Effects Of QKI On The Proliferation And Secretion Factors Of Pig Sertoli Cells

Sertoli Cells(SC)are epithelial cells that initiate sexual function development and maintain spermatogenesis.It is in direct contact with spermatogenic cells and provides nutrients for spermatogenic cells.It also secretes a variety of cytokines and plays an important role in regulating sperm cell differentiation and maintaining spermatogenesis.During spermatogenesis,the SC can phagocytose residual cytoplasm and secrete testicular fluid.As a terminally differentiated cell,SC stops proliferating after sexual maturation.The number of spermatogenic cells that SC nourishment is limited,so the proliferation of SC directly affects the testicular size and spermatogenesis of adult animals.After sexual maturity,SC maintains the homeostasis of spermatozoa,which determines the quality of sperm in animals.Therefore,the proliferation and secretion of SC directly affects the fertility of boars.The RNA-binding protein QKI is a member of the STAR family and has an RNA signal transduction activation function.It is mainly involved in the regulation of various tissue development and cell proliferation and differentiation by specifically binding Pre-mRNA and mature mRNA containing the QRE original(QRE sequence: ACUAAY-N(1-20)-UAAY,Y=U/C).Therefore,this study explores the effect of QKI on the proliferation and secretion of SC by exploring the target genes of QKI in immature SC,and the results provide a basis for the regulation of cell proliferation and function.1.Isolation,Purification and Identification of Marker Proteins of Immature Sertoli CellsFirst,cells were obtained from 21-day-old piglet testes by two-step enzymatic digestion,and the cells were purified by differential adherence.Through morphological observation,the shape of the cells is elliptical or circular,with multiple protrusions.Two satellite nucleosomes around the nucleus can be clearly observed under the microscope at 200 times and 400 times.The purity of the SC was determined to be close to 100% by calculating the staining ratio of the immunofluorescent marker SC marker proteins GATA-4,WT-1 and FASL in the cells.The isolated and purified cells were finally identified as porcine testicular immature SC by the immature SC marker protein KRT-18.2.QKI Target Genes Obtained by RNA Sequence Analysis and RIP-seq VerificationSubsequently,we obtained the 3'UTR of all mRNAs in the pig genome through the Ensemble website,and a total of 5,445 genes contained QRE elements.The complexes of QKI and mRNA were pulled by QKI antibody using the RIP-seq assay.The mRNA after purification was subjected to transcriptome sequencing,and there were 3551 genes differentially expressing more than 2 times.The results of intersection analysis showed that there were 1379 mRNAs containing QRE elements in the transcriptome sequencing,including 7 genes validated in previous studies.Then 10 genes were randomly selected for RT-PCR,which was consistent with the sequencing results of the transcriptome.KEGG and Go analysis showed that QKI-binding mRNA was more abundant in cell cycle pathway,which implied that QKI might affect the cell cycle and proliferation of SC.3.Effect of QKI on proliferation and secretion factors of Sertoli cellsThe overexpression vector and interference vector were transfected into SC respectively,and the content of PCNA mRNA in the overexpression group increased significantly(P<0.01).The content of PCNA in silence group decreased significantly(P<0.01).To detect the effect of QKI on cell cycle,The results showed that when QKI was overexpressed,S phase of SC in pBI-CMV3-QKI group was significantly higher than that in control group(P<0.01),while S phase of SC in shQKI group was significantly lower than that in shNC(P<0.01).Compared with the control group,the cell viability of pBI-CMV3-QKI group increased significantly at 72h(P < 0.01).After QKI interference,the cell viability of shQKI group was significantly lower than that of control group at 72h(P < 0.01).ELISA kit was used to determine the effects of QKI on the secretion of cytokines ABP,EGF,GDNF and IGF-1.When QKI was overexpressed,the secretion of EGF,GDNF and IGF-1 in pBI-CMV3-QKI group was significantly higher than that in pBI-CMV3 group,but there was no significant difference in the secretion of ABP.Conclusion: QKI accelerates cell cycle progression and promotes cell proliferation by binding mRNA of cell cycle related genes in immature porcine SC.At the same time,it has the function of promoting the secretion of GDNF,IGF-1 and EGF.The molecular mechanism of its regulation needs to be further studied.