The Effect And Mechanism Of MCHR1Silencing On Fat Synthesis Of Mouse3T3-L1Cells

Background:Obesity and its related diseases have global epidemic, and discusses themechanism and treatment of obesity in the new method always is a hot spot in thefield of medicine. Melanin concentrating hormone (MCH) secreted by thehypothalamus, is present in the blood, is the only a peptide of reducing the secretioncan lead to weight loss of animals, plays an important role in the central regulation ofenergy, peripheral fat organizations may also play a role in. In rodents, MCH worksthrough its receptor Melanin concentration hormone receptor1（MCHR1）. SilenceMCHR1, blocking MCH pathway for reducing the generation of adipose tissue mayhave important significance. RNA interference (RNAi) such as low cost, reliableeffect, can be used to block specific purpose gene expression. This paper will useRNAi technology to silence the MCHR1of mouse3T3-L1cells, observe the effect onthe fat synthesis of cells and to explore the mechanism, lay the foundation for theobese gene therapy.Objective：Using RNAi technology to silence the MCHR1of mouse3T3-L1cells toobserve the effect on fat synthesis and to discuss molecular mechanism.It will provida new idea for the obese gene therapy.Methods:(1)Mouse3T3-L1cells were cultured and induced differentiation;(2)Usingcloning technique to build three positive and a negative vectors of sh-MCHR1;(3)Using plasmid liposome to transfect the mouse3T3-L1cells;(4) Using RT-PCR,Western blot to filter out the best silent MCHR1vector. The best vector will betransfected the mouse3T3-L1cells, MCH interventions, induced differentiation and oil red O staining photographed and measured A510, to observe the effect on fatsynthesis.(5) Using RT-PCR, Western blot methods to detect fat cell transcriptionfactor peroxisome proliferator-activated receptor (PPARγ), CAAT enhancer bindingprotein α (C/EBPα) and adipokines fatty acid binding protein (aP2) mRNA andprotein expression, to clarify the role and mechanism of silencing of mouse3T3-L1MCHR1in fat synthesis.Results:1、Mouse3T3-L1cells growth in good condition and induced to differentiateinto mature fat cells, amount of lipid droplets increased gradually over time.Therelationship between fat droplets and time is linearity.2、The vectors of sh-MCHR1was successfully constructed. Three sh-MCHR1positive interference and a negative interference vectors detected by DNA sequencingwas completely correct. The four vectors named sh-MCHR1-1、sh-MCHR1-2、sh-MCHR1-3and sh-MCHR1-0.3、Established a plasmid method transfection of mouse3T3-L1cells, thetransfection efficiency reach the highest after72hours，between50%to60%.4、We transfected four vectors for the3T3-L1cells. After72hours, The resultsof RT-PCR suggested that there was no statistical differences between sh-MCHR1-0control and normal control, and the relative expression quantity of MCHR1ofexperimental groups sh-MCHR1-1、sh-MCHR1-2、sh-MCHR1-3were0.13±0.03、0.60±0.05、0.65±0.04，decrease significantly(P<0.05). The results of Western blotsuggested that there was no statistical differences between sh-MCHR1-0control andnormal control, and the expression level of MCHR1of experimental groupssh-MCHR1-1、sh-MCHR1-2、sh-MCHR1-3were0.15±0.03、0.61±0.02、0.64±0.04,decrease significantly(P<0.05). sh-MCHR1-1is the best vector for silencingMCHR1of the mouse3T3-L1cells.The sh-MCHR1-1transfected mouse3T3-L1cells, the mouse3T3-L1cells werebe intervened with1000nmol/L MCH and induced differentiation, after10、12d theRed Oil O A510were0.46±0.02and0.54±0.03, significantly decreased compared with the control group, the difference was statistically significant(P<0.05).5、 The sh-MCHR1-1transfected mouse3T3-L1cells,1000nmol/L MCHintervene and induce differentiation. The relative expression quantity of PPARγmRNA of the groups6、8、10、12d were0.36±0.02、0.45±0.04、0.58±0.04、0.63±0.02,significantly decreased compared with the control group, the difference wasstatistically significant (P<0.05); The relative expression quantity of C/EBPα mRNAof the groups8、10、12d were0.34±0.03、0.42±0.03、0.51±0.01, significantlydecreased compared with the control group, the difference was statistically significant(P<0.05); The relative expression quantity of aP2mRNA of the groups8、10、12dwere0.39±0.02、0.42±0.01、0.48±0.01, significantly decreased compared with thecontrol group, the difference was statistically significant (P<0.05); The relativeexpressionquantityofPPARγ proteinof the groups8、12d were0.46±0.03、0.61±0.04,significantly decreased compared with the control group, the difference wasstatistically significant (P<0.05); The relative expression quantity of C/EBPα proteinof the groups8、12d were0.51±0.03、0.68±0.02, significantly decreased comparedwith the control group, the difference was statistically significant (P<0.05); Therelative expression quantity of aP2protein of the groups8、12d were0.54±0.01、0.68±0.02, significantly decreased compared with the control group, the differencewas statistically significant (P<0.05).Conclusions：Silence the MCHR1of mouse3T3-L1cells could to slow down the process ofthe fat synthesis which be intervened by MCH and induced differentiation.Mechanisms may be weakened by MCH-induced ERK1/2signaling, reducing of theexpression of PPARγ and C/EBPα activation, reduce the fat factors expression suchas aP2, eventually lead to a reduction in the fat synthesis. This research provides anew way of thinking for the obese gene therapy.