| Background and ObjectObesity is a medical condition in which excess body fat has accumulated to theextent that it may have a negative effect on health, leading to reduced life expectancyand/or increased health problems，which is a combination of genetic and environmentalfactors. Genome-wide association analysis confirmed KCTD15(Potassium channeltetramerisation domain containing15)gene mutation has close correlation with obesity.The latest research results shown that the KCTD15gene plays an important role at theearly stages of the adipocyte differentiation (3T3-L1cell lines). The knockdown ofKCTD15gene can inhibit3T3-L1precursor adipocyte differentiation. But the potentialmechanism of KCTD15regulation adipocyte cell differentiation is unclear. In addition,The recent clinical studies have shown that the KCTD15mRNA expression leveldecreased in white adipose tissue in overweight and obese patients compare to the whiteadipose tissue of patients with normal body weight.Therefore, our study using the the3T3-L1precursor adipocyte cell, focus on thefunction and mechanism of KCTD15induced3T3-L1precursor of adipocytedifferentiation. Our result will further clarify KCTD15gene functions, explore itsmechanism of obesity relevance, and provide theoretical basis for new treatment target.Methods1. To cultivate3T3-L1precursor adipocyte cells with cell culture.2. To knock down the KCTD15in3T3–L1precursor adipocyte cells with RNAinterference3. To detect the KCTD15gene expression level with RT-PCR and Western Blot4. To screen the differentially expressed genes with the gene microarray.5. GO enrichment and KEGG pathway analysis6. To verify differentially expressed genes mRNA expression levels with RT-PCR. Result1. Induced the3T3-L1precursor adipocyte cells.2. The RT--PCR and western blot experiments show that RNA interference(RNAi)suppressed the KCTD15gene mRNA level and the overall protein levels.3. Screening the833differentially expressed genes (including386genes up-regulated and453down-regulated genes)with gene microarray experiment.4. The bioinformatics analysis of differentially expressed genes of results:(1)according to the standard of biological processes GO analysis results show that theenrichment of the main functions of differentially expressed genes in the biologicalmembrane assembly, cytoskeleton assembly, chromosome assembly, cell adhesion,adipocyte differentiation and protein metabolism and the immune process;(2) accordingto the difference of gene encoding protein cell positioning GO analysis results show thatthe vast majority of differentially expressed genes encoding proteins in cells on theskeleton and cell membrane;(3) according to the molecular function of differentiallyexpressed genes GO analysis results show that the main enrichment of differentiallyexpressed genes to the combination of the cytoskeleton, adjust the nucleosidetriphosphatase activity, enzyme inhibitor.(4) the KEGG pathway analysis results showthat the main enrichment to differentially expressed genes dicarboxylic acid metabolismand galactose metabolism pathway.5. RT-PCR experiment results showed that ABCA7and CUX1genes expressionlevel increases, MTHFD1gene, gene VPS4B and MDH1gene expression down-regulation, consistent with the expression profile chip test results.Conclusion1. KCTD15gene function not fully relies on the classic adipose differentiationmechanism.2. The function of KCTD15is associated with the cytoskeleton and biofilm andaffect the cell energy metabolism process.3. KCTD15gene inhibited the adipocyte cells differentiation by affected lipidsynthesis and transport, DNA methylation, DNA synthesis and inhibiting andtranscription factors. |
