调Hep G2表达apo(a)。miR-626mimic+miR-626inhibitor处理组apo(a)表达水平显著高于miR-626mimic处理组，miR-626inhibitor处理组apo(a)表达水平显著高于对照组。转染miR-626处理组细胞裂解后荧光强度显著低于对照组。结论：miR-626显著下调apo(a)表达水平；miR-626下调Hep G2表达apo(a)是通过直接和LPAmRNA3’-UTR序列结合，抑制LPAmRNA翻译实现的。
[Abstract]:Aim: lipoprotein (a) [lipoprotein (a), Lp (a)] is composed of apolipoprotein (a) [apolipoprotein (a), apo (a)] and apolipoprotein B-100 (apolipoprotein B-100) via disulfide bonds, which are mainly synthesized in hepatocytes. Many clinical studies have shown that high Lp (a) plasma levels are an independent risk factor for atherosclerotic (Atherosclerosis,As) related diseases such as stroke in early onset cardiovascular diseases. Lp (a) plasma levels are insensitive to many drug and non-drug treatments. Hemodialysis is the only way to reduce Lp (a) levels by more than 50%. In the regulation of apo (a) expression in vivo, there is no other report except that some single nucleotide polymorphisms (single nucleotide polymorphism,SNP) of apo (a) gene LPA can affect the plasma level of Lp (a). A number of miRNAs have been found to be related to atherosclerosis (Atherosclerosis,As) and lipid metabolism. This study aims to screen microRNA, regulating LPA gene by bioinformatics and to test its effect of decreasing apo (a) in hepatocytes. Methods: the secondary structure of LPA3' untranslated region (3'-Untranslated Regions,3'-UTR) sequence was analyzed by bioinformatics, and the miRNAs. that might act on LPA gene was predicted by on-line prediction tool. The high expression apo (a) cell lines were screened by western blot and the transfection efficiency of miRNA mimics was detected by fluorescence. The expression levels of Hep G2LPA mRNA were detected by RT-PCR in the negative control group and in the miRNA mimics treated group at 6 h and 24 h, respectively, and the Hep G2apo (a) expression levels at 24 h, 36 h and 48 h in the miRNA mimics treated group and the negative control group were detected by westernblot. The expression of 48hHep G2apo (a) was detected by western blot in negative control miRNA,miR-626mimic,miR-626mimic miR-626inhibitor and miR-626inhibitor. Luciferase report system was used to verify miR-626 target. Statistical analysis: the experimental data using mean 卤standard deviation (卤SD), using GraphpadPrism5.0.1 to analyze and map the data, select 95% confidence interval, P0.05 as the significant difference. Results: bioinformatics predicted that the miRNAs that might act on LPA was miR-655,miR-590-3p,miR-519a,miR-519b-3p,miR-338-3p,miR-519c-3p,miR-590-5p,miR-425,miR-626.. The expression of apo (a) in Hep G2 cells was high, which could be used for further experiment. There was no significant difference in LPA mRNA expression between miR-655,miR-590-3p,miR-519a,miR-519b-3p,miR-338-3p,miR-519c-3p,miR-590-5p,miR-425 and miR-626mimics treatment group and control group. MiR-655mimic and miR-590-5p mimic could slightly down-regulate the expression of apo (a), in Hep G2, while miR-626mimic could significantly down-regulate the apo (a) expression of Hep G2 in apo (a). MiR-626mimic miR-626inhibitor treatment group, and the apo (a) expression level in miR-626inhibitor treatment group was significantly higher than that in miR-626mimic treatment group, while that in miR-626inhibitor treatment group was significantly higher than that in control group. The fluorescence intensity of miR-626 transfected group was significantly lower than that of control group. Conclusion: miR-626 can significantly down-regulate the expression of apo (a) and miR-626 down-regulates the expression of apo (a) in Hep G2 by directly binding with LPAmRNA3'-UTR sequence to inhibit LPAmRNA translation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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