In this study, our work confirmed that FAM172A could inhibit the polymerization of MTs, and maybe as the novel MAP (Microtubule-associated protein). FAM172A had the binding ability to MTs, while the network of MTs was destabilized in HepG2 cells knockdown of FAM172A. Moreover, cell cycle was promoted in the FAM172A-depleted HepG2 cells exhibited hallmark of MTs. In contrast, the cell cycle delays in cells up-expression of FAM172A were likely the consequences of MTs defects. The modification was associated with mitosis through binding to MTs, which revealed the new regulation mechanism for polymerization of tubulin.
We had found that interference expression of FAM172A could significantly reversed the inhibition of cell mitosis induced by shRNA of β-tubullin, but FAM172A exhibited the role on the contrary. Moreover, shRNA of FAM172A + shRNA of β-tubullin significantly promoted mitotic entry, meanwhile β-tubullin shRNA and FAM172A + β-tubullin shRNA significantly put off mitotic entry, especially in FAM172A + β-tubullin shRNA group. In summary, these results sustained powerfully the FAM172A-β-tubullin interaction is the negative factor in proliferation of hepatoma cells at onset of mitosis.
To ensure the stable of genome, high fidelity must execute at the phase of mitosis in cell cycle. Because the material of gene need to be duplicated, while it must isolate each chromosome into two offspring cells. The genetic material must be separated into each daughter cell, but chromosome segregation defects may be associated with tumorigenesis . The stability of genome is essentially maintained based on precise genetic material replication and its equal distribution into descendant cells. The cell cycle of eukaryote means a series of events, certainly including sequential actions, such as S-phase (DNA synthesis), M-phase (cell division), G1-phase (intervening gap phases) allow cell growth, and G2-phase (check genomic material integrity) during proliferation. The cyclins are the regulatory partners of CDKs activity, the sequential and coordinated rise and fall of CDKs will drive the cell cycle of normal cell. The diverse cyclins modulate varying phases in cell cycle, additionally the respective checkpoints govern the cell cycle transition to block entry into the next phase before genetic or cellular defects are repaired [19, 20].
In our prior studies, we had identified the CDK2 and cyclin A expression were promoted by FAM172A in HepG2 cells . Cyclins E1 and A2 can promote progression and entry of S phase then regulate the cell cycle. The alterations of cyclin E1 or A2 control the aggressive HCC with homogenous entity through characterized transcriptionally activating ATR and E2F pathways combined with high frequency of inactivation PTEN and RB1. The hundreds of templated insertions and tandem duplications was the unique signature of HCC driven by cyclin with activation of TERT promoter and structural rearrangements, which enriched forcefully in regions of chromatin with early-replicated activation. The investigation confirms the novel entity of poor prognosis in HCC, meanwhile the characteristic of rearrangement is associated with the stress of replication .
In this study, expression of FAM172A was interfered, which markedly reversed the inhibition of G1/S phase induced by shRNA of β-tubullin, but the over-expression of FAM172A exhibited the role on the contrary. Besides, shRNA of FAM172A + shRNA of β-tubullin remarkably attenuated the phase of G2/M of HepG2 cells than control group, meanwhile β-tubullin shRNA and FAM172A + β-tubullin shRNA enhanced visibly the G2/M phase, especially in FAM172A + β-tubullin shRNA group. In summary, these results forcefully demonstrated that the interaction of FAM172A with β-tubullin may play a negative regulator of G1/S and a promotor of G2/M transitions.
In previous investigation, our results gave a excellent cognition in FAM172A gene, which played the exquisite affection on regulation of cell cycle, and was as the tumor suppressor of HCC through arresting G1/S phase of cancer cells. Thus, it is crucial to learn about the bio-functions of FAM172A on cell cycle. Then, the binding of taxol-stabilized MTs to FAM172A was performed using spin-down assay of MT proteins. Our data demonstrated that the purified MTs could interact with FAM172A, and the pellets of recombinant FAM172A was as the presence fraction of MTs. Furthermore, the assays of MTs polymerization was performed with recombinant FAM172A in vitro, and the results confirmed that FAM172A strongly suppressed the assembly of tubulin to MTs. Additionally, the pelleting assay was conducted to separate the fraction of P-pelleted, stable MTs and the fraction of S-soluble, dynamic MTs, which identified that FAM172A promoted the stability of MTs. The cells stable knockdown of FAM172A with its shRNA could reduce stabilized levels of MTs relative to control group. In a word, the results indicated that FAM172A was the stabilizing factor of MTs.
In conclusions, FAM172A might be the crucial mediator of polymerization tubulin and development of HCC. It suggested that aim at FAM172A through suppressing the polymerizaton of tubulin maybe a feasible strategy for treatment of HCC.