SMAD4 Interacts With SIVA1,USP9X To Regulate Porcine Follicular Granulosa Cell Apoptosis

SMAD4,an important member of the SMAD family and a nucleoprotein shuttle protein,plays a key role in mediating TGF-? pathway signaling and regulating target gene transcription,and participates in various cellular processes in vivo.Previous studies in our laboratory have confirmed that SMAD4 is an important regulator of porcine follicular atresia and granulosa cell function,which not only promotes granulosa cell proliferation,inhibits granulosa cell apoptosis,but also participates in the regulation of granulocyte steroid hormone synthesis.Further analysis revealed that SMAD4 regulates porcine ovarian granulosa cell function by regulating epigenetic pathways such as microRNAs(miRNAs)production and long non-coding RNAs(lncRNAs)expression.In this paper,we used a transcriptome data of SMAD4 knockdown in porcine ovarian granulosa cells to identify a new pig gene,SIVA1,which encodes a protein among ubiquitin-ligase enzymes.At the same time,it was found that the deubiquitinating enzyme USP9X is regulated by its substrate SMAD4.Therefore,the ubiquitin-ligase enzyme SIVA1 and deubiquitinating enzyme USP9X were used as the research object to analyze the mutual regulation mechanism between SMAD4 and SIVA 1,USP9X,and the mechanism of synergistic regulation of apoptosis of porcine follicular granulosa cells,based on the previous studies in our laboratory,we continued to explore the mechanism by which SMAD4 regulates apoptosis of porcine follicular granulosa cells through the epigenetic pathway of ubiquitination/deubiquitination.The main findings of this paper are as follows:1 SMAD4 interacts with ubiquitin-ligase enzymes SIVA1 to regulate apoptosis of porcine follicular granulosa cellsThis paper analyzed the transcriptome data of SMAD4 knockdown in pig ovarian granulosa cells obtained in the early stage of the laboratory,and found a new transcript that was significantly up-regulated in SMAD4 knockdown porcine follicular granulosa cells,qRT-PCR confirmed that SMAD4 is the suppressor of this new transcription.The full-length mRNA sequence of the new transcript(646 bp in length)was obtained by RACE technology,including a 101 bp 5'-UTR,a 379bp coding region sequence and a 166bp 3'-UTR.The homology alignment revealed that the nucleotide sequence of the coding region of the new transcript was 81.79%identical to the human SIVA1 gene,and the amino acid sequence of the encoded protein was 77.14%respectively,so name it pig SIVA1.Tissue expression profiling revealed that the SIVA1 gene is present in tissues such as porcine ovary,muscle,brain,liver,stomach,heart muscle,large intestine,and spleen,and is highly expressed in ovarian tissues..Fluorescence in situ hybridization and immunohistochemical analysis revealed that SIVA1 was expressed in the porcine follicular wall layer and free granulosa cells at different developmental stages,especially in the atresia follicles.qRT-PCR and Western blot confirmed that the expression levels of SIVA1 mRNA and protein in pig atresia follicles were significantly healthy follicles.FACS analysis showed that the apoptosis rate of porcine follicular granulosa cells decreased after knockdown of SIVA1,while the apoptosis rate of granulosa cells increased after overexpression of SIVA1.These results indicate that the novel gene SIVA1 is a promoter of porcine follicular atresia and granulosa cell apoptosis.In order to analyze the molecular mechanism of SMAD4 inhibition of SIVA1 gene transcription in porcine follicular granulosa cells,the 5' regulatory region of porcine SIVA1 gene was cloned and its core promoter region was successfully identified.Four SMAD binding elements(SBE)were found in the core promoter region of porcine SIVA1 gene.The luciferase reporter gene system and chromatin immunoprecipitation technology(ChIP)confirmed that SMAD4 can bind to SBE in the core promoter region of porcine SIVA1 gene to inhibit the promoter region activity of the SIVA1 gene.FACS analysis and Western blot further confirmed that SMAD4 can inhibit the expression of SIVA1 protein and SIVA1 mediated granulosa cell apoptosis in porcine follicular granulosa cells.After overexpression and knockdown of SIVA1,the level of SMAD4 protein in porcine follicular granulosa cells was significantly down-regulated or up-regulated,indicating that SIVA1 can feedback-regulate SMAD4.Chromatin immunoprecipitation(Co-IP)shows that overexpression of SIVA1 can increase the level of SMAD4 protein ubiquitination in porcine follicular granulosa cells.Conversely,knockdown of SIVA1 can reduce SMAD4 protein level in porcine follicular granulosa cells.These results indicate that SIVA1 regulates SMAD4 protein expression through ubiquitination.The above results indicate that SMAD4 and SIVA1 can form a negative feedback regulatory loop,SMAD4-SIVA1 loop,in porcine follicular granulosa cells.FACS results showed that the SMAD4-SIVA1 loop regulates apoptosis of porcine follicular granulosa cells by mutual regulation.To further explore the molecular mechanism of SMAD4-SIVA1 loop regulation of apoptosis in porcine follicular granulosa cells,we analyzed the downstream factors of SMAD4 and SIVA1,and found that the apoptosis marker gene BCL2 is a common downstream factor,which was down-regulated during porcine follicle atresia.So BCL2 was selected as the target gene(protein)for further study.Western blot confirmed overexpression or knockdown of SMAD4 and SIVA1 can change BCL2 protein levels in porcine follicular granulosa cells showed significantly.ChIP analysis showed that SMAD4 can directly bind to SBE in the porcine BCL2 gene promoter region and promote BCL2 gene transcription.Co-IP analysis showed that overexpression of SIVA1 up-regulated the ubiquitination level of BCL2 protein in porcine follicular granulosa cells,while knockdown of SIVA1 down-regulated the ubiquitination level of BCL2 protein.In addition,the SMAD4-SIVA1 loop regulates the expression of BCL2 in porcine follicular granulosa cells by mutual regulation,and regulates apoptosis of porcine follicular granule cells by BCL2.These results indicate that BCL2 is a common target of the SMAD4-SIVA1 loop,and the SMAD4-SIVA1 loop regulates apoptosis of porcine follicular granulosa cells via BCL2.2 SMAD4 and deubiquitinating enzyme USP9X interact to regulate apoptosis of porcine follicular granulosa cellsPrevious studies in our laboratory found that deubiquitinating enzyme USP9X promoted SMAD4 protein expression in porcine follicular granulosa cells,but the mechanism is still unclear.In this paper,Co-IP analysis showed that the level of SMAD4 protein ubiquitination in porcine follicular granulosa cells was increased after knockdown of USP9X gene.FACS analysis showed that overexpression of SMAD4 inhibited USP9X knockdown-induced apoptosis of porcine ovarian granulosa cells,indicating that USP9X promote deubiquitination of SMAD4 protein to increase its protein level,which in turn affects apoptosis of porcine follicular granulosa cells.In addition,the mRNA and protein levels of endogenous USP9X gene in porcine follicular granulosa cells were significantly up-regulated or down-regulated after SMAD4 overexpression or knockdown,indicating that SMAD4 and USP9X can form a new regulatory loop,SMAD4-USP9X loop,in porcine follicular granulosa cells.Further analysis revealed that SMAD4 can directly act as a transcription factor to bind to SBE on the promoter region of the USP9X gene to regulate the transcriptional activity of the USP9X gene.FACS analysis showed that the specific knockdown of USP9X can effectively reduce the inhibited apoptosis rate of porcine follicular granulosa cells by SMAD4 overexpression,indicating that deubiquitinating enzyme USP9X can mediate the regulation of SMAD4 on apoptosis of porcine follicular granulosa cells.Our previous studies have demonstrated that LRH-1 regulates apoptosis in porcinefollicular granulosa cells via CYP19A1.It was further found that LRH-1 binds directly to the promoter region of the CYP19A1 gene and forms the LRH-1/CYP19A1 axis in porcine follicular granulosa cells.Bioinformatics analysis revealed that the LRH-1 gene promoter region contains SBE,and the luciferase reporter gene system,chromatin immunoprecipitation(ChIP),qRT-PCR and western blot confirmed that SMAD4 can interact with the LRH-1 gene promoter region.The binding of SBEs up-regulates the transcriptional activity of LRH-1 gene and promotes the expression of LRH-1 in porcine ovarian granulosa cells.At the same time,knockdown of USP9X inhibited the LRH-1/CYP19A1 axis in porcine follicular granulosa cells,and Co-IP found elevated levels of LRH-1 protein ubiquitination,indicating that USP9X activates the LRH-1/CYP19A1 axis by promoting deubiquitination of the LRH-1 protein.SMAD4 and deubiquitinating enzyme USP9X can regulate the LRH-1/CYP19A1 axis through each other,and both can regulate the apoptosis of porcine follicular granulosa cells through LRH-1.These results indicate that the LRH-1/CYP19A1 axis is a common target for the SMAD4-USP9X loop,and the SMAD4-USP9X loop regulates apoptosis in porcine follicular granulosa cells via the LRH-1/CYP19A1 axis.miRNAs can be used as an important epiregulatory factor,which can play an important role in porcine follicular atresia and granulosa cell apoptosis.In this paper,214 and 320 potential miRNAs regulating USP9X gene and SMAD4 were predicted by bioinformatics methods,respectively.Among them,14 and 16 were differentially expressed in porcine follicle atresia,and 11 were common miRNAs.Nine miRNAs up-regulated during porcine follicle atresia were found by luciferase activity assay of which miR-26b/27b/30c/195/320a targets both SMAD4 and USP9X.Functional analysis revealed that miR-26b can target inhibit the SMAD4-USP9X loop in porcine follicular granulosa cells and its mediated granulosa cell function(apoptosis).Further analysis revealed that miR-26b inhibits the LRH-1/CYP19A1 axis in porcine follicular granulosa cells by target inhibiting the SMAD4-USP9X loop.These results indicate that SMAD4 and USP9X share a common regulatory miRNAs in porcine follicular granulosa cells that promote apoptosis in porcine follicular granulosa cells by targeting inhibition of the SMAD4-USP9X loop and the LRH-1/CYP19A1 axis.In summary,this paper identified a new gene SIVA1 related to porcine follicular atresia and granulosa cell apoptosis,and found that SMAD4 can form a negative feedback loop with ubiquitin-ligase enzyme SIVA1,and regulate the porcine follicular granule cells with the apoptosis marker gene BCL2.At the same time,this study also confirmed that SMAD4 can form a positive feedback loop with deubiquitinating enzyme USP9X,and regulate the apoptosis mechanism of porcine follicular granulosa cells through LRH-1/CYP19A1 axis.