The Role Of PUF In The Proliferation Of Male Germ Cells And Prostate Cancer Cells

Recent studies have showed that RNA binding proteins(RBPs)could play important roles in development and differentiation.Pumilio-Fem3-binding factor(PUF)proteins represent a highly conserved family of RNA binding proteins that act as factor of post-transcriptional regulation.PUF proteins bind with high specificity to sequences in the 3'UTRs of target mRNAs through their PUMILIO homology domains,resulting in accelerated mRNA turnover and decreased translation.PUF proteins are widespread in eukaryotes,with homologs gene from yeast,worms,fruit flies to mice and human.In flies and worms,they are found to play predominant roles in germ cells development,such as proliferation of primordial germ cells,the self-renewal of germline stem cells(GSCs)and so on.There are two PUF family members,PUMILIOl(PUM1)and PUMILIO2(PUM2),exist in mammals including the man.Studies showed that mouse lacking either Pum1 or Pum2 remain fertile,yet double knockout of both genes led to embryonic lethality,making it difficult to analyze their roles in spermatogenesis in mice.We generated conditional knockout mutant mice via germ cell specific Cre(Vasa-Cre and Stra8-Cre),which could knockout Pum genes in specific time point of spermatogenesis.We found Vasa-Cre and Stra8-Cre induced double knockout mice of Puml and Pum2 showed significantly reduced testis size and male infertility.The testis weight of Vasa-Cre induced double knockout mice was lower than that of wild type mice as early as at postnatal day 6,implicating potential roles in early germ cell development.To understand the cause of infertility of Pum conditional knockout mutant mice,we,first of all,analyze their defect by histology.In the experimental group,a large number of germ cells in the testes were lost.Moreover,the number of elongating and elongated spermatids was significantly reduced.The phenotype of reduced germ cells could be observed in VDKO(Vasa-cre/+;Pum1F/-;Pum2-1-)mice as early as at postnatal day 12,and the staining of VASA,a marker of germ cells,further verified our results.Staining results showed that most of spermatocytes and round spermatids,labeled by two different germ cells marker BOULE and DAZL,were lost in the Pum double knockout mice.A large number of germ cells lost in the adult double mutant mouse testis may be caused by the reduced proliferation or increased apoptosis.We found that the number of spermatogonial stem cells(SSCs)was obviously reduced and many apoptotic cells were mainly spermatocytes.Our finding of Pum's roles in male germ cell proliferation and maintenance supports that PUF family's function in germ cell maintenance in one of the most ancient functions of this family.Given that Pum proteins have a conserved role in germ cell proliferation and that Pum are also expressed in many human tissues besides gonads,we hypothesised that Pum may also regulate cancer cell proliferation at posttranscriptional level.To determine the role of PUMl in tumors,we select prostate cancer as the study object since it is one of the leading causes of cancer death in Europe and the United States.We found that the expression of PUMl protein in prostate cancer was much higher than that in paracancerous tissue.Reduced PUMl protein in prostate cancer cells slowed down the cell proliferation and increased the apoptosis.Moreover,the formation of clones and tumors were significant reduced.In summary,PUMl plays an important role for the maintenance of male germ cells and the growth of prostate cancer cells,which provided new insights for the male infertility and the mechanism of tumor formation.