小鼠初级精母细胞GC-2中TUBB4B的表达及其对NF-κB和MAPK信号通路的调控

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

目的探究微管蛋白(TUBB4B)与胞浆羧肽酶(CCP1)在小鼠初级精母细胞(GC-2)中的互相作用关系,以及TUBB4B在调控GC-2发育中的作用。方法使用慢病毒感染GC-2细胞分别构建TUBB4B基因敲减组(TUBB4B-KD组)和阴性对照组(NCKD组);TUBB4B基因过表达组(TUBB4B-OE组)与阴性对照组(NC-OE组),利用嘌呤霉素筛选稳定细胞株。采用RT-qPCR和Western blot分别在mRNA和蛋白水平上检测细胞模型是否构建成功,并进一步探究在GC-2细胞中TUBB4B与CCP1二者之间相互调控的表达关系。CCK8及流式细胞术检测TUBB4B沉默和过表达后对GC-2细胞增殖及周期的影响;Western blot及细胞免疫荧光实验找出TUBB4B沉默和过表达后发生表达改变的信号通路因子,并在细胞水平上进行标记验证。结果GC-2中与NC-KD(NC-OE)组相比,TUBB4B沉默和过表达后,CCP1在mRNA和蛋白水平上的表达均发生一致的性改变(P<0.05);同样CCP1敲减和恢复表达后与NC组相比,TUBB4B的表达也随之发生一致的性改变(P<0.05)。CCK8和流式细胞术实验发现TUBB4B敲减和过表达对GC-2的增殖速率和细胞的周期无明显改变。Western blot和细胞免疫荧光实验显示TUBB4B敲减和过表达后,细胞中核因子κB(NF-κB)信号通路关键蛋白:p65,p-p65与丝裂原活化蛋白激酶(MAPK)信号通路关键蛋白:ErK1/2,p-ErK1/2会发生相应的明显改变(P<0.05);而CCP1敲减后会明显影响PolyE表达(P<0.05)。结论在GC-2细胞中TUBB4B与CCP1表达相互调控为正向作用,且CCP1对TUBB4B具有去谷氨酰化修饰作用,TUBB4B参与初级精母细胞中NF-κB和MAPK信号通路的调控。

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(NCOE)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 pp65)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.