IFITM3 Regulates NCAPG Through Phosphorylation to Inuence the Invasion and Metastasis of HCC

Background: Several studies have demonstrated that the expression of IFITM3 and NCAPG is closely related to the prognosis of various tumors. However, the mechanism of action of these two is not yet clear. In this study, we have explored the mechanism of action of IFITM3 and NCAPG in the promotion of the invasion and metastasis of hepatocellular carcinoma (HCC). Methods: Specimens of liver cancer and adjacent tissues from 55 HCC patients at the Department of Hepatobiliary Surgery, Second Aliated Hospital of Nanchang University were collected, and the expression of NCAPG and IFITM3 was determined by qRT-PCR and Western blot. Through the analysis of multiple databases, the relationship between IFITM3 and NCAPG was identied and veried by the CO-IP method. Using SiRNA and plasmids to downregulate and up-regulate IFITM3, we observed the expression of STAT3/CDK1, as well as NCAPG mRNA and protein. After downregulating and up-regulating the expression of IFITM3 and NCAPG, the ability of HCC cells to invade and metastasize was determined using a scratch test and Transwell. After using pathway inhibitors and activators, the expression of NCAPG was observed. Results: According to the database, both IFITM3 and NCAPG were highly expressed in Liver hepatocellular carcinoma. We also conrmed that IFITM3 and NCAPG were up-regulated in HCC tissues and cells. Furthermore, the bioinformatics analysis and CO-IP indicated that there was protein interaction between IFITM3 and NCAPG, and that IFITM3 could regulate NCAPG by phosphorylating it. We further conrmed our observations by retrospective experiments. Reuse of pathway inhibitors and activators indicated that IFITM3 could regulate NCAPG through STAT3/CDK1 to promote the invasion and metastasis of HCC. Finally, animal experiments conrmed that our results were also reproducible in vivo. Conclusion: IFITM3 can regulate NCAPG through STAT3/CDK1 to promote the invasion and metastasis of HCC.


Background
Liver cancer is a common malignant tumor, and hepatocellular carcinoma (HCC) is the most common type of liver cancer [1,2]. HCC has a poor prognosis, as it rapidly metastasizes, and by the time diagnosis is made, most of it is already in the advanced stage of cancer [3,4]. Therefore, the identi cation of biomarkers of liver cancer and understanding the mechanism of invasion and metastasis of liver cancer needs to be pursued urgently.
Interferon-induced transmembrane protein 3 (IFITM3) is a member of the interferon-stimulating gene (ISG) family [5,6]. Previous studies have reported that IFITM3 is closely related to infections by several viruses [7][8][9]. For example, studies have shown that restriction of viral entry by IFITM3 inhibits the infectivity of iris virus and Noda virus [10]. Sun et al. have also shown that IFITM3 prevents acute in uenza in mice [11]. The latest research has also reported that IFITM3 is closely related to the development of COVID-19 [12,13]. The BAT SARS WIV1 coronavirus uses the ACE2 of a variety of animals as the receptor and evades IFITM3 through the activation of the membrane fusion protein TMPRSS2 [14].
In addition to being closely related to viral infections, the expression of IFITM3 is also signi cantly positively correlated with the prognosis of several tumors [13,[15][16][17]. For example, IFITM3 up-regulates the expression of c-myc through the ERK1/2 signaling pathway to promote the proliferation of liver cancer cells [8]. Although IFITM3 has been proven to be a key gene affecting disease progression in several elds, its in uence in liver cancer has been relatively scarcely studied, and its speci c mechanism remains to be understood.
The non-SMC condensin I complex subunit G (NCAPG), which organizes the coiled topology of a single chromatid, represents an overexpressed antigen in various types of cancer [18][19][20]. It helps reorganize chromatin into rod-shaped mitotic chromosomes and ensures the separation of sister chromatids during cell division [21,22]. In the earliest research, NCAPG was identi ed as a breeding gene in dairy cows. Subsequent research reported that NCAPG was associated with the progression of various diseases [23,24]. For example, NCAPG was identi ed as a risk factor for psoriasis. It was also involved as a key gene in the progression of various tumors [25][26][27]. For example, NCAPG is overexpressed in colorectal cancer and prostate cancer tissues, and has a close relationship with its prognosis. The latest research has reported that NCAPG plays an extremely important role in HCC, but the speci c mechanism of action is still unknown.
Protein phosphorylation is an important step in the post-translational modi cation of proteins and plays an important role in determining the activity of enzymes and other important functional molecules, the delivery of secondary messengers, and the cascade of enzymes [28,29]. STAT3 is a member of the STAT family. It is an important factor that is involved in the modi cation of phosphorylation and is an important nuclear transcription factor [30,31]. STAT3 can also be activated by HBV, HCV, and various oncogene proteins [32]. An unregulated STAT/SOCS signal can also lead to the activation of STAT3, which then regulates the transcription of downstream genes [30,33,34]. Hepatocellular carcinoma (HCC) tissues show a signi cant overexpression of STAT3, which can result in malignant transformation of hepatocytes and cause cancer. Furthermore, activation of the STAT3 signal and c-Myc, EGFR, TGF, survivin, and VEGF disorders are closely related to the occurrence and development of HCC [35,32].
Cyclin-dependent kinases (CDK) represent a Ser/Thr kinase system that corresponds to the cell cycle process. It is not only regulated by phosphorylation and dephosphorylation but also is affected by oncogenes and tumor suppressor genes. Therefore, identifying and clarifying the relationship between STAT3/CDK1 and HCC is crucial for the diagnosis and treatment of liver cancer.
In our research, we observed that both IFITM3 and NCAPG played an irreplaceable role in the invasion and metastasis of HCC, and IFITM3 could positively regulate NCAPG. Importantly, we discovered for the rst time that IFITM3 could in uence the invasion and metastasis of HCC by changing the level of NCAPG phosphorylation modi cation.

Tissue specimen
The study was carried out on 55 patients with HCC diagnosed between 2015 and 2019. Only patients that had undergone hepatectomy without any treatment before surgery, including radiotherapy or chemotherapy, were included in the study. The liver cancer and adjacent tissue specimens were placed in liquid nitrogen immediately upon collection.

Cell transfection
The SiRNA and plasmids were obtained from Ruibo (Guangzhou), and the HCC cells were transfected using Lipofectamine 3000 (Thermo Fisher Scienti c, Inc), according to the manufacturer's instructions.
All the transfected cells were incubated in complete medium for at least 24 h prior to transfection and rinsed with phosphate buffered saline (PBS, pH 7.4) before transient transfection.

Scratch test
NHCC-97H and HCC-LM3 cells were used for the scratch experiment. First, the cells were seeded in a sixwell plate and transfected in the six-well plate. While transfecting and changing the medium, the six-well plate was scratched using a 200ul sterile tip, marked as 0 h, and a picture was taken. After 24 h, another picture was taken at the same marked location. The blank distance of the two pictures was compared statistically.

Co-immunoprecipitation (CO-IP)
The cells were rst transfected and then harvested to prepare protein samples, using the following steps: 1. Cells were harvested 24-48 h after transfection by adding an appropriate amount of cell lysis buffer (containing protease inhibitors), lysing them on ice for 30 min, and then centrifuging them12000 rpm for 30 min. 2. A microcentrifuge tube was taken for Western blot analysis, 1 µg of the corresponding antibody was added to the remaining lysate, which was added to the cell lysate, and then incubated at 4°C overnight.
3. An aliquot of 10 µL of protein A agarose beads was taken, and an appropriate amount of lysis buffer was used. The solution was washed thrice while centrifuging at 3,000 rpm for 3 min each time. 4. An aliquot of 10 µL of protein A agarose beads pretreated to the cell lysate was incubated overnight with the antibody and then incubated at 4 °C for 2-4 h for coupling of the Protein A agarose beads. 5. After the immunoprecipitation reaction, the mixture was centrifuged at 3,000 rpm for 3 min at 4 to settle the agarose beads at the bottom of the tube, after which the supernatant was carefully aspirated, and agarose beads were washed 3-4 times with 1 ml of lysis buffer. Finally, 15 µL of 2×SDS loading buffer was added and boiled for 5 min. 6. The proteins were then separated by SDS-PAGE and subjected to Western blot analysis.

In vivo experiments
For the in vivo invasion and metastasis assays . First, we divided the nude mice into seven groups, and injected HCC-LM3 cells from different treatments into the tail vein. After four weeks, the mice were sacri ced and the lung tissues of the nude mice were collected for HE staining and immunohistochemistry. Afterward,1×10 7 cells in 100 µL PBS were injected subcutaneously into the sides of male BALB/c-nu/nu mice (6-8 weeks old; n = 6 per group) (Hunan SJA Experimental Laboratory Animal Company) (Hunan, China). After six weeks, the mice were sacri ced (Using cervical dislocation method), and lung tissues were collected for HE staining and immunohistochemistry (IHC). All the animal experiments were approved by the Animal Experiment Ethics Committee of the Second A liated Hospital of Nanchang University and were carried out in accordance with the guidance of the British Animal (Scienti c Procedures) Act, 1986, and the EU Directive 2010/63/EU.

HE staining and immunohistochemistry (IHC)
The prepared tissues were xed with tissue xative, placed in para n blocks, and sectioned using a microtome. The tissue sections were rst dewaxed with xylene and then dehydrated with gradient ethanol. The sections were stained with H&E to identify any changes in their morphology and then rehydrated and microwaved in sodium citrate buffer (10 mmol/L, pH 6.0) to recover the antigen. The sections were incubated with 0.3% hydrogen peroxide/PBS for 30 min and then blocked with serum.
Subsequently, the tissue was incubated with 1:200 diluted rabbit monoclonal antibody NCAPG (ab226805, Abcam, Cambridge, MA, USA) at 4 °C overnight. It was then washed with PBS every 5 min for three times and then incubated with the secondary antibody at 37 °C for 30 min. Subsequently, the sections were stained with diaminobenzidine (DAB) and hematoxylin dye, after which the excess dye was washed with running water, rehydrated with gradient alcohol, and sealed with neutral resin. Finally, the sections were observed under an inverted microscope, and images were taken.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc.). All the data are expressed as mean ±standard deviation (SD). The two groups were compared using the t-test. Pearson's χ2 test was used to analyze the relationship between IFITM3 expression and NCAPG. All experiments in the study were repeated thrice. P values of <0.05 or 0.01 were considered statistically signi cant.

IFITM3 and NCAPG are highly expressed in HCC and signi cantly associated with prognosis
To evaluate the expression of IFITM3 and NCAPG in HCC, we rst analyzed the documented expression of IFITM3 and NCAP in HCC from the TCG1 database. We observed that both IFITM3 and NCAPG were highly expressed in HCC, and both high and low levels of expression were associated with poor HCC prognosis (Fig 1A-1D). We then performed PCR and Western blot on the liver cancer specimens and adjacent tissues from the 55 patients enrolled in our study (Fig 1E,1F,1I), and observed that the results were consistent with those in the database. We also performed PCR analysis on selected liver cell lines HL-7702, and hepatoma cell lines MHCC-97H, HCC-LM3, SMCC-7721, and Huh-7, and the results also indicated that the expression of IFITM3 and NCAPG in HCC cells was up-regulated (Fig 1G,1H). From these results, we suggest that the expression levels of IFITM3 and NCAPG are up-regulated in HCC tissue cells, which affects the prognosis of HCC patients.

The relationship between IFITM3 and NCAPG in HCC
In order to further understand the relationship between IFITM3 and NCAPG, we carried out bioinformatics analysis on multiple databases available online and observed that both IFITM3 and NCAPG were highly expressed in liver cancer tissues, with a positive correlation between the two ( Fig. 2A). This was consistent with our results of qRT-PCR analysis on the tissues (Fig. 2E). Furthermore, we downloaded samples from multiple databases such as TCGA/Oncomine, constructed a volcano map of 5000 gene expressions in LIHC (Fig. 2C), and predicted the target genes downstream of IFITM3 through the mirDIP and starBase databases (Fig. 2D). We then predicted on the String website that there may be a phosphorylation modi cation between IFITM3 and NCAPG (Fig. 2B). Finally, using CO-IP, we con rmed the presence of protein interactions between IFITM3 and NCAPG (Fig. 2F).

IFITM3 can positively regulate NCAPG
After understanding the relationship between IFITM3 and NCAPG, in order to further regulate the relationship between the two, we transfected the interfering fragments of IFITM3 (i.e., SiRNA) and plasmids into MHCC-97H and HCC-LM3, and observed the mRNA and protein expression of NCAPG. Our ndings con rmed that IFITM3 positively regulated NCAPG; thus, when downregulation of IFITM3 reduced the mRNA and protein levels of NCAPG (Fig. 3A-3C), and vice versa (Fig. 3D-3F). This indicated that IFITM3 positively regulated the expression level of NCAPG.

IFITM3 regulates NCAPG and affects the invasion and metastasis of HCC
After determining that IFITM3 could positively regulate NCAPG, we conducted a retrospective experiment to further corroborate our experimental results. We transfected the interfering fragments and plasmids of IFITM3, interfering fragments and plasmids of NCAPG, interfering fragments of IFITM3 and plasmids of NCAPG simultaneously, and plasmids of IFITM3 and interference fragments of NCAPG simultaneously, and compared them with the control group. We observed that when the expression was IFITM3 was upregulated, that of NCAPG was also enhanced, and vice versa (Fig. 4A-4D). Simultaneous transfection of the IFITM3 interference fragment and NCAPG plasmid or the IFITM3 plasmid and NCAPG interference fragment showed levels of NCAPG expression that were close to those of the control group. Regarding the relationship between IFITM3 and NCAPG on the invasion and metastasis ability of HCC cells, we observed through cell scratch experiments and Transwell experiments that the expression of IFITM3 and NCAPG was positively correlated with the ability of cell invasion and metastasis (Fig. 4E-4H). The upregulation of IFITM3 and NCAPG enhanced the ability of invasion and metastasis of the cells, while downregulation of both showed an opposite effect. On the other hand, simultaneous downregulation of IFITM3 and upregulation of NCAPG, or upregulation of IFITM3 and downregulation of NCAPG, showed no signi cant difference in the invasion and metastasis ability, compared to the control group. This indicated that IFITM3 regulated NCAPG to in uence the invasion and metastasis of HCC cells.

IFITM3 regulates NCAPG through phosphorylation modi cation
Regarding the speci c mechanism of action between IFITM3 and NCAPG, our data showed that there may be phosphorylation modi cations between IFITM3 and NCAPG, and IFITM3 could regulate NCAPG through the STAT3/CDK1 pathway (Fig. 2B). To this end, we up-regulated the expression of IFITM3 and observed the expression of STAT3/CDK1. We observed that downregulation of the expression of IFITM3 decreased the expression of STAT3/CDK1 (Fig. 6A-6F), while the upregulation of IFITM3 increased the expression of STAT3/CDK1. Furthermore, we observed that the expression of NCAPG was up-regulated after the use of STAT3/CDK1 activators and downregulated after the use of inhibitors (Fig. 6G-6L). This indicated that IFITM3 regulated NCAPG via STAT3/CDK1.

Con rmation of results through animal experiments
In vitro cell experiments indicated that IFITM3 regulated NCAPG to in uence the invasion and metastasis of HCC. In order to add credibility to our ndings, we performed experiments on animals. We observed that the degree of lung metastasis in mice injected with cells containing up-regulated IFITM3 and NCAPG was signi cantly higher than that in the control group, that in mice injected with cells containing downregulated IFITM3 and NCAPG was signi cantly lower, while injection with cells containing simultaneously up-regulated IFITM3 and downregulated NCAPG, or downregulated IFITM3 and upregulated NCAPG, showed no signi cant difference (Fig. 7A-7B). Thus, in vitro and in vivo experiments reiterate that IFITM3 could affect the expression of NCAPG through phosphorylation modi cation, which in turn in uenced the invasion and metastasis of HCC (Fig. 7C).

Discussion
In this study, we demonstrated that both IFITM3 and NCAPG were highly expressed in HCC tissues and cells, and their expression was closely related to the prognosis and recurrence of liver cancer. We also demonstrated for the rst time that IFITM3 regulated NCAPG through phosphorylation to affect the invasion and metastasis of HCC.
Several reports have suggested the involvement of IFITM3 in the development of cancer [36,37]. For example, studies have reported that IFITM3 up-regulated the expression of c-myc through the ERK1/2 signaling pathway, and promoted the proliferation of liver cancer cells. Studies have also demonstrated that IFITM3rs12252-CC was related to the poor differentiation and progression of HCC. Recently, IFITM3 has been reported to play an irreplaceable role in the development of novel coronaviruses [7,10,13,38]. In our research as well, IFITM3 was observed to play a critical role in HCC. The expression of IFITM3 was directly related to the prognosis of HCC patients, and could affect the invasion and metastasis of HCC. Western blot and qRT-PCR of the collected liver cancer specimens and adjacent tissues revealed that IFITM3 was abnormally expressed in liver cancer tissues. After silencing IFITM3, the invasion and metastatic ability of MHCC-97H and HCC-LM3 decreased, while the opposite was observed after the upregulation of IFITM3. IFITM3 is highly likely to be a novel marker of liver cancer, which represents an important breakthrough for the diagnosis and treatment of liver cancer.
NCAPG has been shown to play a vital role in biological growth and development [19,24,39,22]. For instance, NCAPG plays a role in promoting the recombination of smooth muscle chromosomes and can be used as a breeding gene to increase fertility in cows. NCAPG may also be used as a therapeutic target for the treatment of T2DM. Recent studies have also reported that NCAPG plays a vital role in the genesis and development of tumors. Studies have demonstrated that NCAPG acts as an oncogene in HCC, and promotes cell proliferation and anti-apoptosis by activating the PI3K/AKT/FOXO4 pathway [40][41][42].
Silencing NCAPG inhibits the proliferation of hepatoma cells and induces their apoptosis. We also con rmed by Western blot and qRT-PCR that NCAPG was abnormally expressed in HCC, and was closely related to the invasion and metastasis of liver cancer. Thus, NCAPG can be used as a marker for the diagnosis and treatment of liver cancer.
Phosphorylation is currently the most widely known post-translational modi cation [28,32,31]. It is estimated that nearly one-third of the proteins in eukaryotes can be phosphorylated at any time. At present, 518 kinds of kinases have been identi ed in human cells, while the plants Arabidopsis and rice have been estimated to possess 1000 and 1467 kinases, respectively. Based on the available databases, about 1-2 million proteins and sites have been estimated to be phosphorylated [43,33,35,34]. In actual research, with the help of mass spectrometry, about 62,679 phosphosites on 11,632 proteins in mammals are known to be phosphorylated. Thus, we know that phosphorylation is a highly common form of a posttranslational modi cation. The protein encoded by the STAT3 gene is a member of the STAT protein family. STAT family members are phosphorylated by receptor-associated kinases. Activated STAT3 directly affects the activity of its active proteins, which in turn affects the downstream genes. In our research, we observed that IFITM3 activated STAT3 through phosphorylation modi cation. The activated STAT3 promoted the phosphorylation of CDK1 and then regulated NCAPG, thereby affecting the invasion and metastasis of HCC cells.
In conclusion, our study demonstrated that IFITM3 and NCAPG were highly expressed in HCC, and the upregulation of both indicate poor postoperative prognosis. More importantly, we observed that IFITM3 could regulate NCAPG through STAT3 to affect the invasion and metastasis of HCC. IFITM3 and NCAPG may be used as biomarkers for the diagnosis and treatment of HCC, further enriching the treatment and diagnosis of liver cancer.

Conclusion
We observed that the expression of IFITM3 and NCAPG was signi cantly up-regulated in HCC tissues and cells, and which was strongly associated with the prognosis. Downregulation of IFITM3 and NCAPG decreased the invasion and metastasis ability of HCC cells, while their upregulation showed an opposite effect. More importantly, we can con rm that there is a phosphorylation modi cation between IFITM3 and NCAPG. IFITM3 regulates the expression of NCAPG through STAT3/CDK1, which in turn affects the invasion and metastasis of HCC.

Availability of data and materials
This article contains data to support the ndings of this study. According to reasonable requirements, the data and materials in this study can be obtained from the corresponding author.

Competing interest
The author declares no con icts of interest.

Consent for publication
Not applicable.

Ethics approval and consent to participate
All experiments strictly followed the panel's speci c guidelines for animal feeding, treatment, and euthanasia. All animal experiments were approved in writing by the Animal Experiment Ethics Committee of the Second A liated Hospital of Nanchang University, and all were implemented strictly in accordance with the "Guidelines for the Care and Use of Laboratory Animals." Funding Figure 1 The upregulation of IFITM3 and NCAPG in HCC is related to poor prognosis. A, B: Expression of IFITM3 and NCAPG in HCC in the TCGA database. C, D: Relationship between the expression of IFITM3 and NCAPG in the database and the prognosis of HCC. E, F: Expression of IFITM3 and NCAPG mRNA in liver cancer tissues and adjacent tissues. G, H: Expression of IFITM3 and NCAPG mRNA in liver cancer cell lines (MHCC-97H, HCC-LM3, SMCC-7721, Huh-7) and liver cell lines (HL-7702). I: Abnormal expression of IFITM3 and NCAPG protein in liver cancer tissues and adjacent tissues. Note: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2
Relationship between IFITM3 and NCAPG. A: XY analysis of IFITM3 and NCAPG in the database. B: String Shengxin analysis of the relationship between IFITM3 and NCAPG. C: Volcano graphs of the expression of IFITM3 and NCAPG in HCC analyzed by Shengxin. D: Analysis of the VENE diagram of downstream genes of IFITM3 from the database. E: Relationship between IFITM3 and NCAPG expression in liver cancer specimens. F: Co-immunoprecipitation of IFITM3 and NCAPG in HCC-LM3 and MHCC-97H hepatocellular carcinoma cells. Note: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.    In vitro experiments con rm that IFITM3 regulates NCAPG to promote HCC invasion and metastasis. A: HE staining and IHC of lung tissue of nude mice injected with HCC-LM3 cells. B: The number of lung metastases of nude mice in each group. C: Simulation diagram of the effect in this experiment. Note: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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