Screening And Preliminary Identification On Differentially Expressed Long Non-coding RNA In Mice Cold-exposure Adipose Tissue

Background: Adipose tissue has mainly two different types: white adipose tissue(WAT) that stores energy and brown adipose tissue(BAT) containing abundant mitochondria with uncoupling protein 1(UCP1) under the membrane which can consume excessive energy by mitochondrial oxidative phosphorylation uncoupling, promoting free fatty acid metabolism. Since 2009 adipose tissue had been found in adult body, brown fat has become a hot research.Unlike BAT,WAT is widely distributed, has very strong plasticity, under the cold stimulation, and get brown fat phenotype, which is called the brown fat activity thermogenesis( " brown "). Studies have shown that inguinal white adipose tissue than visceral white adipose tissue is more likely to occur "brown". So far the previous reports on the development of fat focused on the perspective of protein-coding genes and micro RNAs regulatory mechanism, however long noncoding RNA(long non-coding RNAs, lnc RNAs) role in the development process of fat is rarely reported. Therefore, fully understand the mechanism of fat development for the treatment of obesity and metabolic diseases has far-reaching significance.Method: Twenty-four 8-week-old C57BL/6 wild-type mice were randomly divided into two groups : the cold-exposure group(6℃) and the control group(28℃).Mice were placed under 6℃ and 28℃ environment and free feed for 10 days. And then obtained inguinal white adipose tissue(ing-WAT) and interscapular brown adipose tissue(i BAT) of two groups respectively. Extracted total RNA from them and construct 4 mixed pool samples which each mixed pool containing 2 samples, and then numbered BAT-H7, BAT-L3, WAT-MH1 and WAT-ML1. Screening multiple differences( Fold Change > 2 or a Fold Change < 0.5, FDR < 0.05) obviously long non-coding RNA by the transcriptome sequencing. Based on the differentially expressed gene, we applied the Gene Ontology(GO) analysis and Pathway analysis to discover the function and pathway enriched among the differentially expressed genes. Combining with bioinformatics report, further screening long noncoding RNA which may play an important role in the process of the fat development of i BAT and ing-WAT groups. Six randomly selected lnc RNAs(including four cross lnc RNAs)validated by real time fluorescence quantitative PCR, followed by expression profile in spleen, muscle, kidney, duodenum, liver, ing-WAT and i BAT from wild type mice.And then expression profiling of lnc RNA-E030011O05 Rik and lnc RNA-6030408B16 Rik in brown preadipocytes differentiation in 0 day,1 day,3 days,5 days.Results and Conclusion: Compared with the control group, the expression of UCP1 of ing-WAT and i BAT in cold stimulation significantly increased.Immunohistochemistry of ing-WAT and i BAT displayed that the signal of UCP1 in cold stimulation group is more stronger than the control group. These results showed that the adipose tissue “cold stimulating mold “was successful. Consistent with previous studies, it validates that cold stimulation can active BAT and promote WAT "browning." RNA-sequence analysis showed that there were 97 differentially expressed lnc RNAs in i BAT(6℃vs 28℃) and 325 differentially expressed lnc RNAs in ing-WAT(6℃vs 28℃), 28 lnc RNAs in intersection of two groups. The lnc RNAs in intersection mainly located in the 1,2,4,8, 9,10,12,15 and 18 chromosome. GO-Analysis and Pathway-Analysis of differentially expressed genes showed that these genes may participate in the regulation of lipid metabolism and transcription, especially on biosynthesis of unsaturated fatty acids and oxidative phosphorylation of signal transduction pathways. q RT-PCR results showed that the results of six lnc RNAs m RNA up and down expressing in accordance with the sequencing results, verify the validity of the sequencing results. And we predict that lnc RNAs may regulate adipose tissue development, needing further verification. Expression of above lnc RNAs in tissues showed that lnc RNAs have tissue-specific expressing pattern.The m RNA expression of lnc RNA-E030011O05 Rik presented up trend and lnc RNA-6030408B16 Rik up-down trend at 0,1,3,5days in preadipocytes differentiation.;We guess that lnc RNA-E030011O05 Rik and lnc RNA-6030408B16 Rik may regulate preadipocytes differentiation.