Journal of Southern Medical University

Journal of Southern Medical University ›› 2023, Vol. 43 ›› Issue (6): 1002-1009.doi: 10.12122/j.issn.1673-4254.2023.06.16

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Expression of TUBB4B in mouse primary spermatocyte GC-2 cells and its regulatory effect on NF-κB and MAPK signaling pathway

LIU Tongjia, WANG Wanlun, ZHANG Ting, LIU Shuang, BIAN Yanchao, ZHANG Chuanling, XIAO Rui   

  1. Key Laboratory of Molecular Pathology of Inner Mongolia Autonomous Region, Department of Pharmacy, Inner Mongolia Medical University, Huhehaote 010059, China
  • Online:2023-06-20 Published:2023-07-06

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Abstract: Objective To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. Methods Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. Results Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P<0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P<0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P<0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P<0.05). Conclusions TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.

Key words: male sterility; cytosolic carboxypeptidase-like1; TUBB4B; primary spermatocytes; nuclear factor kappa-B pathway; mitogen-activated protein kinase MAPK pathway