The Effect And Mechanism Of MicroRNA-199a In The Development Of Obesity In Mice

ObjectivesTo provide theoretical basis for the obesity mechanism related with adipose differentiation disorder, and to study the effect of microRNA-199a in the development of obesity in mice, we measured the miR-199a expression level between ob/ob mice and C57/BL6J mice by SYBR Green PCR and Taqman probe PCR method.We also observed the relationship between the expression of microRNA-199a and the key gene associated with adipogenesis and differentiation, such as Peroxisome Proliferator Activated Receptor (PPARy2), CCAAT/enhancer binding proteins (C/EBPa), adipocyte fatty acid-binding protein (aP2), sterol regulatory element binding protein-1 (SREBP-1c), etc.MethodsNow 40 male, which include 20 C57/BL6J mice and 20 ob/ob mice, were fed adaptively one week, and then according to the way of 2* 2 factorial design, they were divided into 4 groups:control group (Cl), high-fat group (C2), knock out leptin group (O1), leptin knock out with high-fat fed group (02). Among them, the C2group and 02 group were fed high-fat diet, and the other two groups were fed normal diet. After the consecutive feeding for 12 weeks, they were put to death. Weekly weigh, the body length, water intake, food intake, and other general conditions were measured and recorded. Dual-energy X-ray scanners were used to measure the whole body fat (not including head) in vivo. The blood glucose level were measured by using a blood glucose meter. The OD values of triglyceride (TG)、total cholesterol (TC)、high density lipoprotein cholesterol (HDL -C)、and low-density lipoprotein cholesterol (LDL-C) were measured by using the multifunction microplate reader. We used Folch method to extract lipids from white adipose tissue and ELISA kit to measure free fatty acid (FFA) and triglyceride (TG) enzyme. The hematoxylin - eosin (HE) staining method were used for paraffin sections of the white adipose tissue and the oil red O staining method for frozen sections of the white adipose tissue. The SYBR Green method were used to measure the expression levels of the miR-199a, PPARy2, C/ EBPa, aP2, and SREBP-lc; and the Taqman probe method were used to measure the expression level of miR-199a. From the statistically analyzed by SPSS 18.0 software, there was a significant difference when P<0.05.Results1. Food intake, weekly weight, Lee’s index, and body fat content of each groupThere is a steady increase of food intake and body weight in each group during the period of feeding. The 02 group has the highest score of body weight, Lee’s index and body fat content, and the C1 group has the lowest score of the above indicators. After factorial analysis, leptin gene and high fat diet exist an interaction in body weight and body fat content indicators (P<0.05).2. Glucolipid metabolism levels of each groupBlood glucose levels in each group have no statistical significance (P>0.05); TG,TC,HDL-C,LDL-C and HDL-C/LDL-C levels and FFA and TG content in lipids have a statistical significance in leptin gene level (P<0.05).The index levels in knock out leptin gene groups were higher than that of the non-knockout groups. TG.TC, FFA and TG levels have a statistical significance in diet level (P<0.05).3. The volume of fat cells and fat droplet size of each groupH&E staining showed that obese groups’ cell count number per unit area is lower. compared with non-obese groups There were statistically significant in leptin gene and dietary levels (P<0.05). The oil red O staining showed, the fat droplet size was bigger in obese groups.4. The expression levels of miR-199a、PPARy2、C/EBPα、aP2、and SREBP-lc of each groupThe expression levels of miR-199a、PPARy2、C/EBPα、aP2 and SREBP-lc were higher in obese groups than that of the non-obese groups by using SYBR Green method. The expression level of miR-199a was higher in obese groups by qRT-PCR method, which was a consistent with SYBR Green method. The correlation coefficient was 0.72, and there was a statistical significance(P<0.05).5. The correlation of miR-199a expression level with Lee’s index, body fat content, and FFA and TG levels of each groupThere was a correlation of miR-199a expression level with Lee’s index, body fat content and FFA and TG levels. The correlation coefficient was 0.60,0.64,0.53,0.85, respectively. It showed a positive correlation and there was a statistical significance(P <0.05).Conclusions1. We observed a weight gain, a larger Lee’s index, body fat accumulation, sugar and lipid metabolic disorder, a free fatty acid and triglyceride increase, and volume of fat cells and lipid raise in each group, both high-energy feeding and knock out leptin gene can promote the occurrence of obesity.2. The correlation between the two factors is significant, and there may be a interaction, which was synergistic.3. The expression levels of miR-199a、PPARy2、C/EBPα、aP2 and SREBP-lc were up-regulated. The expression levels between miR-199a and adipose differentiation factor had a high degree of consistency. It showed that miR-199a may participate in adipocyte differentiation to regulate the metabolism of obesity genes.4. It showed a positive correlation between miR-199a expression level and Lee’s index, body fat content and FFA and TG level. It implied that miR-199a participated in the process of lipid accumulation and directly relevant to obesity.