MiR-184和miR-150通过靶向自噬基因促进肾脏系膜细胞衰老

发布时间：2018-05-27 15:08

本文选题：miR-184 + miR-150　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：肾脏是机体衰老比较快的器官之一，衰老过程中伴随着肾功能的逐步下降，老年肾脏病理中会出现肾小球肥大、系膜基质增多、肾小球硬化和小管间质纤维化等，但目前对肾脏衰老机制的研究还不明确，因此，了解肾脏衰老的机制对相关肾脏疾病的治疗将具有重要的意义。微小RNA（microRNA，miRNA）是真核生物中广泛存在的非编码单链小分子RNA，具有高度保守的序列和功能，互补结合靶基因而降解mRNA或抑制mRNA翻译，负调控表达靶基因。在机体发育、免疫、衰老、肿瘤及癌症等多种生理病理过程中，miRNA中发挥重要作用。细胞通过溶酶体对自身受损结构的吞噬降解过程称为自噬，研究表明衰老与自噬关系密切。本研究通过体内外实验，旨在阐明在肾脏组织衰老进程，miRNA差异表达的确切作用及对自噬的影响，及其在调控系膜细胞衰老作用中的分子机制。全文共分三个部分：第一部分：热量限制影响老年大鼠肾脏组织中miRNA的表达变化及其验证通过饮食限制对大鼠衰老进程进行干预，过碘酸-希夫氏碱（PAS）染色，观察大鼠的肾脏病理变化，miRNA芯片检测正常饮食（AL）或热量限制（CR）对大鼠肾脏皮质miRNA表达的影响，最后RT-PCR鉴定差异表达的miRNAs。结果发现：与3月龄大鼠相比，24月龄AL和CR组大鼠肾脏组织均显示出衰老特征，而24月龄CR组与AL组相比，肾小球系膜细胞增生较轻，小管间质炎症细胞浸润和小管萎缩硬化减轻。芯片扫描及聚类分析结果显示CR组与AL组24月龄大鼠肾组织中miRNA-132、miRNA-150和miRNA-184的差异表达具有统计学意义（p0.05），且三种miRNAs均被下调。RT-PCR进一步验证了上述结果，与正常饮食组相比，能量限制组miRNA-132、miRNA-150和miRNA-184分别下调了66.9%，66%和96.7%（p 0.05）。同时，验证了老年大鼠肾脏皮质miRNA-132、miRNA-150和miRNA-184的表达明显高于青年大鼠（p 0.05）。本部分研究结果显示，热量限制可抑制大鼠肾脏组织的衰老，miRNA-132、miRNA-150和miRNA-184的差异表达可能参与了肾组织衰老过程的调控。第二部分：miR-184和miR-150通过调控系膜细胞自噬活性影响氧化损伤和衰老首先通过分离AL组24月龄大鼠肾脏固有细胞（肾小球系膜细胞，肾小球内皮细胞以及肾小管上皮细胞），RT-PCR分析miR-184和miR-150在3种细胞中的表达以确定两种miRNA起主要调控作用的细胞并建立体外研究模型。结果发现，miR-184和miR-150在系膜细胞中的表达显著高于其它两种细胞中的表达（p0.05～0.01），故选择系膜细胞作为体外研究的细胞模型。以从3月龄和24月龄大鼠肾组织分离得到的原代系膜细胞作为模型，通过干预miR-184和miR-150的表达，观察其对系膜细胞胞内自噬水平以及氧化应激损伤和衰老进程的影响。结果发现：24月龄大鼠来源的系膜细胞出现明显的衰老特征，SA-β-gal和衰老相关异染色质（SAHF）阳性率达到95%以上，与3月龄大鼠来源的细胞差异显著（p0.01）；同时与年轻组细胞相比，衰老细胞中氧化损伤产物（MDA，8-OhdG以及羰基化蛋白质）含量显著增加（p0.01），miR-184和miR-150表达升高，自噬作用标记分子LC3表达减少，且泛素蛋白酶降解系统相关蛋白p62表达增加，细胞自噬活性降低，提示，衰老组系膜细胞中miR-184和miR-150表达的增加可能通过抑制自噬活性而诱导细胞衰老。转染外源miR-184和miR-150mimics促使二者高表达后，LC3表达降低，p62表达升高，自噬降低；而在转染miR-184和miR-150inhibitors后，，细胞净自噬水平升高。同时，长效转染miR-184和miR-150mimics后，SA-β-gal和SAHF细胞阳性率升高（p0.05），氧化损伤产物积累增加。本部分结果表明，miR-184和miR-150可能通过抑制系膜细胞的自噬从而调控系膜细胞衰老。第三部分：miR-184和miR-150通过靶向Rab1和Rab31调控细胞自噬活性利用生物信息学软件，比如Targetseans、miRanda和PicTar，预测miR-184和miR-150潜在的靶基因；通过体外实验验证miR-184和miR-150对靶基因表达的影响；进一步采用双荧光素酶报告基因系统检测miR-184和miR-150与靶基因3′-UTR序列的相互作用。结果发现：miR-184可能的靶基因有239-649个，而miR-150有209-2825个，其中与自噬体形成和成熟相关的Rab蛋白家族有很多成员为miR-184和miR-150共同的靶基因，本研究拟选择Rab1和Rab31靶基因进行验证。结果发现转染miR-184和miR150mimics或inhibitors对系膜细胞Rab1和Rab31mRNA水平无显著影响（p0.05），但miR-184和miR150mimics可以显著下调二者蛋白的表达水平（p0.05～0.01），而miR-184和miR150inhibitors分别转染后Rab1表达上调了78%和56%（p0.05），Rab31上调86%和67%(p0.05)。双荧光素酶检测结果显示：miR184和miR-150能通过与Rab1和Rab31mRNA的3′-UTR相互作用而下调荧光素酶活性（p0.05～0.01）。本部分研究证实， miR184和miR-150可以直接作用于Rab家族蛋白信使mRNA3′-UTR而降解或抑制其表达，从而阻止其参与细胞自噬过程的调节。综上所述，本研究发现热量限制老年大鼠与正常饮食老年大鼠肾脏皮质miRNAs的表达存在差异，其中包括miR-184和miR-150，肾小球系膜细胞是miR-184和miR-150差异表达发生的主要肾组织固有细胞。体外研究证实，miR-184和miR-150能作用于共同的靶基因Rab1和Rab31mRNA从而在转录后水平上调节细胞自噬活性，并进一步影响细胞内氧化损伤产物的积累而最终调控细胞的衰老进程。
[Abstract]:The kidney is one of the organs of fast aging, with the gradual decline of renal function in the aging process. There will be glomerular hypertrophy, mesangial matrix increase, glomerulosclerosis and tubulointerstitial fibrosis in the aged kidney pathology. However, the study of renal aging mechanism is not clear. Therefore, the mechanism of renal aging is related to the related mechanisms. The treatment of kidney disease will be of great significance.
Small RNA (microRNA, miRNA) is a non coding single strand small molecule RNA widely existed in eukaryotes, with highly conservative sequences and functions, complementing the target gene to degrade mRNA or inhibit mRNA translation and negatively regulate the expression of target genes. In the physiological and pathological processes, such as body development, immunity, senescence, swelling and cancer, miRNA plays an important role in miRNA The phagocytosis and degradation process of cells through lysosomes on their damaged structures is called autophagy. The study shows that senescence is closely related to autophagy. The purpose of this study is to elucidate the exact role of miRNA differential expression and the effect on autophagy in the renal tissue aging process and the molecules in regulating the senescence of mesangial cells through the experiments in vitro and in vivo. The full text is divided into three parts:
Part one: the effect of caloric restriction on the expression of miRNA in kidney tissues of aged rats and its validation
The effects of dietary restriction on the aging process of rats, periodate Schiff's alkali (PAS) staining, and the pathological changes of kidney in rats were observed. The effect of normal diet (AL) or caloric restriction (CR) on the expression of miRNA in renal cortex of rats was detected by miRNA chip. The final RT-PCR identification of differential expression of miRNAs. found that compared with 3 month old rats, 24 months The renal tissue of rats aged AL and CR showed aging characteristics, and the glomerular mesangial cell proliferation was lighter, tubulointerstitial inflammatory cell infiltration and tubuloatrophy sclerosis reduced in the 24 month old CR group compared with the AL group. The results of chip scanning and cluster analysis showed the differences in miRNA-132, miRNA-150 and miRNA-184 in the kidney tissues of the 24 month old rats of the CR group and the AL group. The expression was statistically significant (P0.05), and three kinds of miRNAs were downregulated by.RT-PCR to further verify the results. Compared with the normal diet group, the energy restriction group miRNA-132, miRNA-150 and miRNA-184 were down 66.9%, 66% and 96.7% respectively (P 0.05). Meanwhile, the expression of renal cortex miRNA-132, miRNA-150 and miRNA-184 in the aged rats was proved to be obvious. It was higher than that of young rats (P 0.05). The results of this study showed that heat restriction could inhibit the aging of renal tissue in rats, and the differential expression of miRNA-132, miRNA-150 and miRNA-184 may be involved in the regulation of renal tissue aging process.
The second part: miR-184 and miR-150 affect oxidative damage and aging through regulating autophagy activity of mesangial cells.
First, by separating the kidney innate cells (glomerular mesangial cells, glomerular endothelial cells and renal tubular epithelial cells) of 24 month old rats, the expression of miR-184 and miR-150 in 3 cells was analyzed to determine the main regulatory role of two miRNA cells and to establish an in vitro study model. The results showed that miR-184 and miR-150 were in the mesangial membrane. The expression in the cells was significantly higher than that of the other two cells (P0.05 ~ 0.01), so the mesangial cells were selected as the cell model in vitro. The primary mesangial cells isolated from the renal tissue of 3 month old and 24 month old rats were used as the model. The intracellular autophagy level of the mesangial cells was observed by interfering with the expression of miR-184 and miR-150. The effects of oxidative stress injury and aging process. The results showed that the mesangial cells of the 24 month old rats had obvious aging characteristics, the positive rate of SA- beta -gal and aging related heterochromatin (SAHF) was more than 95%, and the difference was significant (P0.01) with the 3 month old rat cells, and the oxidative damage in senescent cells compared with the young group cells. The content of MDA, 8-OhdG and carbonylation protein increased significantly (P0.01), the expression of miR-184 and miR-150 increased, the expression of autophagy marker LC3 decreased, and the expression of ubiquitin protease related protein p62 increased and the autophagic activity decreased. The increase of miR-184 and miR-150 in the mesangial cells of the senescent group may pass through the increase of the expression of miR-184 and miR-150. Inhibition of autophagic activity induced cell senescence. After transfection of exogenous miR-184 and miR-150mimics, the expression of LC3 decreased, the expression of p62 increased, and autophagy decreased, and the net autophagy increased after transfection of miR-184 and miR-150inhibitors. Meanwhile, the positive rate of SA- beta -gal and SAHF cells increased after the transfection of miR-184 and miR-150mimics. The results show that miR-184 and miR-150 may regulate the senescence of mesangial cells by inhibiting autophagy of mesangial cells in high (P0.05).
The third part: miR-184 and miR-150 regulate the autophagy activity through targeting Rab1 and Rab31.
Use bioinformatics software, such as Targetseans, miRanda and PicTar, to predict the potential target genes of miR-184 and miR-150; verify the effect of miR-184 and miR-150 on target gene expression through in vitro experiments, and further use the double luciferase reporter gene system to detect the interaction between miR-184 and miR-150 and the target gene 3 '-UTR sequence. It is found that there are 239-649 possible target genes for miR-184, and 209-2825 of miR-150, of which the Rab protein family associated with the formation and maturation of autophago is a common target gene for miR-184 and miR-150. This study is to select the Rab1 and Rab31 target genes for verification. The results were found to be transfected with miR-184 and miR150mimics or inhibitors to the mesangial membrane. The levels of Rab1 and Rab31mRNA were not significantly affected (P0.05), but miR-184 and miR150mimics could significantly decrease the expression level of two proteins (P0.05 ~ 0.01), while Rab1 expression was up to 78% and 56% (P0.05) after miR-184 and miR150inhibitors, and Rab31 up 86% and 67% (P0.05). Luciferase activity (P0.05 ~ 0.01) was down regulated by the interaction with 3 '-UTR of Rab1 and Rab31mRNA. This part of this study confirmed that miR184 and miR-150 could directly act on the Rab family protein messenger mRNA3' -UTR and degrade or inhibit its expression, thus preventing its involvement in the regulation of autophagic pathway.
To sum up, the study found that there was a difference in the expression of miRNAs in the renal cortex of the aged rats and the normal diet old rats, including miR-184 and miR-150, and the glomerular mesangial cells were the main renal tissue innate cells of the differential expression of miR-184 and miR-150. In vitro studies have proved that miR-184 and miR-150 can be used to target the common target. Gene Rab1 and Rab31mRNA regulate autophagic activity at post transcriptional level, and further affect the accumulation of intracellular oxidative damage products and ultimately regulate cell aging process.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

【相似文献】