QKI-5 Suppress Porcine ST Cell Apoptosis Through Regulation WT1 And Caspase8

The sertoli cells are located in the basal part of seminiferous tubules and form a fine seminiferous tubules wall with various developmental states of spermatogenic cells.Sertoli cells provide nutrients for spermatogenic cells,initiate differentiation of spermatogonial stem cell,maintain spermatogenesis,and remove residual cytoplasm during phagocytosis of spermatids.The proliferation of sertoli cells determines testicular size and the quality & quantity of sperm after maturation.Moreover,the number of mature sertoli cells depends on the number of immature support cells.Thus,immature sertoli cells play important roles in animal testicular development and spermatogenesis.RNA-binding protein QKI is a highly conserved member of the STAR family.QKI mainly expresses three isomers named QKI-5,QKI-6 and QKI-7.QKI participate in cell proliferation,apoptosis and differentiation by binding to the QREs of target m RNAs.The aim of this study was to investigate the effect of QKI-5 on ST cell apoptosis and the post-transcriptional regulation of QKI-5 on WT1 and Caspase8.First,in order to clarify that there are QKI-5,QKI-6 and QKI-7 isoforms in pigs.Specific primers were designed using sequences of human QKI-5,QKI-6 and QKI-7,and PCR was performed using porcine c DNA as template.The results showed that three sequences could be successfully cloned in porcine tissue and has the specificity C terminal of QKI-5,QKI-6 and QKI-7,respectively.And QKI-5 have a typical nuclear localization signal(NLS).Subsequently,the effects of QKI-5 on ST cell apoptosis was studied.First,overexpression vector of QKI-5 was constructed and four interfering vectors(sh RNAs)were synthesized.The results showed that the number of cells when QKI-5 overexpression were significantly higher than that of the control group,however the number of cells was significantly decreased when QKI-5 was silenced.The results of flow cytometry showed that the apoptosis rate was 2.32±0.11% when QKI-5 overexpression,and the apoptosis rate was 25.64±0.68% when QKI-5 silence.It proved that QKI-5 had the function of inhibiting ST cell apoptosis.Then,in order to verify the molecular mechanism of QKI-5 inhibite apoptosis.Bioinformatics analysis results showed that a QRE(1009bp-1014 bp,ACTAAC)was in the 3’UTR of the Caspase8 and the QRE-1(2046bp-2052 bp,ACTAAC)and QRE-2(2211bp-2217 bp,ACTAAC)were in WT1 3’UTR.In order to verify whether QKI binds to 3’UTR of Caspase8 and WT1,we constructed the wild-type luciferase reporter gene Caspase8-3’UTR-WT and the mutant gene Caspase8-3’UTR-MUT;and WT1 Wild-type gene WT1-3’UTR-WT and three mutant genes WT1-3’UTR-MUT1,WT1-3’UTR-MUT2 and WT1-3’UTR-MUT3.Luciferase activity assay showed that QKI-5 binds to Caspase8-3’UTR-WT,However,QKI-5 binds to WT1’s QRE-1 only.Finally,QKI-5 was overexpressed and silenced in ST cells demonstrating the post-transcriptional regulation of QKI-5 on Caspase8 and WT1.The results showed that QKI-5 contribute to the stability of WT1 m RNA during the period of 0-2h to 6-8h after actinomycin D treatment and QKI-5 significantly promoted the expression of WT1 and its downstream gene Bcl-2.The results appear opposite when QKI-5 was silenced.QKI-5 accelerates the degradation of Caspase8 m RNA during the addition of actinomycin D 0-2h to 6-8h.QKI-5 also significantly inhibit the expression of Caspase8 and Caspase3 levels.The results appear opposite when QKI-5 was silenced.Conclusion: QKI-5 has the effect of inhibiting the apoptosis in ST cells.The molecular mechanism is that QKI-5 maintain WT1 m RNA stability,promote the expression of WT1 and Bcl-2;and accelerate the degradation of Caspase8 m RNA.significantly inhibit the expression of Caspase8 and Caspase3 by post-transcriptional regulation.