Preliminary Study Of The Function And Mechanism Of TUBB4B In Mouse Primary Spermatocyte GC-2 Cells

Objective: Tubulin in the cytoskeleton plays an important role in testicular development and spermatogenesis.This study explores the role of tubulin β-4b chain(tubulin β-2C)in the development of primary spermatocytes and its regulatory mechanism.Method:Mouse primary spermatocyte GC-2 with Tubb4 b gene knockout and overexpression was obtained by lentivirus-mediated gene editing technique.In Tubb4 b knockdown and overexpressed GC-2 cells and Cytosolic carboxypeptidase-like 1(CCP1)knockdown and reexpressed GC-2 cells,The expression of Tubb4 b and CCP1 on mRNA and protein levels were detected by Reverse transcription Quantitative Real-time PCR(RT-qPCR)and Western blot,respectively.The effects of Tubb4 b knockdown and overexpression on the proliferation and cycle of GC-2 cells were detected by cell counting kit-8 and flow cytometry,and the expression of cell cycle factors was detected by RT-qPCR.Detection of Nuclear factor kappa-B(NF-κB)and Mitogen-activated protein kinase by Western bolt and cellular immunofluorescence assay The expression of MAPK signaling pathway factors.Transcriptomic sequencing was used to screen the differentially expressed genes in GC-2 cells after Tubb4 b knockout,and bioinformatics was used to enrichment and analysis the functions of differentially expressed genes and the possible signaling pathways involved,and RT-qPCR was used to verify the differentially expressed genes.Results: After lentivirus-mediated siRNA-Tubb4 b infection with GC-2,compared with negative control group,the expression of Tubb4 b and CCP1 on both mRNA and protein levels was significantly decreased(P < 0.05).After lentivirus infected with GC-2 carrying Tubb4 b overexpression plasmid,compared with negative control group,The mRNA and protein levels of Tubb4 b and CCP1 were significantly increased(P < 0.05).The expression of Tubb4 b in GC-2 cells knocked down by CCP1 was decreased with the decrease of CCP1 expression(P < 0.05).After the restoration of CCP1 expression,the expression of Tubb4 b was increased with the increase of CCP1 expression(P < 0.05),and the expression of Tubb4 b was restored to a level similar to that in the control group.The proliferation of CCK8 cells showed that Tubb4 b knockdown and overexpression had no significant effect on the proliferation of GC-2 cells.Flow cytometry showed that the cycle of Tubb4 b knockdown and overexpression GC-2 cells had no significant change compared with the control group.After Tubb4 b knockdown,the expressions of CDK-1,CDK-2,Cyclin B1 and Cyclin D1 were significantly increased(P < 0.05),while the expressions of Cyclin A1 and Cyclin E1 were significantly decreased(P <0.05).After Tubb4 b overexpression,the expressions of CDK-1,CDK-2,Cyclin B1,Cyclin A1 and Cyclin E1 were significantly increased(P < 0.05),while the expression of Cyclin D1 was significantly decreased(P < 0.05).Western blot assay showed that compared with the control group,the expression of p65,a key protein of NF-κB signaling pathway,was significantly decreased in GC-2 cells after Tubb4 b knockout(P< 0.05),and phosphorylated p-P65 was not significantly changed(P >0.05).There were no significant changes in the key proteins of MAPK signaling pathway ErK1/2 and p38,and the phosphorylated P-Erk1/2 expression level was significantly increased(P < 0.05),while PolyE was significantly decreased(P < 0.05).On the contrary,the expressions of P38 and ErK1/2 in GC-2 cells after overexpression of Tubb4 b were not significantly changed,while the expressions of P65,p-P65 and PolyE were significantly increased(P< 0.05),while the expressions of p-Erk1/2 were significantly decreased(P < 0.05).After the restoration of CCP1 expression,the expression of ErK1/2 decreased with the increase of CCP1 expression level,and reached a level similar to that of control group(P < 0.05).Immunofluorescence assay showed the position of expression of related factors in the signaling pathway changes at the cellular level.By transcriptomic sequencing,a total of 1328 genes were up-regulated after Tubb4 b silencing,and 562 genes were down-regulated.Biogenic analysis showed that Tubb4 B was involved in extracellular matrix,extracellular space,cell adhesion,biological attachment,cell migration,neural active ligand-receptor metabolism,axon orientation and other processes.Functional enrichment was found to be highly correlated with tumor necrosis factor signaling pathway,tumor/cancer metabolic pathway and calcium ion signaling pathway.Finally,the expression of Cadm1,Armcx4,Lama5,Dync2h1 and other male sterility related genes was significantly decreased after Tubb4 b knockout by RT-qPCR(P < 0.05),which was consistent with the transcriptomic sequencing results.Conclusion Tubb4 b and CCP1 are positively regulated in primary spermatocyte GC-2 cells,and CCP1 can deglutamylation Tubb4 b.The decreased expression of Tubb4 b can regulate the NF-κB and MAPK signaling pathways and affect the expression of male reproductive related genes such as Cadm1,Armcx4,Lama5 and Dync2h1.Conclusion:In primary spermatocyte GC-2 cells,the expression of TUBB4 B and CCP1 regulates each other positively,and CCP1 may affect the transcription,translation and post-translational modification of Tubb4 b through the cleavage of the carboxyl terminal or the polyglutamic acid at the end of the side chain;The decreased expression of Tubb4 b can regulate the NF-κB and MAPK signaling pathways and affect the expression of male reproductive related genes such as Cadm1,Armcx4,Lama5 and Dync2h1.