The Molecule Mechanism Of Mir-26B Regulates Apoptosis Of Porcine Ovarian Granulosa Cells By Targeting SMAD4

In female mammals, there about70%-99%folliculars became atresia, the current studies found that ovarian atresia essence was granulosa cell apoptosis. Studies have shown that the Smad4（SMAD family member4） is the only common mediated type Smad （Common-mediator Smad, Co-Smad） in the TGF-β/Smad signal path and BMP/Smad signaling pathway, it played an important role in mammalian ovarian granulosa cells proliferation, differentiation, follicular development and reproductive ability. Studies have found that cumulus cells exhibit abnormal especially granulosa cells abnormal manifest early luteal when we specific knockout mouse ovaries ovarian Smad4gene. In our experiment, we use the Erhualian pig as the research object, by cloning and sequencing technology to obtain the Erhualian pig Smad4gene coding sequence; at the same time we used bioinformatics correlation method to analysis Smad4gene sequence characteristics and protein structure; we detected the expression characteristics of Smad4gene expression in tissue characteristics and ovarian tissue cells by RT-PCR and immunohistochemistry; we through the real-time PCR and Western blot technology analysis the differences about Smad4gene and protein expression levels in ovarian tissue between Erhualian and commercial pig; RNAi technology has been used to analysis the effect of Smad4gene in porcine ovarian granulosa cell apoptosis; by constructing Smad4-3’UTR fluorescent luciferase reporter vector to detect the role of miR-26b targeting on the Smad4gene; used AnnexinV-PI staining combined with Flow Cytometry Technical to analysis miR-26b targeting Smad4to regulate the apoptosis of granulosa cells. Our study had provided a reference and theoretical basis for the molecular mechanism to reveals Erhualian high fecundity traits. The results of this paper as flowing: 1. The cloning and sequence analysis about Smad4gene in Erhualian pigWe obtained Smad4gene coding region sequence of Erhualian, the total length were1659bp, by cloning and sequencing. Found Smad4gene located on chromosome1,104431104486kb （GenBank serial number:NC₁01443.3） in pig. By homology analysis we found that Smad4gene of Erhualian nucleotide sequence was coincidence with other mammals more than86%. Smad4gene of Erhualian encodes a protein containing552amino acids, the molecular mass is60.488kD, coincidence with mammalian is84-98%. The prediction Erhualian Smad4protein Domain contains three domains, MH1domain, SAD domain and the MH2domain, they are conserved in human, bovine and other mammals. The predicted tertiary structure of Erhualian Smad4protein contains seven alpha helices,12beta folding and several random coils. We built a mammalian NJ phylogenetic tree Zebrafish as an outgroup, and found that the clustering results were concordance to the classical taxonomy anastomosis. The results show that Smad4gene is quite conservative in evolution and has a high homology with other mammalian species, so we presumed that Smad4gene play an important role on fecundity and granulosa cell apoptosis in Erhualian and other mammalian species.2. Smad4gene mRNA expression level in tissue and protein expression pattern in Erhualian ovarianWe detected Smad4mRNA expression in small intestine, liver, brain, heart, kidney, spleen, uterus and ovarian tissue of Erhualian pig by RT-PCR method found that Smad4gene mRNA was expressed in8Erhualian organization, show tha Smad4gen was extensive expression in pig. Immunohistochemical analysis of Smad4protein was expressed in every porcine follicular developmental stages and found it mainly distributed in the oocyte and the granulosa cells. The results of qRT-PCR technology in ovarian tissue of Erhualian show Smad4gene mRNA expression were extremely significantly higher than commercial pigs, extremely significant difference,（P<0.01）. Western blot showing tha Smad4gen protein expression were extremely significantly high than commercial pigs, significant difference （P<0.01）3. The fuction of Smad4in porcine follicular granulosa cell apoptosisSmad4-siRNA transfected porcine ovarian granulosa cells, after48h, using qRT-PCR to detect Smad4mRNA expression, found Smad4-siRNA can effectively inhibit Smad4gene expression and mRNA levels decreased72.44percent, extremely significant difference,（P<0.01）. After72h detected Smad4protein levels it decreased by41.67%, extremely significant difference,（P<0.01）, illustrating that Smad4-siRNA could inhibit granulosa cell Smad4gene expression in pig, effectually. The apoptosis related genes Bcl-2gene mRNA expression levels were significantly lower significant difference （P<0.05）, but Bax gene expression levels did not change significantly. Detected the apoptosis rate significant increase, by Flow Cytometry. The results show an important role in the regulation of Smad4gene in pig granulosa cell apoptosis.4. Samd4gene was a miR-26b target gene in pigCombined with Bioinformatics website forecast and our research group miRNA microarray detection of porcine follicular atresia process miRNA expression profiling in early days, found that miR-26b is one of candidate regulation miRNAs on the Smad4gene. miR-26b mimics, mimics NC and Smad4gene3’UTR luciferase reporter gene vectors were co-transfected into Hela cells, to detected the luciferase activity after transfected24hours, found that transfected with miR-26b mimics group reporter gene activity was significantly lower than the control group （mimics NC group）（P<0.01）, show that miR-26b could extremely significant inhibited the activity of the reporter gene, it’s a strong proof to prove Smad4is a target gene of miR-26b.5miR-26b targeting pig smad4gene to regulated granulosa cells apoptosisIsolated culture of pig granulosa cells, when the cells was a good state using LipofectamineTM RNAiMAX transfected miR-26b mimics and negative control into porcine granulosa cells, detected the Smad4gene mRNA expression after48hours. Found smad4transcriptional level decrease extremely significantly,（P<0.01）, granulosa cells apoptosis-related genes Bcl-2mRNA expression levels were significantly reduced,（P<0.05）, but the Bax gene expression levels did not change significantly. After72h Smad4protein expression levels significantly decreased,（P<0.01）, about80.38%, by Western blot analysis. After48hours, Annexin V-PI staining and Flow Cytometry to detect apoptosis rate significant increase,（P<0.05） So miR-26b can regulated ovarian granulosa cell apoptosis by targeting to pig Smad4gene.