FAM172A suppresses polymerization of tubulin and development of HCC (Hepatocellular Carcinoma)


 BackgroundThe polymerization of tubulin play the vital role in development and pathogenesis of cancer. Our study will explain the role of FAM172A in tubulin polymerization of HCC cell line.MethodsFAM172A-interacting proteins were screened from cDNA library of human liver with the system of yeast two-hybrid, the combined protein of FAM172A was further identified with system of mammalian two-hybrid, CO-IP experiment, and assay of GST-pull down. Then cell line HepG2 of human hepatocellular carcinoma was transfected with AAV-FAM172A or FAM172A-shRNA, and then proliferation ability and cell cycle were assessed. The expression of FAM172A and β-tubulin polymerization were determined. The effect of FAM172A on development of hepatocellular carcinoma associated with polymerization of tubulin was studied, and we used xenograft mice in vivo experiment.ResultsSeveral clones were positively screened from library, which included β-tubulin cDNA. Further studies confirmed FAM172A could combine with β-tubulin. FAM172A suppressed polymerization of tubulin, meanwhile the proliferation and cell cycle of HepG2. Besides, AAV-FAM172A could inhibit the development of HCC in xenogarft mice. ConclusionsOur results indicated FAM172A might be the crucial mediator of polymerization tubulin and HCC development. It suggested that aim at FAM172A through suppressing the polymerizaton of tubulin maybe the viable strategy for treatment of HCC.


Introduction
Microtubules are hollow tubular structures that are widely present in various eukaryotic cells. Α-and βtubulin are the major components of the microtubule structure, which bind to form a heterodimer, and several heterodimers are connected end to form a raw silk. The assembly of heterodimers can lengthen the length of the microtubules, which in turn can be shortened by depolymerization of the microtubules into heterodimers. This dynamic polymerization and depolymerization process is essential for microtubule function. It is known that the three major drug binding sites of tubulin: paclitaxel, vinblastine and colchicine binding sites are combined with β-tubulin, while β-tubulin is isotyped and point mutations are critical in the resistance mechanisms of paclitaxel and vincristine [1,2].
The lamentous, cylindrical, and hollow structures formed from MTs (Microtubules) with the behaviour characteristics of highly dynamic [1]. MTs have the vital function in controlling motility, division and growth of cell, cell machinery, regulating intracellular tra cking, and preserving shape of cell [2]. The characteristics of MTs is dynamic equilibrium through non-covalent combined with dimers of tubulin, subsequently forming the increasing MTs and then returned dimers after depolymerization [3]. During the process of cell division, the highly crucial structure of mitotic spindle is generated from MTs, which correctly segregate cells chromosomes in eukaryote [4]. Through interfering the dynamic equilibrium will cause deadly injury of cells, such as stabilizing disassembly of MTs and polymerization suppression of tubulin. After dealt with the agents which could bind to tubulin, cells would appear the cytological hallmark and be arrested in phase of mitosis. These cells display absence or deform of mitotic spindle, missing nuclear membrane, and condensed chromosomes [5]. By interfering the dynamic equilibrium with antimitotic compounds in clinic, which will be the feasible way to explore anticancer drugs [6].
In recent ten years, the developing interfere agents which protect against cell division by combining with polymers, oligomers, ro deimers of α, β-tubulin show greatly frequency report [7]. The antimitotic products of synthetics, semisynthetics or nature include a large number of agents with different chemical structures, but combining with same region of tubulin. These drugs include two major types, the one is assembly inhibitors of MTs, which could bind either Vinca domain or CLC site through performing alkylate the sulfhydryl groups of tubulin. The another is MTs stabilizers, which combines the polymerized heterodimer of α, β-tubulin with high a nity [7]. The polymerization of tubulin exhibit the key role in development and metastasis of HCC [8][9][10][11][12].
Our previous experiment data demonstrated FAM172A might be the suppressor of HCC through regulating the endoplasmic reticulum stress, cell cycle associated with of Notch 3 and NF-κB signaling pathway [13,14]. In the present study, we will clarify the bio-function of FAM172A in tubulin polymerization and HCC progression.

Materials And Methods
Cell culture and transfections of plasmids, recombinant proteins The Homo sapiens cell line HepG2 (ATCC® HB-8065™) of Hepatocellular Carcinoma (HCC) was obtained from American Type Culture Collection (ATCC), which was cultured with DMEM and FBS. The transfection agent jetPRIME (Polyplus-transfection, Illkirch-Graffenstaden, France) was used to perform the stable over-expression and knockdown β-tubullin or FAM172A.
FAM172A-3 × FLAG-6 × HIS was constructed with the modi ed vector of pcDNA5/FRT/TO, which was used to over-expression of FAM172A. The interfere plasmid of FAM172A shRNA was used, which was constructed in our previous work [13,14]. The recombinant proteins of FAM172A was used, which was prepared in our previous work [13,14].

RNAi targeting sequences
Primers for shRNA were designed following the manufacturer's instructions and cloned into the pSilencer2.1-neo vector to construct the shRNA against FAM172A. Primers for shRNA were designed following the manufacturer's instructions and cloned into the pSUPER.retro puro and pSUPER-nGFP vector to construct the shRNA against FAM172A. The target sequence in FAM172A is 5' -GAUAUGGAGUAAUAGUACUTT-3'. To establish stable knockdown cell lines, the transfected HeLa cells with the corresponding vectors of shRNA were selected with neomycin. FAM172A-knockdown cells were transfected with FAM172A-Myc and FAM172A-ΔLIR-Myc expression plasmids and selected with zeocin to establish stable rescued cell lines. FAM172A-knockout cells were established using CRISPR/Cas9 system and veri ed through immunoblotting analysis. The β-tubullin-knockout cells were established as above method, too. The target sequence in β-tubullin is 5' -GGCCTGAAGATGGCTTCCACTTCAAGAGTGTGGAAGCCATCT TCAGGCCCTTTTTG-3'.
Yeast two-hybrid and mammalisn two-hybrid assay The cDNA library of human liver (contains around four million transformants) was screened with the bait plasmid of pGBKT7-FAM172A using the lack medium (lacking histidine, adenine, leucine, and tryptophan) but containing 3-aminotriazole (1 mM). Next, the obtained positive clones were further veri ed with single yeast hybrid and analysis of mammalian two-hybrid using the CheckMate™ system of Promega.

Western blot analysis and CO-IP (co-immunoprecipitation)
The proteins were resolved with electrophoresis of SDS-PAGE (sodium dodecylsulphate-polyacrylamide) gel, then transferring membranes were performed. Moreover, membranes were blocked with skim milk and probed using the primary antibody. The following antibodies were used in this study: GAPDH (Santa Cruz, Santa Cruz, CA, USA; sc-47724-WB 1:10 000), FAM172A (Abcam; ab 121364: WB 1:2,000; IF 1:300), and β-tubulin (Abcam; ab6046: WB 1:500; IF 1:1,000). Then the detection of chemiluminescence-based were conducted according to the secondary antibody conjugated horseradish peroxidase (HRP) with exposed lm. The assays of co-immunoprecipitation (CO-IP) were immunoprecipitated with Suspension Agarose beads with Protein G Plus/Protein A.

The assay of GST pull-down
The fusion proteins tagged to His-or GST-expression were puri ed by agarose of Ni-NTA (GE) and beads with glutathione-sepharose (4B, GE), respectively. The transcription and translation plasmid of β-tubullin over-expression in vitro was used with system of TNT (Promega), then the puri ed fusion proteins tagged His-or β-tubullin protein was incubated with fusion protein contained GST and combined with beads of glutathione-sepharose. Furthermore, the beads were analyzed using western blot assay.
The synchronization of HepG2 cells, then labelled with BrdU for detection of mitotic index At rst, cell synchronization was performed with block method using double thymidine, which was the reversible arresting agents for cell cycle. After that, HepG2 cells were arrested at the early phase of S. Besides, the M-phase arrest HepG2 cells were blocked with nocodazole (Sigma) at early S phase.
Subsequently, the synthesis of DNA was evaluated using label of BrdU, the positive HepG2 cells stained with antibody of anti-BrdU were counted manually using microscope of immuno uorescence. Moreover, the events of mitotic were also scored based on the staining of DNA using DNA dye (Hoechst 33258) and time-lapse videomi croscopy (captured with Openlab software). The morphological changes of HepG2 cells were observed, too. Which included the condensation of DNA and morphology of nucleus.
Furthermore, the analysis of ow cytometric after staining phospho-H3 was used to assess the index of mitotic.

Cell cycle
The harvested HepG2 cells were numbered and then xed with ice cold 70% ethanol at -20 °C. Then, the con-stained cells with propidium iodide and mitotic marker H3pS10 were secondly dying DNA using PI and determined with the ow cytometry of BD FacsCalibur.

The puri cation of microtubules (MTs), binding experiments of spin-down and assays of MTs polymerization
For the binding experiments of MAPs and MTs, which were rstly puri ed, respectively. Secondly, the above puri ed MTs were pelleted and mixed with the recombinant proteins of FAM172A in PME buffer containing paclitaxel (10 µM) to allow the occur of binding between puri ed MTs and recombinant protein of FAM172A. Finally, the samples of binding experiments for MTs spin-down were performed using western blot experiment. The uorescence-based kit for Polymerization of Tubulin (Cytoskeleton Inc, Denver CO, Cat. # BK011P) was used to perform the assays of MTs polymerization.

Intracellular stability analysis of MTs
Intracellular stability assay of MTs was conducted in HepG2 cells stable knockdown of FAM172A with its shRNA. The fraction of S (supernatant) and P (pellet) contained solubilized tubulin and sedimented polymerized tubulin, respectively. All samples were detected using western blot method.

Animals
The xenograft mice model was conducted with nude mice (Laboratory Animal Sciences, Southern Medical University, Guangzhou, Guangdong Province, China), which were housed in the Laboratory Animal Center of Southern Medical University (Guangzhou, Guangdong Province, China in the environment of pathogen-free with standard laboratory diet and 12 h light/dark cycle at temperature controlled (20-24 ºC). The experimental protocol was approved by the licensing committee of Southern Medical University (Guangzhou, Guangdong Province, China). All experiments related to animals were performed based on Ethical Committee of Southern Medical University (Guangzhou, Guangdong Province, China) in accordance with relevant guidelines of Chinese.
The HepG2 suspension was subcutaneously injected into right anks of nude mice. When the size of tumor reached 3-8 mm, the recombinant FAM172A proteins were initiated given (1.0 mg/ml) once every three days for six times. All mice were sacri ced at the 21th day, tumor mass was determined, the expression of FAM172A and β-tubulin in tumors tissues were detected with immunohistochemical staining and western blot assay, respectively.

Statistics
All data was represented with mean ± standard deviation (SD). Between the two groups, the Student's ttest was used to analyze their differences. Abnormally distributed data of groups was analyzed with Kruskal-Wallis ANOVA. The statistical analyses were performed using SPSS software (version 18.0). P < 0.05 is signi cant statistically.

Results
Verify the interacting protein of FAM172A using yeast twohybrid For verifying the novel interacting proteins of FAM172A, the yeast two-hybrid system was used to conduct the screening from cDNA library of human liver (4 million transformants) with bait of FAM172A sequence. Consequently, several positive clones were isolated, which encoded β-tubullin cDNA (Fig. 1A). It indicated FAM172A interacts with β-tubullin.
Identi ed the interaction betweenβ-tubullin with FAM172A in vitro and in vivo The system of mammalian two-hybrid (CheckMate, Promega) was used to test our hypothesis about the interaction between FAM172A and β-tubullin in vivo. The co-transfections of pBIND-FAM172A and pACTβ-tubullin plasmids were used conducted as the experiment group, while the single plasmid transfection group with blank, pBIND, pACT, pACT+pBIND-FAM172A, pACT-β-tubullin+pBIND was used as negative control, respectively. Moreover, the co-transfections of pACT-MyoD and pBIND-Id control vectors were used as positive control. Next, the assays of luciferase were conducted with Promega Assay System of Dual-Glo, while the e ciency of transfection was corrected with luciferase of Renilla. The level of luciferase in group co-transfections with pBIND-FAM172A and pACT-β-tubullin was 2.73-fold higher than control group (P<0.05) (Fig. 1B). Moreover, the FAM172A and β-tubullin protein expression in the above cells was determined using western blot assay (Fig. 1C). To con rm β-tubullin interacting with FAM172A, the CO-IP assay was further conducted through transfection with FAM172A tagged Myc or β-tubullin tagged Flag, control vector. These results demonstrated the particular interaction between Myc-FAM172A and Flag-βtubullin (Fig. 1D). Besides, the GST-pull down experiment in vitro was performed, which result identi ed the truly interaction between GST-FAM172A and β-tubullin (Fig. 1E). In addition, the confocal image of HepG2 cells co-transfections with pDS-RED1-N1-β-tubullin and pEGFP-C1-FAM172A was photographed using microsope of Zeiss 510 META (Fig. 1F). The results indicated the not only β-tubullin protein (red) localized at the nucleus and cytoplasm of HepG2 cells, but FAM172A (green) localized in the cytoplasm and nucleus. The nucleus were stained with DAPI (blue). Finally, the overlaid images identi ed β-tubullin partly overlapped with FAM172A at cytoplasm and nucleus. Our data indicated the interaction between βtubullin and FAM172A in vitro and in vivo.
Mapping of the interaction regions between FAM172A and β-tubullin Moreover, experiment of co-immunoprecipitation was used to con rm the required interacted area with FAM172A in β-tubullin, which was reconstructed for the four deletion mutants through cleavage with caspase-3 at three sites D231, D290, and D318 ( Fig. 2A). Both the full-length β-tubullin and C1 fragments of it at carboxy-terminal could interact with FAM172A, but the N-terminal β-tubullin fragment with residues 1-231 at amino-terminal could not show the interaction between the two proteins ( Fig. 2A). Besides, the C1 fragment of β-tubullin with residues 232-451 could speci cally bind to FAM172A, however the N-terminal of β-tubullin fragment with residues 1-231 at amino-terminal, C2 β-tubullin fragment with residues 290-451 and C3 β-tubullin fragment with residues 318-451 or Flag did not show the interactions. It is demonstrated that the required region of β-tubullin for bonding to FAM172A is 232-290 region of βtubullin (Fig. 2B). The C1fragemnts cleaved from C-terminal of β-tubullin appeared the highest a nity to FAM172A, which indicated the important for the β-tubullin-FAM172A interaction between residues 232-290 region (Fig. 2B). Furthermore, the proteins of β-tubullin were puri ed to con rm the FAM172A-βtubullin interaction in vitro, which was agree to the our results of Co-IP.
Interaction of β-tubullin with FAM172A inhibits mitotic entry of HepG2 cells Additionally, the shRNA of β-tubullin was used to inhibit the expression of β-tubullin protein in HepG2 cells (Fig. 3A). The cell mitosis of released after using block with double thymidine to synchronize cell at the G1/S phase, then the synthesis of DNA was evaluated with BrdU. Incorporation of BrdU into the control, the mitotic cells transfected with shRNA of β-tubullin decreased obviously than control. Moreover, interference expression of FAM172A could signi cantly reversed the inhibition of cell mitosis induced by shRNA of β-tubullin, but the role of FAM172A exhibited on the contrary (Fig. 3B). Our results indicated that the interaction between β-tubullin and FAM172A may control the DNA synthesis, and then transition of G1/S boundary or mitosis in hepatoma cells.
The ow cytometry and stain of phospho-H3 was additionally used to con rm the role of FAM172A-βtubullin interaction in cell mitotic entry (Fig. 3C). The shRNA of FAM172A+shRNA of β-tubullin signi cantly promoted mitotic entry, meanwhile β-tubullin shRNA and FAM172A+β-tubullin shRNA signi cantly 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.
The FAM172A-β-tubullin interaction attenuates G1/S phase, and accentuates G2/M transitions Moreover, we performed analysis for cell cycle (Fig. 4). As shown in Fig. 3, the G1/S phase of cells transfected with shRNA of β-tubullin (Fig. 4C) decreased obviously than control cells (Fig. 4B). Furthermore, interference expression of FAM172A (Fig. 4A) could signi cantly reversed the inhibition of G1/S phase induced by shRNA of β-tubullin (Fig. 4C) , but the over-expression of FAM172A exhibited the role on the contrary (Fig. 4D). Our results indicated that the interaction between β-tubullin and FAM172A may affect synthesis of DNA, and then the transition of G1/S phase in hepatoma cells.

FAM172A modulate microtubules (MTs)
The cells knockdown of FAM172A showed the reduced phenotype of entry into mitosis and S-phase [13], and is corresponding to the dynamics of destroyed MTs [15]. Recently, the FAM172A associated cytoskeletal proteins were identi ed with IP-mass spectrometry methods by our research team. According to these data, between the network of MTs and FAM172A, the constructed relationship was wellcharacterized, then we investigated interestingly whether FAM172A was MAP (bona de microtubuleassociated protein). For testing our hypothesis, the confocal images were rstly obtained in cells stained with FAM172A and the primary MTs component β-tubullin (Fig. 1F). The results indicated β-tubullin colocalized with FAM172A in the cell fraction of nuclei and cytoplasm (Fig. 1F), which con rmed the intracellular co-localization and the interaction between FAM172A and β-tubullin. The machines of macromolecules of the cytoskeletal structures, primarily comprised of MT polymers, were that coordinate the cleavage furrow orientation and segregate chromosomes, Given these data, FAM172A may dynamically interact with cytoskeleton and chromatin. Moreover, a series of researches had identi ed interestingly the link between dynamics of MTs and RNA or nucleolus, which was related to mitotic spindle and executed by a great deal of ribosomal and nucleolar factors, such as RPS3, nucleolin, and NPM1 [16,17]. These proteins could regulate the dynamics of MTs. Therefore, FAM172A was hypothesized by our team that may play the regulation role in dynamics of MTs.

FAM172A attenuates polymerization of microtubules (MTs)
For trying out the above hypothesis, the binding of taxol-stabilized MTs to FAM172A was performed using spin-down assay of MT proteins. Our data demonstrated that the puri ed MTs could interact with FAM172A, and the pellets of recombinant FAM172A was as the presence fraction of MTs (Fig. 5A). Furthermore, the consequences of function induced by the interaction between FAM172A and MTs that was determined. The assays of MT polymerization was performed with recombinant FAM172A in vitro, and the results con rmed FAM172A strongly suppressed the assembly of tubulin to MTs (Fig. 5B). Additionally, the pelleting assay was conducted to separate the fraction of P-pelleted, stable MTs and the fraction of S-soluble, dynamic MTs, which identi ed that FAM172A promoted the stability of MTs. The cells stable knockdown of FAM172A with its shRNA could reduce stabilized MTs levels relative to control group ( Fig. 5C and 5D). In conclusion, the results indicated that FAM172A was the stabilizing factor of MTs.

FAM172A restrained xenografts of HCC
The xenografts mice model of human HCC was established using HepG2 to detect the activity of antitumor with FAM172A in vivo. These data gave evidence of rapidly growth of xenografts in control mice. But after treating xenografts with recombinant FAM172A protein in experiment mice, the xenografts grew slowly ( Fig. 6A and 6B). Besides, the xenografts' weight of experiment mice was markedly less than control mice (Fig. 6A and 6B). Moreover, the average volume of tumor in control mice was remarkedly bigger than control mice at the terminal of trial endpoint (Fig. 6B). Therefore, these results con rmed that the recombinant FAM172A protein could dramatically suppressed proliferation of tumors and the process of HCC development. As the results of proteins expression levels through immunohistochemical staining (Fig. 6C) and western blotting (Fig. 6D) in xenograft, the recombinant protein FAM172A upregulated itself expression and donwregulated expression of β-tubullin. These data identi ed FAM172A recombinant protein remarkably restrained HCC development through controlling the polymerization of microtubules in vivo.

Discussion
In this study, our work con rmed 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 modi cation 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 signi cantly 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 signi cantly promoted mitotic entry, meanwhile β-tubullin shRNA and FAM172A + β-tubullin shRNA signi cantly 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 delity 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 [18]. 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 identi ed the CDK2 and cyclin A expression were promoted by FAM172A in HepG2 cells [14]. 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 con rms the novel entity of poor prognosis in HCC, meanwhile the characteristic of rearrangement is associated with the stress of replication [21].
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 puri ed 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 con rmed 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 identi ed 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.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.  Interaction of β-tubullin with FAM172A in vitro and in vivo. The yeast two-hybrid system was used to conduct the screening from cDNA library of human liver with bait of FAM172A sequence. Consequently, the positive clones were isolated that encoded β-tubullin cDNA (A). The system of mammalian two-hybrid was used to test our hypothesis about the interaction between FAM172A and β-tubullin in vivo. The level of luciferase in group co-transfections with pBIND-FAM172A and pACT-β-tubullin was 2.73-fold higher than control group (P<0.05) (B). Moreover, the FAM172A and β-tubullin protein expression in the above cells was determined using western blot assay (C). The CO-IP assay was further conducted through transfection with FAM172A tagged Myc or β-tubullin tagged Flag, control vector. These results demonstrated the particular interaction between Myc-FAM172A and Flag-β-tubullin (D). Besides, the GSTpull down experiment in vitro was performed, which result identi ed the truly interaction between GST-FAM172A and β-tubullin (E). In addition, the confocal image of HepG2 cells co-transfections with pDS-RED1-N1-β-tubullin and pEGFP-C1-FAM172A was photographed using microsope of Zeiss 510 META (F). The results indicated the not only β-tubullin protein (red) localized at the nucleus and cytoplasm of HepG2 cells, but FAM172A (green) localized in the cytoplasm and nucleus. The nucleus were stained with DAPI (blue).

Figure 2
Mapping of the interaction regions between FAM172A and β-tubullin. Moreover, experiment of coimmunoprecipitation was used to con rm the required interacted area with FAM172A in β-tubullin, which was reconstructed for the four deletion mutants through cleavage with caspase-3 at three sites D231, D290, and D318 (A). Both the full-length β-tubullin and C1 fragments of it at carboxy-terminal could interact with FAM172A, but the N-terminal β-tubullin fragment with residues 1-231 at amino-terminal could not show the interaction between the two proteins (A). Besides, the C1 fragment of β-tubullin with residues 232-451 could speci cally bind to FAM172A, however the N-terminal β-tubullin fragment with residues 1-231 at amino-terminal, C2 β-tubullin fragment with residues 290-451 and C3 β-tubullin fragment with residues 318-451 or Flag did not show the interactions (B). The C1fragemnts cleaved from C-terminal of β-tubullin appeared the highest a nity to FAM172A (B).

Figure 3
Interaction of β-tubullin with FAM172A inhibits mitotic entry of hepatoma cells . Additionally, the shRNA of β-tubullin was used to inhibit the expression of β-tubullin protein in HepG2 cells (A). The mitotic cells transfected with shRNA of β-tubullin decreased obviously than control. Moreover, interference expression of FAM172A could signi cantly reversed the inhibition of cell mitosis induced by shRNA of β-tubullin, but the role of FAM172A exhibited on the contrary (B). The ow cytometry and stain of phospho-H3 was additionally used to con rm the role of FAM172A-β-tubullin interaction in cell mitotic entry (C). The shRNA of FAM172A+shRNA of β-tubullin signi cantly promoted mitotic entry, meanwhile β-tubullin shRNA and FAM172A+β-tubullin shRNA signi cantly put off mitotic entry, especially in FAM172A+βtubullin shRNA group.  FAM172A attenuates microtubule polymerization. The puri ed MTs could interact with FAM172A, and the pellets of recombinant FAM172A was as the presence fraction of MTs (A). The assays of MT polymerization was performed with recombinant FAM172A in vitro, and the results con rmed that FAM172A strongly suppressed the assembly of tubulin to MTs (B). FAM172A promoted the stability of MTs. The cells stable knockdown of FAM172A with its shRNA could reduce stabilized MTs levels relative to control group (C and D).