Molecular Mechanism Of MiR-26a-5p Regulating VEGFA Expression In Chicken Follicular Granulosa Cells

Follicular development in chickens is a complex and highly coordinated physiological process,and the proliferation and decline of the vascular system is accompanied by the entire growth process of the follicles.As the most abundant and powerful angiogenic factor in ovarian tissue,vascular endothelial growth factor(VEGF)promotes angiogenesis and improves blood vessel permeability,and plays an important role in maintaining ovarian function and follicular development.We found that miR-26a-5p was significantly expressed in chicken's ovarian tissues via high-throughput sequencing in our earlier study,and overexpression of miR-26a-5p in chicken-grade follicular membrane cells could screen out many differentially expressed genes related to vascular development.It was speculated that miR-26a-5p may be involved in the regulation of chicken follicular blood vessel development.This study analyzed the molecular mechanism of miR-26a-5p regulating VEGFA expression in chicken-grade follicular granulosa cells.The results are as follows:1?We revealed the genes regulated of miR-26a-5p in chicken follicle granule cells by transcriptome sequencing.Overexpression of miR-26a-5p in granulosa cells of chicken grade follicles and sequencing of the transcriptome.The without over expression of miR-26a-5p in granulosa cells was used as a control.686 differentially expressed genes were screened from grade follicle granulosa cells over-expressing miR-26a-5p,of which 326 were significantly up-regulated and 360 were significantly down-regulated.Bioinformatics analysis found that differentially expressed genes were enriched in apelin signaling pathway,TGF-? signaling pathway,vascular smooth muscle contraction and other signal pathways related to follicular development and vascular development.The expression changes of 14 DEGs including SEMA3 A,TLR-2,VEGFA,GSK-3B,FZD1,Wnt4,Wnt5 a and CTNNB1 were verified by qRT-PCR.VEGFA was a vital factor involved in vascular development and played an important regulatory role in the development and selection of follicular blood vessels.Therefore,VEGFA was selected as a candidate gene to study the development of blood vessels.2?miR-26a-5p targeted NLK to down-regulate VEGFA.We have demonstrated that miR-26a-5p could bind to the 3'UTR of the NLK gene in our previous research.In this study,overexpression of miR-26a-5p in GCs,it was found that NLK was significantly down-regulated in mRNA and protein levels by qRT-PCR and Western Blot,and then validated the targeted regulatory relationship between miR-26a-5p and VEGFA in depth.Transfecting miR-26a-5p mimic,inhibitor and NLK siRNA into GCs,the results showed that the high expression of miR-26a-5p could significantly inhibit the expression of VEGFA at the mRNA and protein levels,while downexpression of miR-26a-5p can significantly increase the expression of VEGFA at the mRNA and protein levels.After interfered with the expression of its target gene NLK,VEGFA was significantly down-regulated in mRNA and protein levels,which was consistent with the overexpression of miR-26a-5p.Those results indicated that miR-26a-5p could down-regulate VEGFA by targeting NLK.3?miR-26a-5p targeted NLK regulates VEGFA by down-regulating ?-catenin which was an important gene of Wnt/?-catenin classic signaling pathway.Transfection of miR-26a-5p mimic,inhibitor and NLK siRNA to GCs to detect the expression of ?-catenin in mRNA and protein levels.It was found that the overexpression of miR-26a-5p positive regulated the expression of ?-catenin,and the inhibition of miR-26a-5p significantly reduced the ?-catenin.However,?-catenin was significantly up-regulated when NLK was interfered,which was consistent with the result of overexpression of miR-26a-5p,and accompanied by negatively regulated the expression of VEGFA.The ?-catenin overexpression vector was constructed and transfected into GCs to verify the expression of VEGFA at the mRNA and protein levels.The results showed that with the increase of ?-catenin expression,the expression of VEGFA decreased significantly.4?Regulation of VEGFA by miR-26a-5p and Wnt.Differentially expressed genes of Wnt-related were also screened in transcriptome sequencing results,so we analyzed the effect of miR-26a-5p on Wnt related genes.Overexpression of miR-26a-5p could significantly up-regulate the expression of Wnt5 a in GCs,but inhibition of miR-26a-5p was largely unaffected.miR-26a-5p could significantly up-regulate Wnt4,and inhibiting the expression ofmiR-26a-5p,Wnt4 was also significantly down-regulated.While when NLK was silenced,Wnt4 had an upward trend(P>0.05).The experimental results showed that miR-26a-5p could activate Wnt4,but not through the regulation of NLK.VEGFA was significantly up-regulated after overexpressing of Wnt4,and interference with Wnt4 would down-regulate VEGFA(P>0.05).The above results indicated that the expression of Wnt4 would affect the expression of VEGFA,that is,VEGFA might be affected by the classical Wnt pathway.In order to further verify the effect of the Wnt pathway on VEGFA,?-catenin,NLK,and VEGFA were significantly down-regulated after GCs were treated with IWR-1(Wnt pathway inhibitor).The results indicated that the expression of VEGFA was regulated by Wnt pathway.5?The effect of miR-26a-5p on the proliferation and apoptosis of chicken grade follicle granule cells.Overexpression of miR-26a-5p and interference with NLK in chicken GCs respectively to detect cell apoptosis,and the results showed that the count of apoptosis in GCs showed a downward trend.After over-expression of miR-26a-5p,the expression of apoptosis-inhibiting gene BCL-2 was significantly up-regulated,and the expression of pro-apoptotic gene Caspase-3 was significantly down-regulated.Combining with the results of proliferation experiments in our previous study,miR-26a-5p could promote the proliferation of GCs.