The Research On Inhibition Of Atherosclerosis By MiR-126Using Conditional Knockout Technique

In recent years, along with rapid economics development and improving living standards,cardiovascular disease has become an significance cause threat to people’s health, andatherosclerosis is a most common of all. As an inflammatory vascular disease, atherosclerosisprocess involve in many endogenous small molecules. Although people’s comprehensivebecome deeper and deeper on the pathogenesis of this disease while medical researchdeveloping, there is still lack of understanding on the effect of endogenous small moleculesfrom microscopic point of view currently. MicroRNAs（miRNAs）are a class of smallnon-coding RNA molecules which are found in recent years. They are capable of regulatinggene expression on post-transcription level, for binding to the target mRNA recognition siteand leading to translation repression or degradation. MiRNAs are highly conserved amongspecies, and approximately1/3of genes are modulated by miRNAs, which suggested thepresence of miRNAs universality. In pathological process of atherosclerosis, the expression ofmiRNAs have changed, causing pathological changes involved in regulation of lipidmetabolism, endothelial cells injury, fiber proliferation, plaque formation and rupture.However, numbers of miRNAs families are so large that currently only a small number ofthem have been studied. The expression change, targets regulating and pathologicalmechanism of majority of miRNAs in development, physiology and disease are still unclear.Our study demonstrated the microarray result of miRNAs expression profiles inendothelial cells, and detected the expression profile of miR-126in mouse tissues. Then weestablished the strain of conditional endothelial cell miR-126knockout mouse, and examinedthe knockout. Using this experimental platform, we studied the impact of miR-126onatherosclerosis. This study focused on using the conditional knockout mouse to reveal thefunction of miR-126on atherosclerosis and endothelial cell injury, and to provideexperimental evidence on treating atherosclerosis related diseases.1. Establishment and verification of miR-126knockout mouse strainObjective To establish conditional vascular endothelial cell miR-126knockout mouselines for providing an ideal experimental platform to study the relationship of miR-126andendothelial cells.Methods （1）Microarray was used to analyze expression profile of miRNA in HUVEC; （2）Real-time RT PCR and Northern blot were used to detect the expression profile ofmiR-126in brain, kidney, lung, liver, heart, intestine and spleen;（3）Establish conditionalvascular endothelial cell miR-126knockout mouse strain by crossing miR-126floxp/+andTie2-Cre+mouse;（4）Real-time RT PCR validated acquirement of miR-126knockout mouse.Verify the knockout mouse line is single gene knockout by PCR;（5）Histological analysiswas used to find phenotypic changes of knockout mouse embryo.Results MiR-126was the most abundant of miRNAs in HUVEC, and expression ofmiR-126was higher in the heart, lung and other organs rich in vascular tissue; We obtainedhomozygous knockout mouse by miR-126floxp/+and Tie2-Cre+mouse hybrid, whose genetypewas miR-126floxp/floxpTie2-Cre+. Real-time RT PCR results suggested miR-126was knockedout in the lung of this mouse; By designing primers, PCR was performed to identify the hostgene of miR-126--EGFL7was not affected. This knockout mouse was a single gene knockoutline; knockout embryos displayed lethality and significant bleeding tendency, as well therewere extensive cell swelling, fusion and red blood cells leakage in liver.2. The research on promotion of atherosclerosis by knockout miR-126Objective To observe basis life parameters and pathology changes induced by high fatdiet in conditional vascular endothelial cell miR-126knockout mouse. In addition, to findexperimental evidence of miR-126involved in lipid metabolism, endothelial cell injury andatherosclerosis, and to clarify the role of miR-126in endothelial cell injury.Methods（1）Mouse were given high fat diet for9months, then the liver tissue was stainwith HE, while collecting plasma to detect total cholesterol, triglyceride levels and collectingaorta for oil red O staining, all of which was to establish atherosclerotic model;（2）Usingconditional vascular endothelial cell miR-126knockout mouse platform, we gave the wildtypeand miR-126knockout mouse high fat diet for9months, then counted the survival rate andbodyweight change trend, and collected plasma, liver, aorta tissue;（3）For assessing lipidmetabolism, we detected plasma total cholesterol and triglyceride levels. In addtion, livertissue frozen sections were stained with HE. Aortic globally oil red O staining and frozensections of the aortic sinus HE staining for observing lipid plaque formation;（4）The aorticfrozen sections of knockout mouse were immunofluorescence stained to detect endothelial cellinjury maker VCAM-1.Results High fat diet for9months resulted in liver extensive fatty degeneration, increased lipid metabolism level of plasma （Total cholesterol212.76±45.87mg/dl vs142.80±43.75mg/dl, Triglyceride201.09±61.98mg/dl vs98.02±51.91mg/dl）, and formation of aorta lipid plaque（11.13±8.21%vs4.69±2.36%）; After feding9months, survival rate of miR-126knockout mouse decreased compared with wildtype（60%vs80%）, but the weight change trend of the2groups was consistent; Pathology ofliver and plasma lipid metabolism index indicated no statistically significant differencebetween2groups （Total cholesterol251.94±46.25mg/dl vs232.75±46.10mg/dl,Triglyceride201.44±54.89mg/dl vs161.37±73.03mg/dl）, and aortic global oil redO staining showed lipid plaque on the inner aorta wall of knockout mouse was more thanwildtype（29.32±7.39%vs12.70±7.54%）; Aortic immunofluorescence staining showed thatVCAM-1fluorescence of knockout mouse was located in the inner walls of vessels, and wassignificantly stronger than the wildtype.Conclusions（1） MiR-126was the highest expression of miRNA in endothelial cells;（2）Using genetic engineering technique, we obtained conditional vascular endothelial cellmiR-126knockout mouse（genetype was miR126floxp/floxpTie2-Cre+）by miR-126floxp/+andTie2-Cre+hybrid;（3） There was serious bleeding tendency in this conditional vascularendothelial cell miR-126knockout mouse embryos, which suggested endothelial cell barrierfunction was injured;（4） Endothelial cell miR-126knockout promoted the formation ofaortic lipids plaque, and accelerated the pathological processes of atherosclerosis induced byhigh fat diet, but did not affect bodyweight and plasma lipid metabolism;（5）Knockout ofmiR-126resulted in aortic endothelial cell injury, and the expression of endothelial cell injurymaker VCAM-1upregulated in aortic intine, which demonstrated that endogenous miR-126may play inhibition role to atherosclerosis and endothelial cell injury.