【摘要】：目的：脂蛋白（a）[lipoprotein(a),Lp(a)]由載脂蛋白（a）[apolipoprotein(a),apo(a)]和載脂蛋白B-100（apolipoprotein B-100,apoB100）通過二硫鍵連接而成，主要在肝細(xì)胞中合成。許多臨床研究表明高Lp(a)血漿水平是動(dòng)脈粥樣硬化(Atherosclerosis,As)相關(guān)疾病如早發(fā)性心血管疾病中風(fēng)等的獨(dú)立危險(xiǎn)因子。Lp(a)血漿水平對許多藥物和非藥物的治療方法不敏感，血液透析是唯一能將Lp(a)水平降低50%以上的方法。在機(jī)體內(nèi)調(diào)節(jié)apo(a)表達(dá)機(jī)制方面，除發(fā)現(xiàn)apo(a)基因LPA的一些單核苷酸多態(tài)性（single nucleotide polymorphism，SNP)能影響Lp(a)血漿水平外，未見其它報(bào)道。目前已經(jīng)發(fā)現(xiàn)多個(gè)miRNAs和動(dòng)脈粥樣硬化（Atherosclerosis,As）以及脂質(zhì)代謝相關(guān)，本研究擬通過生物信息學(xué)的方法篩選調(diào)控LPA基因的microRNA，然后在肝細(xì)胞中驗(yàn)證其降apo(a)效用。 方法：通過生物信息學(xué)分析LPA3’端非翻譯區(qū)（3’-Untranslated Regions,3’-UTR）序列中的二級結(jié)構(gòu)，并通過在線預(yù)測工具預(yù)測可能作用于LPA基因的miRNAs。用western blot篩選apo(a)高表達(dá)細(xì)胞株；通過熒光檢測miRNA mimics轉(zhuǎn)染效率。分別通過RT-PCR檢測轉(zhuǎn)染陰性對照miRNA mimic和轉(zhuǎn)染預(yù)測所得miRNA mimics處理組6h、12h和24h Hep G2LPA mRNA表達(dá)水平；通過westernblot檢測轉(zhuǎn)染陰性對照miRNA mimic和轉(zhuǎn)染預(yù)測所得miRNA mimics處理組24h、36h和48h Hep G2apo(a)表達(dá)水平。通過western blot檢測轉(zhuǎn)染陰性對照miRNA、miR-626mimic、miR-626mimic+miR-626inhibitor和miR-626inhibitor處理48hHep G2apo(a)表達(dá)水平。使用熒光素酶報(bào)告系統(tǒng)進(jìn)行miR-626靶標(biāo)驗(yàn)證實(shí)驗(yàn)。統(tǒng)計(jì)學(xué)分析：實(shí)驗(yàn)所得數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(±SD)表示，用GraphpadPrism5.0.1對數(shù)據(jù)進(jìn)行分析和作圖，選取95%可信區(qū)間，P0.05為差異有顯著性意義。 結(jié)果：生物信息學(xué)預(yù)測可能作用于LPA的miRNAs為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細(xì)胞apo(a)表達(dá)水平較高，可用來做后續(xù)實(shí)驗(yàn)。miR-655、miR-590-3p、miR-519a、miR-519b-3p、miR-338-3p、miR-519c-3p、miR-590-5p、miR-425和miR-626mimics處理組與對照組相比LPA mRNA表達(dá)水平均差異無統(tǒng)計(jì)學(xué)意義；miR-655mimic和miR-590-5p mimic能輕微下調(diào)Hep G2表達(dá)apo(a)，而miR-626mimic能顯著下調(diào)Hep G2表達(dá)apo(a)。miR-626mimic+miR-626inhibitor處理組apo(a)表達(dá)水平顯著高于miR-626mimic處理組，miR-626inhibitor處理組apo(a)表達(dá)水平顯著高于對照組。轉(zhuǎn)染miR-626處理組細(xì)胞裂解后熒光強(qiáng)度顯著低于對照組。 結(jié)論：miR-626顯著下調(diào)apo(a)表達(dá)水平；miR-626下調(diào)Hep G2表達(dá)apo(a)是通過直接和LPAmRNA3’-UTR序列結(jié)合，抑制LPAmRNA翻譯實(shí)現(xiàn)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 鹵standard deviation (鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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