| The Wilms tumor1gene (WT1) encodes zinc finger proteins that function astumor suppressors and play important roles in the development of the genitourinarysystem and several other tissues. WT1has been reported to play a role in RNAprocessing, and it is most well-known and documented as a transcription factor. Thehuman Wilms tumor1gene was associated with a deletion at the11p13locus, whichis linked to WAGR syndrome that includes Wilms tumor predisposition, aniridia,genitourinary abnormalities and mental retardation. In mice, low levels of WT1expression are first detected at embryonic day9in the intermediate mesoderm andlater in the metanephric mesenchyme, from which the nephrons and stroma of adultkidneys are derived. Importantly, a kidney phenotype is attributable to abnormalitiesin the WT1-null mesenchyme as it fails to differentiate when co-cultured withwild-type ureteric bud cells. It is possible that WT1primarily plays a permissive rolethat allows the differentiation of mesenchymal cells upon receiving signals from theureteric bud. In this case, a failure to differentiate might result in the apoptosis ofthese renal precursors. Furthermore, the gonads are absent and the retinas areabnormal in WT1-null mice. The absence of WT1may lead to the upregulation ofapoptosis-inducing factors and/or the downregulation of survival factors.However, the role of the WT1gene in the development of the kidney, testis andother organs in pigs, which are an attractive, large animal model for the study ofcertain human diseases or the assessment of therapeutic applications of tissue/organtransplantation, is still unclear. To determine WT1’s role in the development ofporcine tissues and organs, PFF, PKF and ST cells were used as in vitro models. WT1was downregulated and upregulated in these cells, and cell apoptosis,apoptosis-related genes and development-related genes were investigated in this study.The main results were as follows:1. WT1was expressed in PFF, PKF and ST cells; Immunocytochemical stainingshowed that the expression of WT1is mainly expressed in the cytoplasm of PFF and PKF cells, in both the cytoplasm and nucleus of ST cells. This expression pattern maybe specific to different cell types and cell cycle stages.2. Here, we successfully constructed a pLV3-WT1shRNA vector and infectedPFF, PKF and ST cells at high efficiency. The pLV3-WT1shRNA dramaticallydecreased WT1expression at both the transcription and protein levels.3. We found that WT1downregulation led to significant cell apoptosis in bothPFF, PKF and ST cells. And when the examination time-point delayed, there wereincreased apoptosis ratio in both PFF, PKF and ST cells. Because apoptosis wassignificantly increased in WT1-downregulated ST cells, therefore, we examined theexpression of the anti-apoptotic gene Bcl-2and the proapoptotic gene Bax in thisstudy. We found that Bcl-2expression was decreased while Bax expression wasincreased in WT1-downregulated ST cells, which suggests that WT1facilitates thebalance between the anti-apoptotic protein Bcl-2and the proapoptotic protein Bax;through this mechanism, WT1may help to maintain the survival of ST cells.4. To determine whether the overexpression of WT1had an opposite effect on thein vitro maintenance of ST cells, we successfully constructed a WT1-overexpressionST cell line by stably transfecting ST cells with pIRES2-WT1-EGFP. The survival ofthese cells were not affected by WT1overexpression.5．Gdnf is positively regulated by WT1in ST cells but negatively regulated inPKF cells, which suggests that its role is tissue/organ specific. Sox-9was regulated byWT1similarly to Gdnf. Sf-1is highly expressed in PKF and ST cells, which isconsistent with its crucial role in the embryonic development of the adrenal glandsand gonads; no apparent effects were observed when WT1was overexpressed. |
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