| Alternative polyadenylation(APA) is an important precursor RNA processing mechanism in eukaryotes.The selection of Poly(A)site determines the length of the m RNA 3’UTR(3’untranslated region),and the longer 3’UTR contains more cis-acting elements that bind to miRNAs(micro RNAs)and/or RBPs(RNA binding proteins).Therefore,APA regulates gene expression by regulating the length of 3’UTR,thereby affecting the stability,translation and localization of m RNA,and finally leads them to have different biological functions.CXCL14(C-X-C Motif Chemokine Ligand 14)is a small signaling protein produced by cells in vivo,usually with a size of 8-10 k Da.Currently,the functions of CXCL14 gene in human and mouse fat synthesis,lipid metabolism and related diseases have been reported in a few literatures,but the post-transcriptional regulation of CXCL14 and its mechanism in adipocyte proliferation and differentiation remain unclear.SAPAS high-throughput sequencing technology was used to analyze the changes and characteristics of APA before and after differentiation of bovine preadipocytes(subcutaneous and intermuscular preadipocytes)in different parts;Based on the results of SAPAS sequencing,CXCL14 gene was focused on the post-transcriptional regulation of different APA isoforms,and the functions of bovine CXCL14 gene and its UTR-APA isoforms in the process of preadipocyte proliferation were preliminarily analyzed.The main results are as follows:Using SAPAS sequencing technology to sequence bovine intermuscular adipocytes and subcutaneous adipocytes before and after differentiation,a total of 29307 million initial sequences were obtained,of which 29258 million sequences had poly(A)sites,accounting for 99.8% of the total sequences.The analysis found that 52.3% of the sequences were located in the bovine genome database;the poly(A)site and APA site conversion events during the differentiation process were analyzed,and it was found that more than 590 genes had APA site conversion during the differentiation process,and369 genes had significantly shorter 3’UTR and 221 genes had significantly longer 3’UTR.The differential genes before and after preadipocyte differentiation were statistically analyzed,a total of 5527 differentially expressed genes were found before and after preadipocyte differentiation,among which the up-regulated genes accounted for 51%.According to the SAPAS high-throughput sequencing results and related literature reports,the CXCL14 gene was selected for research.Using 3’RACE technology,two UTR-APA isoforms of bovine CXCL14 gene were obtained with sizes of 125 bp and1158 bp,which were consistent with the SAPAS high-throughput sequencing results.Then,the analysis of dual-luciferase reporter gene experiments showed that different isoforms of bovine CXCL14 gene UTR-APA had different expression levels,and the luciferase activity of short 3’UTR was higher.Finally,the overexpression recombinants of different APA isoforms of CXCL14 were constructed and transfected into HEK293 T cells respectively.The qRT-PCR and Western blot analysis showed that the m RNA and protein expression levels of the short 3’UTR isoforms were included.Both were significantly higher than the long 3’UTR isoforms,which were consistent with the results of the dual-luciferase reporter gene assay.In order to further analyze the reasons for the differential expression of APA isoforms of different lengths of the CXCL14 gene,6 miRNAs that can bind to the long 3’UTR isoforms of bovine CXCL14 were predicted through the miRNAs prediction analysis website: miR-17-5p,miR-150,miR-217-5p,miR-671-5p,miR-340-5p,miR-874-3p.Through dual-luciferase reporter gene assay,qRT-PCR and Western blot analysis,the results all showed that miR-17-5p,miR-150,miR-217-5p down-regulated the expression level of bovine CXCL14 long 3’UTR isoform,while short 3’UTR isoform of CXCL14 gene escaped the inhibition of miR-17-5p,miR-150,miR-217-5p due to the lack of their binding sites,and the expression level did not change significantly.To preliminarily study the effect of CXCL14 gene on adipocyte proliferation,CXCL14 was overexpressed in the preadipocyte model 3T3-L1.Through the analysis of CCK-8 cell proliferation experiments,the results showed that compared with the negative control(NC)group,the absorbance value of the overexpression group was significantly increased.The qRT-PCR analysis showed that compared with NC,the expression of cell proliferation marker genes Cyclin E、cullin3 and Cyclin D1 in the overexpression group were significantly up-regulated.To further explore the effect of the long and short 3’UTR isoforms of CXCL14 gene on the proliferation of preadipocytes,p CMV-Flag,p CMV-CXCL14 S and p CMV-CXCL14 L were transfected into 3T3-L1 preadipocytes,respectively.Both CCK-8 and qRT-PCR results indicated that p CMV-CXCL14 S had a significant effect on promoting the proliferation of preadipocytes than p CMV-CXCL14 L.In conclusion,this study used SAPAS high-throughput sequencing technology to analyze the changes of APA and the characteristics of APA during preadipocyte differentiation at the omics level.Focused on differential post-transcriptional regulation of APA isoforms of bovine CXCL14 gene: CXCL14 produces two UTR-APAs through APA,and it was found that the short APA isoform of the CXCL14 gene has a higher expression level.Further analysis found that the short 3’UTR isoforms were upregulated by escaping the inhibition of miR-17-5p,miR-150,and miR-217-5p.Meanwhile,overexpression of CXCL14 can promote the proliferation of 3T3-L1 preadipocytes,and the short 3’UTR isoform had a more significant effect on the proliferation of preadipocytes than the long3’UTR isoform.These results provide a theoretical basis for further research on the mechanism of bovine CXCL14 gene in the process of proliferation and differentiation. |
