Mechanism Of CircBTBD7 Regulating The Growth And Development Of Immature Porcine Sertoli Cells

Spermatogenesis is a complex biological process,which is regulated by many specific molecules and cells.As a new non coding RNA,circRNA has the function of regulating gene expression.Based on previous studies,this study explored the role of ce RNA in the regulation of miR-222 and mir-24-3p with circBTBD7 as the breakthrough point,and further expanded the research content and direction.The results of this study can provide new enlightenment and theoretical basis for further revealing the mechanism of noncoding RNA regulating the growth and development of immature porcine Sertoli cells.The results are as follows:(1)Overexpression of circBTBD7 promoted the proliferation and inhibited the apoptosis of immature porcine Sertoli cellsBased on the previous RNA-seq identification results,circBTBD7 from BTBD7(BTB/POZ domain containing protein 7)gene was screened out,located in the region Chr7 :121258975-121260967 of the porcine genome,containing 2 exons and 1 intron,with a total length of 1992 nt.In order to clarify the function of circBTBD7,PCE-RB-MAM-EGFP overexpression vector of circBTBD7 was constructed and transfected into immature porcine Sertoli cells.RT-PCR、agarose gel electrophoresis and Sanger sequencing were used to verify its cyclization and overexpression in porcine immature Sertoli cells.Flow cytometry was used to detect cell cycle,and q RT-PCR was used to detect cell cycle related genes(C-MYC,CNNE1,CNND1 and CDK)and proliferation related genes(BMP,IGF,FGF,GDNF,PCNA and EGF)expression level,CCK8 and Ed U kits were used to detect cell proliferation,ATP kit was used to detect ATP level,annexin V-FITC/PI staining kit was used to detect cell apoptosis,Western blot was used to detect the expression of apoptosis related genes(BCL2,BAX and Caspase-3).The results showed that after overexpression of circBTBD7,the cell cycle process was accelerated,and the cells in G1 phase significantly moved to S and G2 phases;the expression of cycle and proliferation related genes were increased,cell proliferation activity was enhanced,and ATP level was increased;the proportion of early apoptosis and late apoptosis were significantly decreased,the protein expression of pro-apoptotic related genes BAX and Casepase-3 decreased,while the protein expression of anti-apoptotic related gene Bcl2 increased.(2)CircBTBD7 regulates the growth of immature porcine Sertoli cells by absorbing miR-222The binding sites of circBTBD7 and miR-222 were predicted online by integrating the previous omics data and using the RNA hybrid software,11 candidate miRNAs were screened,including miR-222,miR-744,miR-425,miR-34 a,miR-30 c,miR-219 a,miR-143-5p,miR-139-3p,miR-24-3p,miR-345-5p,and miR-139-5p.The team’s previous research results showed that miR-222 could be involved in regulating the growth of immature porcine Sertoli cells,it was found that the PI3K/ Akt signaling pathway was inactivated by down-regulating the expression of GRB10 gene,thus inhibiting the growth of immature porcine testicular Sertoli cells.Therefore,we preliminarily studied miR-222 to explore whether circBTBD7 could be involved in the growth regulation of immature porcine testicular Sertoli cells as a competitive endogenous RNA.Circ RNA-pull down and real-time quantitative PCR were used to determine that miR-222 could be adsorbed by circBTBD7.The results of Fluorescence In Situ Hybridization(FISH)showed that the expression of circBTBD7 in cytoplasm was higher than that in the nucleus,which preliminarily proved that circBTBD7 may act as a ce RNA to regulate gene expression at the post-transcriptional level.In order to further clarify the regulation of circBTBD7 on the growth of immature porcine Sertoli cells by the absorption of miR-222,a co-transfection experiment between circBTBD7 and miR-222 was designed in this study.The transfection consisted of A)circBTBD7-PCE-RB-MAM-EGFP vector + miRNA mimic,B)circBTBD7-PCE-RB-MAM-EGFP vector + mimic NC,C)blank-PCE-RB-MAM-EGFP vector + mimic NC,the proliferation and apoptosis of immature porcine Sertoli cells were detected by the above methods,and the expressions of miR-222 and GRB10 genes in the immature Sertoli cells of porcine testis were detected by real-time fluorescence quantitative PCR.The results showed that circBTBD7 could partially rescue the effect of miR-222 on cell biological function,which may be induced by increasing the expression of downstream target gene GRB10.(3)Circ BTBD7 regulates the growth of immature porcine Sertoli cells by adsorbing miR-24-3pAmong the 11 candidate miRNAs,the results of circRNA-pull down experiment showed that miR-24-3p could be significantly adsorbed by circBTBD7.In order to clarify the targeting relationship between circBTBD7 and miR-24-3p,dual luciferase reporter gene vectors were constructed according to the binding sites of circBTBD7 and miR-24-3p,which were co-transfected with miRNA or NC respectively into immature porcine Sertoli cells to detect the fluorescence activity.The miRNA-pull down and other techniques were used to further reverse verify the binding relationship,and the experiments proved that circBTBD7 could directly bind to miR-24-3p.In order to further confirm the regulation of circBTBD7 by adsorbing miR-24-3p to the growth of immature Sertoli cells in porcine,the same co-transfection experiment as miR-222 was designed in this study,the proliferation and apoptosis of immature porcine Sertoli cells were detected by the above methods.The results showed that circBTBD7 regulates the growth of immature porcine Sertoli cells by adsorbing miR-24-3p.(4)miR-24-3p targets MAPK7 gene to inhibit the proliferation and promote apoptosis of immature porcine Sertoli cellsTo further explore the targeting relationship and regulation mechanism between miR-24-3p and m RNA.Firstly,miR-24-3p and m RNA omics data were integrated to analyze the expression level relationship between miR-24-3p and target genes.Target Scan,miRtarbase,miRDB,miRWalk,RNA hybrid and other online software were used to predict the binding sites of miR-24-3p and target genes.Target genes were integrated to participate in KEGG signaling pathway analysis to screen out MAPK7 gene.In order to determine the regulatory relationship between miR-24-3p and MAPK7,the m RNA and protein expression levels of the target gene MAPK7 were detected by q RT-PCR and Western blot,respectively,in the immature Sertoli cells of porcine testis with overexpression and inhibition of miR-24-3p.In addition,miRNA-pull down experiment can also verify whether the expression of MAPK7 gene exists in the purified RNA,and detect the proliferation and apoptosis of immature Sertoli cells of pig testis by using the above various experimental methods.The results showed that after the overexpression of miR-24-3p,the m RNA expression of MAPK7 gene was decreased and the protein level was significantly decreased;At the same time,the expression of MAPK7 gene in the purified RNA of wild-type biotin-miR-24-3p transfected cells was significantly higher than that of the mutant group.After overexpression of miR-24-3p in immature Sertoli cells of porcine testis,the cell growth was inhibited,the cell apoptosis rate was significantly increased and the relative content of ATP was decreased.The experimental results of interference with MAPK7 gene were consistent with the results of overexpression of miR-24-3p,while the results of inhibition of miR-24-3p expression were contrary.In order to further investigate the mechanism of miR-24-3p and MAPK7 genes in immature porcine testis Sertoli cells,three groups of co-transfection experiments were designed.The transfection components were: A)miR-24-3p inhibitor + MAPK7 siRNA,B)miR-24-3p inhibitor + siRNA NC,and C)inhibitor NC + siRNA NC.The proliferation and apoptosis of immature porcine Sertoli cells were detected by various experimental methods above.The results showed that compared with the other groups,the CCK8 and EDU proliferation experiments of miR-24-3p inhibitor + siRNA NC group showed increased cell activity.Flow cytometry showed that the cell cycle progression was accelerated,q RT-PCR verified that the expression levels of proliferating genes were increased;The cell apoptosis rate was decreased,and the relative content of ATP was increased.The effect was consistent with the inhibition of miR-24-3p expression,while the effect was opposite in the transfection group with miR-24-3p inhibitor + MAPK7 siRNA.In conclusion,circBTBD7 can act as a ce RNA to regulate the growth and development of immature porcine Sertoli cells,and enhance the expression of downstream target genes GRB10 and MAPK7 by adsorption of miR-222 and miR-24-3p,thereby promoting the proliferation and inhibiting the apoptosis of immature porcine Sertoli cells.