CHAF1A–PCNA interaction promotes cervical cancer progression via the PI3K/AKT/FoxO1 signaling pathway

An increasing number of studies demonstrate that histone chaperones play critical roles in tumorigenesis and development. In previous research, we conrmed that CHAF1A was highly expressed in cervical cancer (CC) and was correlated with poor prognosis. However, the biological function and specic mechanism of CHAF1A in the development of CC have not been reported. Methods CHAF1A knockdown in SiHa and HeLa.cells by lentivirus vector is veried by RT-PCR and Western blot analysis.CCK-8, ow cytometry assays, colony formation, cell migration assay and real-time cell analysis assay were performed to determine the cellular function of CHAF1A in CC.Tumor xenograft assay was conducted on nude mice to assess the effect of CHAF1A in vivo. Cell immunouorescence and co-immunoprecipitation were applied to examine the interaction between CHAF1A and PCNA. We found that CHAF1A knockdown in SiHa and HeLa cell lines inhibited proliferation and promotedits apoptosis. Further research indicated that CHAF1A promoted proliferation and inhibited apoptosis in CC by activating the PI3K/Akt/FoxO1 signaling pathway. Moreover, CHAF1A directly interacts with PCNA and co-promotes CC progression. The discovery the mechanism CC may new for 48, 72, 96, stand for 1 The 450 nm


Introduction
Cervical cancer (CC) is one of the most common malignant tumors worldwide. It is the second most common malignant tumor in females (1). However, despite signi cant advancements in CC therapies, such as surgical resection, chemotherapy, and radiotherapy, the prognoses of patients with CC remain poor. CC continues to be a major cause of death from cancer worldwide (1) . Persistent human papillomavirus infectionis the main etiological factor for CC development. CC is a multistep process involving the deregulation of multiple genes. The mechanisms of carcinogenesis are quite complex, and both genetic and epigenetic alterations can lead to carcinogenesis. In the last decade of cancer research, abnormalities in epigenetics have been consistently observed. Aberrations of epigenetics in cancer mainly involve aberrant histone modi cations and altered DNA methylation (2,3) .
Accumulating evidence suggests that cancer initiation, progression and the involvement of histone variants and their partners are closely linked (4,5) . CAF-1 is a trimeric complex protein that includes P150, P60, and p48. CAF-1 is important for chromatin assembly during DNA synthesis, and interfering with CAF expression may affect DNA synthesis, leading to DNA damage or even programmed cell death (6-11). As the largest subunit of CAF-1 and a major component exercising biological functions, CHAF1A plays a very important role in DNA mismatch repair, epigenetic regulation of embryonic stem cells, and diverse biological behaviors such as cell division and, proliferation, among others (7,(11)(12)(13)) .
An increasing number of studies have con rmed that the dysregulated expression of CHAF1A is associated with the development and progression of several tumors, such as: glioblastoma, hepatocellular carcinoma, colorectal cancer, and gastric cancer (14)(15)(16)(17)(18)(19)(20)(21)(22). Our previous study veri ed its high expression in CC by immunohistochemistry and association with the poor prognosis of CC (23). However, the speci c biological functions and mechanisms of action of CHAF1A in cervical cancer have not been reported. In the present study, we clar ed that CHAF1A could promote cell proliferation in CC, thereby inhibiting apoptosis and enhancing migration. Moreover, CHAF1A and proliferating cell nuclear HeLa cells were maintained in DMEM, supplemented with 10% fetal bovine serum (FBS); HaCat cells were cultured in the RPMI-1640 medium; and SiHa, C33A, and Caksi were cultured in MEM with 10% FBS. All cells were cultured with 1% penicillin-streptomycin antibiotics (Beyotime, China).
The multiplicity of infection was determined before lentiviral transfection. The cells were passaged and seeded at 2 × 10 4 cells / well in 6 well plates. After the cells were plated for 24 h, the original medium was discarded and the fresh medium containing lentiviral particlees and polybrene was added (the concentration of polybrene used was in accordance with the amount recommended by the manufacture).The medium was then replaced with completely fresh medium after 24 h.After the cells were allowed recover and grow further for 24 h, they were switched to 2.5 ug/mL of puromycin for weeklong culture and selection of cells successfully transfected with the lentivirus.The culture was further maintained for a week with purinomycin, and the culture medium was replaced with the complete medium.
The cells that were successfully transfected with lentiviral particles were subcultured for subsequent experiments.

RT-PCR
RT-PCR was performed using a RT-PCR kit (Takara, Shiga, Japan) in accordance with the instructions provided by manufacturer. Total RNAs from cells were extracted using TRIzol Reagent, and then reversely transcribed into cDNA by using the Prime Script RT Reagent Kit (TaKaRa), RT-qPCR was performed with the S1000 Real-Time System (Bio-Rad, Hercules, California, USA). SYBR Green (SYBR Premix Ex Taq TM II; TaKaRa) was used for uorescent quanti cation. The following cycling conditions were used for RT-PCR: pre-denaturation (95 °C, for 30 s), denaturation ,95 °C, for 5 s, and 40 cycles; annealing 60°C for 30 s), The relative mRNA expression was calculated using the 2−∆∆Ct method. All RT-PCR results were repeated at least 3 times. The primers used are listed in Table 1.

Flow cytometry
The effects of CHAF1A knockdown on cell cycle and apoptosis were assessed by ow cytometry. When the total number of cells reached 1.0 × 10 6 in the culture ask, the cells were trypsinized. Cells were collected after centrifugation at 1000g for 5 min, washed three times with PBS, and resuspended in 100 µL of PBS solution. Subsequently 500µL pre-cooled 75% alcohol in a lysis buffer containing 1 mg / mL RNase, 0.05% Triton X-100, 1 mg / mL. RNase, and 50 mg / mL propidium iodide (Sigma, Aldrich, St. Louis, MO, USA) was xed in their solution, incubated in the dark at room temperature for 30 min, and immediately analyzed using a FACS Calibur instrument (Becton Dickinson, CA, USA). Apoptotic samples were resuspended in 500 μL of PBS without ethanol. The cell instrument was analyzed and the results were recorded.

Colony Formation Assay
Cells (500 cells/well) were seeded in a 6-well plate and then incubated for the next 14 d until cell colonies appeared. The cells were subsequently xed with 4% paraformaldehyde for 15 min and then stained with Giemsa for 20 min. The rate cell clone formation was ultimately calculated.

CCK8 Assay
Cell Counting Kit-8 (CCK8) (Boiss, Beijing, China) was used to determine cell viability. The cells were counted after digestion and the concentration was adjusted to 2000 cells / 100 UL, Two sets of control and experimental samples with ve replicate wells per set were then set up. The plates were seeded for 24, 48, 72, 96, and 120 h and left to stand for 1 h, in accordance with the instructions provided by the manufacturer, The 450 nm was then determined.

Real-time cell analysis (Proliferation)
Baseline values were rst veri ed by adding 100 UL of the complete medium per well onto a dedicated 16 well electronic plate.The HeLa and SiHa cells were digested, centrifuged, resuspended, and seeded onto a baseline 16 well electronic plate containing 5000 cells / well with gold microelectrode arrays attached.The cell index was monitored using the xCELLigence system (Roche, Mannheim, Germany) for 96 h with measurements taken every 15 min.The results were analyzed using the RTCA software (version 2.0).

Cell Migration Assay
Cell migration was quanti ed using the in vitro scratch wound assay. HeLa and Siha cells (1 × 10 6 /well) were seeded in a 6-well plate. After adhesion, a linear wound was made using a 10 µL micropipette tip. The scratched wells were washed whith PBSseveral times to remove the detached debris caused by scratching. Images of the wounds (0, 24 h) were taken with a light microscope (20× magni cation). After 24 h images were again recorded. The number of migrated cells was then determined.

Real-time cell analysis (Migration)
The baseline values were determined. The mediumwas placed in the lower chamber of a 16-well electronic plate, and a cell culture medium containing 10% FBS was added. The upper chamber was mounted, and 30 µL of the serum-free medium in the upper chamber electronic well plate for 1 h at 37 °C and 5% CO2 incubator. 100 UL of serum-free medium (100 UL containing 50000 cells) was seeded into each well of the upper chamber of the cell migration plate. The cell migration index was recorded every 15 min for approximately 90 h.

Western blot analysis
Proteins were extracted from cervical cancer cells and prepared using RIPA lysis and an extraction buffer was supplemented with a protease and phosphatase inhibitor cocktail for 30 min on ice. Cell lysates were ultrasonically disrupted three times for 10 s each, followed by centrifugation at 12000 g for 15 min at 4°C . Supernatants were collected, denatured by boiling after the addition of the Laemmli sample buffer without a reducing agent, and then assayed. Protein samples after quanti cation were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di uoride membranes (Millipore, Bedford, MA). The membranes were then blocked and.then incubated with a speci c primary antibody diluent in a room at 4 °C overnight. Details of the primary antibodies are listed in Table 1. The membranes were then washed with TBST 3 times and then incubated with a secondary antibody (1:7500 dilution) for 1 h. The blots were washed 3 times in TBST. The immunoblots were detected with an ECL kit and visualized using an imaging system (Fusion, Germany).

Cell immuno uorescence
The CC cell lines SiHa and HeLa were seeded in 24-well plates. The well surfaces were covered with 13 mm glass coverslips. Immuno uorescence assay was performed when the cell con uence reached 90%. The cell culture medium was discarded. The cells were washed three times with PBS, xed in 4% paraformaldehyde for 10 min at room temperature, washed again with PBS three times , permeabilized with 0.3% Triton X-100 for 10 min, and blocked with 5% goat serum for 30min. The samples were incubated with the primary antibody (mouse polyclonal PCNA antibody, 1:2400; rabbit polyclonal CHAF1A antibody, 1:200) overnight in a room at 4 °C. The samples were washed 3 times with PBS solution and then incubated in the dark at room temperature for Alexa Fluor 488 conjugated Goat anti rabbit (Abcam, ab150115) and Alexa Fluor 647 conjugated goat anti mouse antibodies (Abcam, 150077) (both 1:1000). DAPI containing uorescence quenched coverslips were added and then observed by confocal laser scanning microscopy.

Co-immunoprecipitation
Protein immunoblotting and co-immunoprecipitation were performed to investigate the interactions of the proteins. Protein interaction was veri ed using the Pierce Co Immunoprecipitation Kit (Thermo Scienti c, 26149). Speci c experimental procedures were conducted, strictly following the instructions provided in the kit. Cells were lysed using a kit-speci c cell lysate for 15 min, sonicated for 10 s three times, and centrifuged at 12000 × g for 10 min. Upper protein lysate was aspirated, and the protein was incubated with 10 μg corresponding antibody overnight at 4 ° C. About 50 μ g of a mock antibody (Rabbit Anti mouse IgG) was used as a control. The next day the resin bound to the protein was washed 4 times following the instructions provided in the kit. Speci cally, 50 μL of the eluate was used. Eluted proteins were immediately boiled and denatured for Western blot analysis.

Tumor xenografts
Ten BALB/c female nude mice ((aged 5 weeks, with an average weight of 20 g).) were provided by China Chongqing Ensville Biotechnology Co. Ltd. The mice were divided into two groups consisting of ve mice each .All mice were given water and chow ad libitum. Control cells and cells with stable CHAF1A knockdown (shCHAF1A) were suspended in cold PBS.The right forelimb of each nude mouse was subcutaneously injected with 1.0 × 106 cells.
On Day 5 of modeling, the width (W), weight and length (L), of the tumor were measured for 3 d (volume [V] = W 2 × L × 0.52). The nude mice were sacri ced using 2% iso urane anesthesia.High concentration of CO2 asphyxia led to death by asphyxiation 21 d after injection, and the transplanted tumors were removed. Part of the tumor was sent for immunohistochemistry. The research protocol for this animal study conformed to the ethical guidelines in laboratory animal research and the Declaration of Helsinki.A approval was obtained from the ethics committee of the First A liated Hospital of Chongqing Medical University (accession number 2021-11).

Statistical analysis
All statistical analyses were performed using GraphPad Prism 7.0. The data are expressed as mean ± standard deviation (SD). Between-group comparisons were conducted using a 2-tailed paired Student t-test to test for statistical signi cance. Statistical differences between groups were determined using one-way ANOVA. P ≤ 0.05 was considered statistically signi cant in all cases. All experiments were performed in triplicate.

Knockdown of CHAF1A inhibits cervical cancer cell proliferation
The expression of CHAF1A in cervical cancer cell lines was signi cantly higher than that in HaCat cells(P 0.05 Fig.1A).To evaluate the functional proliferation of CHAF1A overexpression in CC, we performed CHAF1A knockdown in CC cells and evaluated the cellular outcomes. CHAF1A cells were depleted in SiHa and HeLa cells by RNA interference, and CHAF1A knockdown e ciency after lentiviral transfection was veri ed by qRT PCR and Western blot analysis. Knockdown e cacy in both SiHa and HeLa cells is shown in Figs. 1.B and 1.C. The stable transfected cells were selected for the subsequent experiment. Cell proliferation was observed using the CCK8 assay. The reduced CHAF1A expression in the HeLa and SiHa cells was found to inhibit cell proliferation. Moreover, the colony formation assay and RTCA presented a trend similar to that of the CCK8 assay after CHAF1A knockdown (P <0.05, Figs. 1D-1F).

Induction of cell cycle arrest and promotion of apoptosis in cervical cancer cells upon inhibition of CHAF1A expression
We further validate the biological function of CHAF1A in CC cells by conducting additional in vitro experiments. The cell cycle was analyzed by ow cytometry. CHAF1A knockdown resulted in a signi cant increase in the percentage of cells in the peak G1 / G0 phase and a decrease in the percentage of cells in peak S phases. CHAF1A inhibition was found to induce G1 arrest in CC cells (Fig. 1G). The number of apoptotic cells in the cells of the CHAF1A knockdown group was signi cantly higher than that in the cells of the control group (Figs.2A,2B). On the basis of these ndings, we speculated that CHAF1A could regulate the abnormal proliferation of CC by interferingwith the cell cycle to inhibit apoptosis.

CHAF1A promotes the migration of cervical cancer
To examine the migratory properties of SiHa and HeLa, we conducted a scratch wound assay. Using a scratch assay, we found that CHAF1A silencing resulted in decreased cell migration (Figs. 2C, 2D). Meanwhile, RTCA assays were used to examine SiHa and HeLa cell migration after transfection with the sh-CHAF1A. The results indicated that the migration of SiHa and HeLa cells was inhibited by CHAF1A knockdown (Fig. 2E).

CHAF1A promotes cervical cancer progression via the PI3K / Akt signaling pathway
Activation of the PI3K / Akt signaling pathway mainly promotes cell growth and survival.We detected the proteins related to PI3K / Akt / FoxO1 by Western blot assay.The results showed that the phosphorylated PI3K (tyr458) and Akt (ser473) levelswere decreased after CHAF1A knockdown,whereas PI3K and Akt levels remained unchanged in the total protein relative to the control group (Fig. 3E).This nding suggested that the high expression of CHAF1A could induce CC cell proliferation and invasionvia the PI3K / Akt pathway.

CHAF1A interaction with PCNA promotes tumor progression
Localization was determined by confocal immuno uorescence microscopy, which revealed the colocalization of CHAF1A and PCNA proteins in the nucleus (Fig. 3A). These proteins were localized in the nucleus (Fig. 3A), suggesting that they could interact with each other in the nucleus. This interaction was con rmed by co-immunoprecipitation. We rst co-precipitated the PCNA protein with the CHAF1A antibody (Fig. 3B), and the CHAF1A protein was precipitated with PCNA (Fig. 3B). We also found that CHAF1A expression was decreased after PCNA knockdown, which was veri ed by both mRNA and protein levels (Figs. 3C, 3D).

Effects of CHAF1A on tumorigenicity in vivo
To further validate the biological role of CHAF1A in vivo, HeLa cells with control and lentiviral knockdown were injected into the right ank of nude mice (Fig.4A). The diameter of the tumor was measured every 3 d to observe tumorigenesis and the average tumor volume was calculated. The mice were sacri ced 20 d after subcutaneous injection, Subsequently, the size and weight of the tumors were determined. The tumor volumes (Fig. 4B) and tumor weights (Fig. 4C) of the nude mice were signi cantly higher than those of the mice in the control group. Moreover, the tumorigenicity assay of the nude mice and immunohistochemistry showed that CHAF1A knockdown inhibited cell proliferation in the tumor. Ki67 and PCNA immunostaining indicated high proliferative indexes within these control groups (Figs. 4D,   4E).

Discussion
Increasing evidence suggests that histone chaperones play an important role in tumor development. The anti-silencing function 1A(ASF1A), Aprataxin and PNK-like factor, and the FAcilitates Chromatin Transcription complex were highly expressed in tumor tissues and poor prognosis was predicted (24)(25)(26). The histone chaperone CAF-1 is similarly involved in multiple biological processes and plays an irreplaceable role (6-11). CAF-1 has been shown to cause severe loss of cell viability, as well as delay S-phase progression and impaired DNA synthesis in human cells if they are unable to perform poor prognosis of their normal function (12,27,28).CHAF1A, as the core component of CAF-1, participated in the progression of various tumor diseases, tumor proliferation, differentiation, and invasiveness. High expression of CHAF1A in a variety of human tumors is strongly associated with poor tumor outcome (14)(15)(16)(17)(18)(19)(20)(21)(22). In our previous research, we con rmed by immunohistochemical experiments that CHAF1A and PCNA were highly expressed in CC tissues; moreover, they were positively correlated, and their high expression was associated with poor prognosis (23) . We demonstrated through numerous experiments the biological function of CHAF1A in CC. Finally, we found that overexpressed CHAF1A is involved in tumor cell proliferation and migration, and inhibits apoptosis. We used CRISPR / Cas9 technology for CHAF1A knockdown and then inhibited the activation of the PI3K / Akt signaling pathway. Thus, we con rmed that CHAF1A overexpression promotes tumorigenesis and the progression of cervical cancer. This nding was further con rmed by our tumorigenesis experiments in nude mice. Upon lentiviral knockdown of CHAF1A, the growth of tumor cells was signi cantly slower than the negative control. The transplanted tumor tissue was removed from the nude mice and the immunohistochemical expression levels of the proliferation markers Ki67 and PCNA were lower in the experimental group than in the control group. To further explore the speci c mechanism of CHAF1A in CC, we rst con rmed that in the SiHa and HeLa cells, both CHAF1A and PCNA were localized in the nucleus, as determined by cell immuno uorescence experiments. This result suggests, their direct interaction. We precipitated the protein extracted from HeLa cells with CHAF1A and PCNA antibodies and then detected the protein precipitated by the immunoprecipitation (IP) antibody with another antibody. The results showed that both antibodies could precipitate the other protein, con rming the interaction between CHAF1A and PCNA in HeLa cells. This conclusion is consistent with previous studies (29)(30)(31)(32). After the PCNA gene expression was knocked out by CRISPR/Cas9 technology, CHAF1A expression was signi cantly reduced. This decrease suggests that PCNA acts directly on CHAF1A and regulates CHAF1A expression. Previous studies have reported that PCNA-driven DNA synthesis can also attract CAF-1 to the S site of DNA repair, such as nucleotide excision repair (33,34). Meanwhile, speci c PCNA mutation weakens the role of CAF-1 in telomeric silencing in Saccharomyces cerevisiae, and PCNA mutations reduce its interaction with CAF-1 (32,35) . Whether they act directly and interact with the target of the other has yet to be determined.
Overall, the present study con rmed that the oncogenic role of CHAF1A in CC. CHAF1A overexpression could promote CC cell proliferation and inhibit cell apoptosis, which predicted a poor outcome in CC patients. The mechanism of CHAF1A in CC was thoroughly discussed, which could provide a new direction for precisely targeted therapy for cervical cancer. Meanwhile, the interaction of CHAF1A with PCNA promotes DNA replication and cell proliferation (9). Studies have reported that CHAF1A can be used as a marker for cell quiescence and proliferation(36-38). The combined results of this study and previous studies, indicate that CHAF1A may serve as a biomarker for predicting postoperative recurrence and malignancyin cervical cancer.

Declarations
Ethics approval and consent to participate Our study was approved by the Ethics Review Board of the First A liated Hospital of Chongqing University (number 2021-11).

Consent for publication
All authors agreed to publish the manuscript.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request..  This histogram shows the statistical apoptosis rate in (B). The calculation was as follows: Apoptosis rate = Q1-LR + Q1-UR(P < 0.05). (C) An invasive wound healing assay was performed to evaluate the in uence of CHAF1A on cell migration ability. This technique showed that CHAF1A silencing signi cantly delayed the mobility of SiHa and HeLa cells (magni cation, ×40). The histogram indicated a statistically signi cant difference (P <0.05). (D) Cell migration in real time was further analyzed using the xCELLigence RTCA system.