Construction Of MCHR2 SiRNA Adenovirus Vector,Cell Lines Stably Expressing MCHR2 And Proteomics Study Of MCHR2

Obesity,a nutritional metabolic disease,has become a global health problem and is a significant risk factor for developing many serious diseases,including cardio-and cerebro-vascular diseases,diabetes, hypertension,dyslipidemias and some cancers.In recent years,it has been well established that obesity is associated with the neuroendocrine control of the hypothalamus.The identification of the hypothalamic orexigenic neuropeptide melanin-concentrating hormone(MCH) and its two receptors (MCHR1 and MCHR2) provides new targets for treatment of obesity. Major advances have been made in identifying the relevance of MCH and MCHR1 to obesity and energy homeostasis by transgenic animals and gene knockout animals.However,the function of MCHR2 and its relevance to obesity is still indistinct.In this study,on one hand,we construct the recombinant adenovirus vector for siRNA targeting human melanin-concentrating hormone receptor 2(MCHR2) gene in order to study the function of MCHR2 in the animal models;On the other hand,we construct two cell lines stably expressing human MCHR2——SHG-44-MCHR2 and SH-SY5Y-MCHR2,and use these cell lines to do the proteomics analysis of MCHR2 in order to evaluate the function and its mechanism of MCHR2 from the whole protein level of cells.This study includes three parts:1.Construction and identification of human MCHR2 gene siRNA recombinant adenovirus vector.Methods:The siRNA sequence targeting MCHR2 gene was synthesized and cloned into the shuttle plasmid p SES-HUS to form the vector p SES-HUS-MCHR2siRNA.It was homogenous recombinated with adenovirus backbone plasmid pAdeasy-1 in E coli BJ5183.Then the recombinant adenovirus vector was transfected into HEK293 cells and adenovirus was packaged and amplified.The CHO cells stably expressing MCHR2 gene named CHO-MCHR2 were infected by the adenovirus.The target gene was detected by RT-PCR and Western Blot.Results:The p SES-HUS-MCHR2siRNA was digested by Kpn I and EcoR V into one fragment,about 8500kb and the sequence of MCHR2 siRNA was correct.The results show that we have successfully constructed p SES-HUS-MCHR2siRNA.Two fragments(about 4.5kb and 30kb) were got after digested by Pacâ… ,the restriction analysis confirmed that correct recombinant adinoviral plasmid was constructed.The fluorescence was observed in 293 cells after transfection.After 48h of treatment by Adeasy-MCHR2siRNA(50pfu/cell),the expression of MCHR2 in CHO-MCHR2 cells was significantly inhibited detected by RT-PCR and Western Blot(compared with the group of Adeasy-SES-HUS).Conclusion: The recombinant adenovirus Adeasy-MCHR2siRNA has been successfully constructed and significantly inhibits the expression of MCHR2 in CHO-MCHR2 cells,which can provide a valid tool to study the function of MCHR2 in the animal models.2.Establishment of SHG-44-MCHR2 and SH-SY5Y-MCHR2 cell lines expressing MCHR2 stably.Methods:â‘ The full-length MCHR2 cDNA fragment was amplified from the human fetal brain cDNA library by PCR. It was cloned into pcDNA3.1(+) and the eukaryotic expressing vector pcDNA3.1(+) MCHR2 was constructed.Then the vector was transduced into SHG-44 cells by Lipofectamine^TM.After screening culture by G418, SHG-44 cells expressing MCHR2 stably named SHG-44-MCHR2 were established,and the transcription and expression of MCHR2 was identified by RT-PCR,Western blot and immunofluorescence.The flow of intracellular Ca²⁺ was detected by Fura-2 Ratio method.â‘¡Human MCHR2 cDNA amplified from the human fetal brain cDNA library by PCR was cloned into pEGFPN1 and the eukaryotic expressing vector pEGFPN1-MCHR2 was constructed.Then the vector was transfected into human neuroblastoma cells SH-SY5Y by Lipofectamine^TM.After screening culture by G418,the SH-SY5Y cells expressing MCHR2 stably named SH-SY5Y-MCHR2 were established.The transcription and translation of MCHR2 were identified by RT-PCR and Western blot.The location of MCHR2/EGFP was observed by laser confocal microscopy.Results: â‘ The full-length of MCHR2 cDNA fragment was amplified and the eukaryotic expression vector pcDNA3.1(+)/MCHR2 was constructed successfully.The expression of MCHR2 was detected by RT-PCR,Western blot successfully and the protein exactly localized on the cytomembrane in the SHG-44-MCHR2 cells detected by immunofluorescence.MCH induced a clear and transient increase of intracellular calcium,and this procedure was completed within 30～50s in SHG-44-MCHR2 cells.â‘¡The eukaryotic expression vector pEGFPN1-MCHR2 was constructed successfully.The expression of MCHR2 was detected by RT-PCR and Western blot successfully and the fusion protein MCHR2/EGFP exactly localized on the cytomembrane in SH-SY5Y-MCHR2 cells.Conclusion:SHG-44-MCHR2 and SH-SY5Y-MCHR2 cell lines have been established successfully,which provides solid experimental foundation for further proteomics study on the function of MCHR2.3.Proteomics analysis of human MCHR2 gene function.Methods: Total proteins of MCH treated SHG-44-MCHR2 and SHG-44-mock cells, SH-SY5Y-MCHR2 and SH-SY5Y-mock cells were extracted and separated by two dimensional gel electrophoresis(2-DE).Conditions(gel strip: pH3-10NL/pH4-7;protein content:200/300μg) were adjusted.The differentially expressed proteins between SH-SY5Y-MCHR2 and SH-SY5Y-mock cells treated with MCH(gel strip:pH3-10NL;protein content:200μg) were analyzed with PDQuest software,and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALD1-TOF-MS) and Mascot database searching.The mRNA alterations of INSIG2,ACOT8,IDH3A,PCK1 and PFKFB4 were confirmed by semi-quantitative RT-PCR,IDH3A and PFKFB4 proteins were confirmed by Western blot.Results:2-DE patterns of SH-SY5Y-MCHR2 and SH-SY5Y-mock cells treated with MCH with high-resolution and reproducibility were obtained.A total of 43 differentially expressed proteins were visualized by 2-DE and silver stain.Of these,34 proteins were identified via MALDI-TOF-MS analysis.21 were up-regulated(eg, IDH3A,PCK1,PFKFB4,ACSM5) and 13 were down-regulated(eg, INSIG2,ACOT8,RAB2B).At the mRNA level,IDH3A,PCK1,PFKFB4 were up-regulated and INSIG2,ACOT8 were down-regulated in MCH treated SH-SY5Y-MCHR2 cells;IDH3A and PFKFB4 proteins were up-regulated in MCH treated SH-SY5Y-MCHR2 cells.Conclusion: Several proteins which participate in some signal pathways of lipid metabolism,glycometabolism,energy metabolism and protein transportation,etc,express differentially between SH-SY5Y-MCHR2 and SH-SY5Y-mock cells both treated with MCH.All of these implicate that MCHR2 may participate in the regulation of energy balance and may have some relationship with obesity and diabetes.