| VDR is a ligand-activated transcription factor belonging to the nuclear receptor family,which mainly regulates biological functions such as calcium and phosphate metabolism,bone development,lipid metabolism,cell proliferation and differentiation,nervous system development,immune regulation,anti-tumor and reproductive regulation through the combination with VitD3.In the early stage of our laboratory,VDR knockout mice were constructed,accompany with the decrease of reproductive ability and the fat synthesis.Based on this research,Illumina Hiseq platform was used to perform transcriptome sequencing analysis on 6-month-old and 12-month-old VDR-/-and WT mouse testis,with a view to screening genes related to lipid metabolism.After obtaining the lipid metabolism gene SCD-1,tissue expression profiling of the SCD-1 gene was carried out with RT-q PCR technology,then the expression level and localization of SCD-1 in different types of testicular cells were analyzed using cellular immunofluorescence staining.Finally,RT-q PCR and Western blot technology were used to explore the intermodulation relationship according to the VDR and SCD-1's expression changes from the perspective of overexpression and interference with the two genes in mouse testicular stromal cells respectively.In this study,the genes of lipid metabolism regulated by VDR in mouse testis were obtained,and the relationship between VDR and SCD-1 was preliminarily analyzed.Research provides basic data.The results are coming after as follows:1.The Q30 value of transcriptome sequencing data quality is above 89%;the number of assembled bases is 3.5 × 109 bp;the coverage rates in Gene ID,UniProtKB,GO,KEGG,and eggNOG databases are 93.93%,92.25%,94.12%,60.76% and 74.58% respectively,and the total number of reads compared to the exon region is more than 92%.2.VDR knockout significantly affected gene expression in mouse testis.There were 23 differentially expressed genes shared between six-month-old and twelve-month-old WT and VDR-/-mice.3.The analysis of GO and KEGG showed that the differentially expressed genes caused by VDR knockout participate in a variety of biological processes,among which the mostinvolved differential genes are metabolism,organism systems and human diseases.Among the top 20 metabolic pathways enriched the differentially expressed genes,and the most significant enrichment in the 6-month-old group is PPAR signaling pathway related to lipid metabolism,and the most significant enrichment in the 12-month-old group is Steroid hormone biosynthesis.4.The analysis found that the differential gene for lipid metabolism in the testis tissues of the 6-month-old group and the 12-month-old group is the SCD-1 gene involved in the PPAR signaling pathway.After the VDR gene knockout,the expression level of SCD-1decreased significantly.5.Analysis of mouse SCD-1 gene tissue expression profile found that the gene expressed in a variety of tissues,the highest expression is adipose tissue,the rest are liver,muscle,lung,skin,testis,kidney,heart,thymus,spleen and small intestine.After VDR knocked out,the expression of SCD-1 in heart,skin,fat,muscle,intestine and thymus all was significantly up-regulated,and the expression of SCD-1 in liver and testis decreased significantly.6.By analyzing the expression of SCD-1 in testicular stromal cells(TM3),spermatogonia cells(GC-1)and spermatogonial stem cells(C18-4),it was found that SCD-1was expressed the highest in TM3 cells,while the expression in C18-4 cells was relatively lower,and SCD-1 is mainly distributed in the cytoplasm.By studying the intermodulation relationship between VDR and SCD-1 in TM3 cells,it was found that VDR guarantees the normal expression of SCD-1 gene in TM3 cells,and there is a phenomenon of negative feedback regulation of VDR by SCD-1,and excessive expression of SCD-1 will inhibit VDR's expression. |
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