Screening Of Differential Expression Profiles Of MicroRNAs During Vascular Calcification And Its Dynamic Changes

Part one Identification of vascular calcification modelObjectiveVascular calcification in animal and cell models were identified, to ensure that theresearch models is reliable for observing the changes of miRNA expression profilingin the process of vascular calcification.Method1. Identification of animal model: SPF4-week-old and12-week-old osteoprotegeringene knockout (OPG-/-) mice and the same week-old wild-type mice were assigned forthis study. We picked their eyeballs and took blood samples to detect ALP and Runx2levels in serum, After these mice were excuted the aortas were dissected, fixed by10%formalin and embedded by paraffin. HE staining was used to observe the aorticcalcification, and immunohistochemistry was used to detect the expression of ALPand Runx2.2. Identification of in vitro cell model: The mouse vascular smooth muscle cells(MOVAS) were incubated with10mmol/L β-glycerophosphate (β-GP) for0,14,21and38days respectively (0day was used as the control group), and then we detectedcalcification by Alizarin Red S staining.Result1. Animal model1.1HE staining: We didn’t observe aortic calcification in4-week-old and12-week-old wild-type mice, as well as the4-week-old OPG-/-mice. However, the12-week-old OPG-/-mice developed significant pathological calcification in aortas.1.2Immunohistochemistry: The expression of ALP and Runx2is higher in aortas of12-week-old OPG-/-mice than the same week-old wild-type mice, and especially onthe calcified region.1.3ELISA detection: The levels of ALP and Runx2in serum were significantlyincreased in4-week-old and12-week-old OPG-/-mice compared with the same week-old wild-type mice (P<0.05).2. In vitro cell model: Some calcified nodules were observed in MOVAS after β-GPtreatment for21days, and multiple calcified nodules scattered after β-GP treatmentfor38days.Conclusion1. The serum ALP and Runx2levels in4-week-old OPG-/-mice were significantlyincreased, but no aortic calcification were observed; While12-week-old OPG-/-micedeveloped aortic calcification apparently, accompanied by the abnormally elevatedlevels of ALP and Runx2in serum and aortas.2. Calcification occurred in MOVAS after β-GP treatment for21days，the cell modelof vascular calcification was being successfully induced. Part two Dynamic changes of the miRNA expression profile invascular calcification processObjectiveThe miRNA microarray was used to screen the differentially expressed miRNA andobserve the dynamic changes of miRNA expression profile during vascularcalcification, and this may lay a foundation for search the key miRNA which mayregulate vascular calcification.Methods1. Animal model: Aortas of4-week-old OPG-/-mice （didn’t observed aorticcalcification) and12-week-old OPG-/-mice(with obvious aortic calcification) bypathological identification, including aortas of the same week-old wild-type mice,total RNA were extracted respectively, then miRNA microarray technology was usedto detect the miRNA expression profile of each sample, and the data on the array wasanalyzed by ratio and hierarchical cluster analysis etc.2. In vitro cell model: MOVAS were treated with β-GP for0d (control group),21d(obviously calcified group), and total RNA were extracted respectively, then miRNAmicroarray technology was used to detect miRNA expression profile of each sample,and the data on the array was analyzed by ratio and hierarchical cluster analysis etc.Result1. Differentially expressed miRNAs in animal model1.14-week-old mice group: The amount of total miRNA which differentiallyexpressed in aortas of OPG-/-mice was370compared with wild-type mice (the foldchange>1.5),162being up-regulated including miR-125b-5p, miR-126-3p, etc, and208being down-regulated including miR-320, miR-2861, etc. Of which56hadsignificant differences (p<0.05),33being up-regulated including miR-139-5p,etc; and23being down-regulated including miR-18a-5p,etc.1.212-week-old mice group: There were101miRNAs differentially expressed inaortas of OPG-/-mice compared with wild-type mice (fold change>1.5),74of those miRNAs being up-regulated and27down-regulated. Of which16miRNAs hadsignificant differences (p<0.05),8being up-regulated including miR-425-5p,miR-125b-5p, etc, and8being down-regulated including miR-29a-5p, miR-187-3p,etc.2. Differentially expressed miRNAs in vitro cell modelMOVAS treated with β-GP for21days showed92differentially expressedmiRNAs compared with the control group (fold change>1.5), of which57wereup-regulated including miR-30a-5p, and35were down-regulated includingmiR-29a-5p.ConclusionThe differentially expressed miRNA profile at two different stages of vascularcalcification were obtained by miRNA microarray, and it is the first time revealing thedynamic changes of some miRNA such as miR-125b expression during vascularcalcification.