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Interaction of STIL with FOXM1 regulates SF3A3 transcription in the hepatocellular carcinoma development

Cell Division volume 20, Article number: 1 (2025) Cite this article

Abstract

Background

Dysregulation of SF3A3 has been related to the development of many cancers. Here, we investigated the functional role of SF3A3 in hepatocellular carcinoma (HCC).

Methods

SF3A3 expression in HCC tissues and cell lines was examined using RT-qPCR. Changes in malignant behavior of HCC cells after downregulation of SF3A3 were assessed by EdU, colony formation, flow cytometry, wound healing, and Transwell invasion assays. Multiple datasets were combined to identify the upstream modifiers of SF3A3. The binding relationship between STIL and FOXM1 was explored by co-IP assay, and the effect of STIL and FOXM1 on the binding of FOXM1 at the SF3A3 promoter was detected by ChIP-qPCR assay. A xenograft tumor model was established to explore the changes of tumors in vivo, and the expression of Ki67, GPC3, and p53 in tumor tissues was detected by immunohistochemistry.

Results

SF3A3 and STIL were overexpressed in HCC tissues and cells, and downregulation of SF3A3 or STIL inhibited the malignant behavior of HCC cells by promoting the expression of p53. An interaction between STIL and FOXM1 regulated the SF3A3 expression in HCC cells. Knockdown of FOXM1 further enhanced the anti-tumor effects of STIL loss on HCC cells in vitro and in vivo, whereas SF3A3 overexpression overturned the impact of STIL loss on HCC cells in vitro and in vivo.

Conclusions

Our findings indicate that STIL/FOXM1 expedites HCC development by activating SF3A3, which highlights the importance of SF3A3 as a promising prognostic marker and therapeutic target for HCC.

Background

Liver cancer is a major global health burden and is projected to disturb more than 1 million individuals annually by 2025, and hepatocellular carcinoma (HCC) is the most common subtype of primary liver cancer (90%) [1]. The identification of powerful biomarkers for surveillance and early HCC diagnosis is still lacking, with available serum biomarkers showing low sensitivity and specificity [2]. Several online algorithms have been developed to identify prognostic biomarkers for the study of molecular mechanisms of tumorigenesis and progression and the discovery of important therapeutic targets [3]. In the study here, splicing factor 3 A subunit 3 (SF3A3) was revealed as the most outstanding prognostic biomarker in HCC in the UALCAN database (http://ualcan.path.uab.edu/index.html).

SF3A3, together with other two subunits, SF3A1 and SF3A2, constitutes the SF3a complex of human U2 snRNP [4]. Even though SF3A3 has been identified as a DNA repair-related gene that predicted poor prognosis in liver cancer [5], its functional role in HCC has not been well described. Interestingly, loss of SF3A3 has been reported to reduce the growth of bladder cancer cells, and the transcription factor E2F6 recruited KDM5C, a H3K4me2/3 demethylase, to the SF3A3 promoter, resulting in high SF3A3 expression [6]. It reminded us to explore a similar mechanism regarding SF3A3 in HCC. The combined dataset query showed that SCL-interrupting locus protein (STIL) was one of the SF3A3-correlated and differentially expressed genes with prognostic value in HCC. STIL is a cell cycle-regulated protein recruited at the mitotic centrosome to facilitate the duplication of centrioles in dividing cells [7]. More relevantly, it was found to be highly expressed in HCC and may serve as an independent prognostic indicator and a potential oncogene in HCC [8]. STIL has been revealed to interact with forkhead box protein M1 (FOXM1) to enhance its transcriptional activity and consequently regulate tumor metastasis and stemness in lung cancer [9]. FOXM1 potentiated the progression of cancer by activating the transcription of its targets, and multiple proteins or RNA have been revealed to interact with FOXM1 and stabilize FOXM1 expression in cancer cells [10, 11]. Since overexpression of FOXM1 was found to be related to aggressive tumor features and dismal prognosis of HCC [12], we conjectured that the interaction between STIL and FOXM1 induced the SF3A3 expression in HCC cells, contributing to the progression of HCC. In the current study, we have investigated the mechanism of how STIL/FOXM1 complex is involved in the oncogenic role of SF3A3 in HCC focused on understanding the relationship between SF3A3 and STIL/FOXM1 axis.

Results

SF3A3, an outstanding prognostic biomarker, is highly expressed in HCC tissues and cell lines

We downloaded the list of predicted prognostic markers in HCC from the UALCAN database [13], in which we found SF3A3 to be the first-ranked prognostic marker (Supplementary Material). Both transcription and protein expression of SF3A3 were elevated in HCC tissues in the UALCAN database (Fig. 1A, B), while overexpression of SF3A3 was related to lower survival of HCC patients (Fig. 1C).

Fig. 1
figure 1

SF3A3 is upregulated and links to unsatisfactory outcomes of HCC patients. (A) Transcription level of SF3A3 (transcript per million) in primary tumor tissues and normal tissues of HCC patients in the UALCAN database. (B) The protein expression of SF3A3 in primary tumor tissues and normal tissues of HCC patients in the UALCAN database. (C) The survival of HCC patients with high SF3A3 expression (n = 90) or medium/low SF3A3 expression (n = 275). (D) SF3A3 mRNA expression in tumor and adjacent tissues from HCC patients using RT-qPCR (n = 30). (E) SF3A3 mRNA expression in HCC cell lines Hep3B and Huh7 and normal hepatocyte cell line THLE-2 using RT-qPCR. All data are expressed as mean ± SD from three experiments. In panel D, significance is determined by paired t-test, and in panel E, significance is determined by one-way ANOVA. **p < 0.01, ****p < 0.0001

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SF3A3 in tumor and adjacent tissues of HCC patients (n = 30) was evaluated using RT-qPCR assay and observed that SF3A3 expression was augmented in tumor tissues (Fig. 1D). RT-qPCR was also conducted to assess SF3A3 expression in HCC cell lines Hep3B and Huh7 and a normal hepatocyte cell line THLE-2. The expression of SF3A3 was much higher in HCC cells than in THLE-2 cells (Fig. 1E).

Downregulation of SF3A3 inhibits the malignant behavior of HCC cells and activates the p53 expression

We downregulated SF3A3 expression in the Hep3B and Huh7 cells, and the success of SF3A3 downregulation was examined by RT-qPCR (Fig. 2A). The proliferative capacity of the cells was assessed by EdU assay and colony formation assay, and the proliferative capacity of the HCC cells was decreased after SF3A3 downregulation (Fig. 2B, C). The results of the flow cytometry assay revealed increased apoptosis in Hep3B and Huh7 cells in response to KD-SF3A3 (Fig. 2D). Cell migration and invasion were analyzed using wound healing assay as well as Transwell invasion assay. SF3A3 downregulation significantly reduced the migration and invasion of both Hep3B and Huh7 cell lines (Fig. 2E, F).

Fig. 2
figure 2

Downregulation of SF3A3 inhibits malignant behavior in HCC cells. (A) SF3A3 mRNA expression in HCC cell lines Hep3B and Huh7 treated with KD-SF3A3 or KD-NC using RT-qPCR. Evaluation of cell proliferation using EdU assay (B) and colony formation assay (C) in Hep3B and Huh7 cells. (D) Detection of apoptosis using flow cytometry in Hep3B and Huh7 cells. Cell migration and invasion were assessed by wound healing assay (E) and Transwell invasion assay (F) in Hep3B and Huh7 cells. (G) The p53 protein expression in Hep3B and Huh7 cells after KD-SF3A3 was assessed by western blot analysis. (H) The protein expression of Cleaved-caspase3, Bax, Ki67, and GPC3 in Hep3B and Huh7 cells after KD-SF3A3 alone or in combination with the p53 inhibitor was assessed by western blot analysis. All data are expressed as mean ± SD from three experiments. In panels A-H, significance is determined by two-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001

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It has been reported that SF3A3 knockdown can induce the expression of classic tumor suppressor signal p53 in non-small cell lung cancer cells [14]. We examined the effect of knockdown of SF3A3 on p53 expression in HCC cells. A significant increase in protein expression of p53 was observed after knockdown of SF3A3 (Fig. 2G). HCC cells with knockdown of SF3A3 were treated with the p53 inhibitor Pifithrin-β hydrobromide (PFT-β). Knockdown of SF3A3 elevated expression of apoptotic proteins Cleaved-caspase3 and Bax and decreased expression of proliferative markers Ki67 and GPC3 in HCC cells, which was reversed by the p53 inhibitor treatment (Fig. 2H).

STIL is highly expressed in HCC patients and knockdown of STIL inhibits the malignant behavior of HCC cells

To investigate the cause for the overexpression of SF3A3 genes in HCC, we downloaded the list of SF3A3 positively correlated genes in the UALCAN database and analyzed the transcriptome analysis of HCC and adjacent non-tumor liver tissues (n = 10) from the GSE202853 dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE202853). The differentially expressed genes (adj. p. value < 0.01 and |logFC| > 2), genes positively correlated with SF3A3 (Pearson correlation coefficient (CC) > 0.7), and prognostic markers in HCC (p. value < 0.01) were intersected. There were four intersecting genes: CDCA8, KIF2C, CDC20, and STIL (Fig. 3A). The roles of CDCA8 [15, 16], KIF2C [17, 18], and CDC20 [19, 20] in HCC have been fully confirmed. The UALCAN database showed that STIL expression was enhanced in tumor tissues of HCC patients (Fig. 3B) and was related to dismal patient prognosis (Fig. 3C).

Fig. 3
figure 3

STIL is overexpressed in HCC and knockdown of STIL inhibits the malignant behavior of HCC cells. (A) The intersection of SF3A3-positively correlated genes and prognostic markers in the UALCAN database, and differentially expressed genes in the GSE202853 dataset. (B) Transcription level of STIL (transcript per million) in primary tumor tissues and normal tissues of HCC patients in the UALCAN database. (C) The survival of HCC patients with high STIL expression (n = 90) or medium/low STIL expression (n = 275). (D) STIL mRNA expression in tumor tissues and corresponding adjacent tissues from HCC patients using RT-qPCR (n = 30). (E) STIL mRNA expression in HCC cell lines Hep3B and Huh7 and normal hepatocyte cell line THLE-2 using RT-qPCR. (F) STIL mRNA expression in HCC cell lines Hep3B and Huh7 treated with KD-STIL or KD-NC using RT-qPCR. (G) SF3A3 mRNA expression in HCC cell lines Hep3B and Huh7 treated with KD-STIL or KD-NC using RT-qPCR. (H) STIL mRNA expression in HCC cell lines Hep3B and Huh7 treated with KD-SF3A3 or KD-NC using RT-qPCR. Evaluation of cell proliferation using EdU assay (I) and colony formation assay (J) in Hep3B and Huh7 cells. (K) Detection of apoptosis using flow cytometry in Hep3B and Huh7 cells. Analysis of cell migration and invasion by wound healing assay (L) and Transwell invasion assay (M) in Hep3B and Huh7 cells. All data are expressed as mean ± SD from three experiments. In panel D, significance is determined by paired t-test; in panel E, significance is determined by one-way ANOVA; in panel F-M, significance is determined by two-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001

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We detected by RT-qPCR in tumor and adjacent tissues from our cohort and revealed that the STIL expression was much higher in tumor tissues (Fig. 3D). STIL expression was also higher in Hep3B and Huh7 cells than in THLE-2 cells (Fig. 3E).

In Hep3B and Huh7 cells, the expression of STIL was downregulated artificially, and the success of STIL downregulation was verified using RT-qPCR (Fig. 3F). The mRNA expression of SF3A3 was also significantly decreased after STIL downregulation (Fig. 3G). However, there was no significant difference in STIL expression in cells after the knockdown of SF3A3, as detected by RT-qPCR (Fig. 3H). The proliferative capacity of the cells was assessed using the EdU assay and colony formation assay. The proliferative capacity of the cells was significantly decreased after STIL downregulation (Fig. 3I, J). Flow cytometry showed increased apoptosis after STIL downregulation (Fig. 3K). Wound healing assay and Transwell invasion assay revealed that cell migration and invasion were reduced by STIL downregulation (Fig. 3L, M).

STIL interacts with FOXM1 to induce SF3A3 expression

STIL has been reported to enhance its transcriptional activity of downstream targets through protein interaction with the transcription factor FOXM1 in lung cancer [9]. Interestingly, FOXM1 is a positively associated gene for SF3A3 (Pearson CC = 0.61) in the UALCAN database. Figure 4A shows multiple binding sites for FOXM1 at the promoter of SF3A3 from the hTFtarget database (http://bioinfo.life.hust.edu.cn/hTFtarget#!/). The GEPIA database (http://gepia.cancer-pku.cn/index.html) showed that both FOXM1 and STIL were significantly and positively correlated with SF3A3 expression in HCC (Fig. 4B, C).

Fig. 4
figure 4

An interaction between STIL and FOXM1 induces SF3A3 expression. (A) The presence of binding sites for FOXM1 at the promoter of SF3A3 was predicted in the hTFtarget database. The positive correlation between FOXM1 (B) and STIL (C) with SF3A3 expression in the GEPIA database. (D) The binding relationship between STIL and FOXM1 was analyzed using co-IP. (E) FOXM1 mRNA expression in HCC cell lines Hep3B and Huh7 treated with KD-FOXM1 or KD-NC using RT-qPCR. (F) The effect of KD-STIL alone or in combination with KD-FOXM1 on FOXM1 binding at the SF3A3 promoter was analyzed using ChIP. (G) Changes in SF3A3 promoter luciferase activity were analyzed using dual-luciferase reporter assays. (H) SF3A3 mRNA expression after downregulation of STIL alone or combined downregulation of FOXM1 and STIL detected by RT-qPCR. (I) FOXM1 protein expression in Hep3B and Huh7 cells after knockdown of STIL. All data are expressed as mean ± SD from three experiments. In panels E-I, significance is determined by two-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

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We then confirmed the binding relationship between STIL and FOXM1 by Co-IP experiments (Fig. 4D). Successful FOXM1 downregulation in Hep3B and Huh7 cells was examined by RT-qPCR (Fig. 4E). The results of ChIP-qPCR experiments showed that knockdown of STIL inhibited the binding of FOXM1 at the SF3A3 promoter, and even fewer SF3A3 promoter fragments were observed in Hep3B and Huh7 cells with further knockdown of FOXM1 (Fig. 4F). We examined the regulation of SF3A3 transcription by STIL and FOXM1 using dual-luciferase reporter assays which demonstrated that knockdown of STIL reduced the luciferase activity of SF3A3 promoter luciferase reporter vector, and further silencing of FOXM1 downregulated the luciferase activity (Fig. 4G). A decrease in SF3A3 mRNA expression was found after the knockdown of STIL, which was further reduced after the combined knockdown of FOXM1, as revealed by RT-qPCR (Fig. 4H). It should be noted that FOXM1 protein expression showed no alteration in the cell lines after the knockdown of STIL (Fig. 4I). STIL does not affect the expression of FOXM1 but rather binds to it to influence the expression of SF3A3.

Combined knockdown of STIL and FOXM1 regulates SF3A3 transcription to suppress malignant behavior in HCC cells

To further determine whether SF3A3 is a downstream effector of the STIL/FOXM1 complex in HCC, Hep3B and Huh7 cells were treated with KD-STIL + KD-FOXM1 (KD-STIL + KD-NC as control) or KD-STIL + OE-SF3A3 (KD-STIL + OE-NC as control). Firstly, RT-qPCR was conducted to detect the expression of SF3A3 in Hep3B and Huh7 cells treated with KD-STIL + OE-SF3A3 or KD-STIL + OE-NC (Fig. 5A). Using EdU assay and colony formation assay, it was found that the EdU-positive cells and colonies formed were further inhibited by KD-FOXM1, while enhanced by OE-SF3A3 (Fig. 5B-C). Flow cytometry assay revealed increased apoptosis after KD-FOXM1 and decreased apoptosis after OE-SF3A3 in the presence of KD-STIL in HCC cells (Fig. 5D). Furthermore, decreased migration and invasion were observed after KD-FOXM1, and amplified cell migration and invasion were noted after OE-SF3A3 in cells (Fig. 5E, F).

Fig. 5
figure 5

Combined knockdown of STIL and FOXM1 regulates SF3A3 transcription to suppress malignant behavior of HCC cells. (A) SF3A3 mRNA expression in HCC cells treated with KD-STIL + OE-NC or KD-STIL + OE-SF3A3 was assessed using RT-qPCR. Evaluation of cell proliferation using EdU assay (B) and colony formation assay (C) in Hep3B and Huh7 cells. (D) Detection of apoptosis using flow cytometry in Hep3B and Huh7 cells. Analysis of cell migration and invasion by wound healing assay (E) and Transwell invasion assay (F) in Hep3B and Huh7 cells. All data are expressed as mean ± SD from three experiments. In panels A-F, significance is determined by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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KD-FOXM1 strengthens and OE-SF3A3 weakens the anti-tumor effects of KD-STIL in vivo

Stably transfected Huh7 cells with KD-STIL or KD-SF3A3 alone (KD-NC as control for both) or with KD-STIL + KD-FOXM1 (KD-STIL + KD-NC as control), KD-STIL + OE-SF3A3 (KD-STIL + OE-NC as control) were subcutaneously injected into mice to form xenograft models. Over the 30 days, the tumors formed by Huh7 cells with KD-SF3A3 and KD-STIL were smaller than those formed by KD-NC-treated Huh7 cells (Fig. 6A). In the presence of KD-STIL, the knockdown of FOXM1 further reduced tumor volume, and overexpression of SF3A3 enlarged tumor volume (Fig. 6B). The weight of tumors weighed on day 30 showed consistent trends where KD-SF3A3 and KD-STIL contributed to lighter tumors (Fig. 6C). Meanwhile, the simultaneous knockdown of FOXM1 resulted in the most pronounced effects on lowering tumor weight, whereas overexpression of SF3A3 further increased tumor burden (Fig. 6D). The mRNA expression of SF3A3 was determined in the tumor tissues of mice with different treatments. As expected, the knockdown of SF3A3 or STIL both downregulated the expression of SF3A3 (Fig. 6E). Further decline or gain of SF3A3 mRNA expression was observed in tumor tissues from mice with KD-FOXM1 or OE-SF3A3, respectively (Fig. 6E). Lastly, the expression Ki67, GPC3, and p53 in the tumor tissues was examined using immunohistochemistry. It was found that the positive staining of Ki67 and GPC3 was reduced, while the expression of p53 was increased following the knockdown of STIL or SF3A3 (Fig. 6F). Still, the KD-FOXM1 further downregulated the expression of Ki67 and GPC3 and enhanced p53 expression. Overexpression of SF3A3 restored the expression of Ki67 and GPC3 and repressed p53 in the presence of KD-STIL (Fig. 6G).

Fig. 6
figure 6

Combined knockdown of STIL and FOXM1 regulates SF3A3 transcription to suppress HCC cell growth in vivo. (A) Measurement of tumor volume changes in mice injected with KD-NC-, KD-SF3A3-, and KD-STIL-treated Huh7 cells. (B) Measurement of tumor volume changes in mice injected with KD-STIL + KD-NC-, KD-STIL + KD-FOXM1-, KD-STIL + OE-NC-, and KD-STIL + OE-SF3A3-treated Huh7 cells. (C) Changes in tumor weight in the KD-NC, KD-SF3A3, and KD-STIL groups. (D) Changes in tumor weight in the KD-STIL + KD-NC, KD-STIL + KD-FOXM1, KD-STIL + OE-NC, and KD-STIL + OE-SF3A3 groups. (E) The mRNA expression of SF3A3 in the tumor tissues of mice in the seven groups. (F) Expression of Ki67, GPC3, and p53 in the KD-NC, KD-SF3A3, and KD-STIL groups was detected by immunohistochemistry. (G) Expression of Ki67, GPC3, and p53 in the KD-STIL + KD-NC, KD-STIL + KD-FOXM1, KD-STIL + OE-NC, and KD-STIL + OE-SF3A3 groups was detected by immunohistochemistry. All data are expressed as mean ± SD (n = 5). In panels A-B, significance is determined by two-way ANOVA; in panels C-G. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Discussion

We observed overexpression of SF3A3 and STIL in HCC tissues and cells in the study here. Mechanistically, we found that STIL interacted with FOXM1 and activated the transcription of SF3A3. Furthermore, we identified the anti-tumor effects of STIL knockdown were strengthened by FOXM1 knockdown and blunted by SF3A3 upregulation.

Consistent with what we predicted from the UALCAN database, SF3A3 was found to be one of the genes significantly associated with overall survival in HCC [21]. Hakobyan et al. revealed that mutations in some of these genes of the splicing machinery, including SF3A3, were significantly enriched in melanomas as compared to benign nevi [22]. García-Cárdenas et al.. also showed that SF3A3 was a central element of a spliceosome-related cluster involving RNA-binding proteins and cancer driver genes in breast cancer [23]. Functionally, the knockdown of SF3A3 has been recently revealed to suppress the growth of acute promyelocytic leukemia cells, along with G1/S cell cycle arrest [24]. Olst et al. demonstrated the silencing of SF3A3 contributed to the activation of p53 in human non-small cell lung cancer cells [14]. Here, the loss-of-function assay using shRNA targeting SF3A3 also showed that the malignant aggressiveness of HCC cells was hampered after SF3A3 silencing, which was related to the activation of p53 since the p53 inhibitor led to enhanced expression of Ki67 and GPC3 in the presence of SF3A3 knockdown. p53 is known for its central role in triggering various types of cell death and intrinsic apoptosis is initiated by the direct interaction of p53 with anti-apoptotic Bcl-2-family members [25]. The counterpart of SF3A3, SF3A2 has been recently reported to participate in the regulation of extrinsic and intrinsic apoptosis, resulting in cisplatin resistance in triple-negative breast cancer cells [26]. These findings suggested the knockdown of SF3A3 might be a novel target for the p53-mediated apoptosis induction in HCC. However, whether SF3A3 exerts a similar anti-apoptotic function in HCC in a p53-independent manner deserves further study.

Under the condition of breast cancer, Ciesla et al. revealed that MYC promoted SF3A3 translation via an eIF3D-dependent mechanism [27]. In the present study, STIL was identified as a possible regulator of SF3A3 and was demonstrated to elicit tumor-promoting properties in HCC. STIL has been summarized to be enhanced in most cancer types relative to their adjacent tissues, and STIL overexpression was linked to worse outcomes and promoted the progression of malignancies [28]. As for its functional role, osteosarcoma cell proliferation, migration, and invasion decreased, while apoptosis increased after silencing of STIL [29]. We also observed similar effects of knockdown of STIL in HCC cells. Mechanistically, Ito et al. found that STIL formed a ternary complex with ARHGEF7 and PAK1, and the knockdown of STIL impeded the accumulation of ARHGEF7-PAK1 complex within membrane ruffles and attenuated the phosphorylation of PAK1 substrates and cortical actin remodeling at the migrating front [30]. Moreover, a robust CEP85-STIL binding is necessary for PLK4 activation and directional cancer cell migration [31]. The present study showed that FOXM1 was a positively correlated gene with SF3A3 and shared multiple binding sites on the SF3A3 promoter. We subsequently substantiated that STIL knockdown reduced the binding between FOXM1 and the SF3A3 promoter.

FOXM1, a member of forkhead transcription factors, has emerged as an essential contributor to cancer progression, including HCC [32], and the protein and RNA interactions of FOXM1 have been summarized [33]. For instance, long non-coding RNA-encoded peptide PINT87aa has been revealed to bind to the DNA-binding domain of FOXM1 to regulate the expression of PHB2, and FOXM1 gain-of-function partially reduced the proportion of senescent HCC cells and enhanced mitophagy [34]. Kurahashi et al. showed that hepatocyte FOXM1 served as a critical modulator to orchestrate liver inflammation in hepatocarcinogenesis [35]. GPC3 is a cell-surface glycoprotein that is frequently overexpressed in HCC [36], and GPC3 and Ki67 are two of the strongest predictors of xenograft engraftment [37]. Our in vivo evidence also demonstrated that the silencing of FOXM1 further downregulated the protein expression of GPC3 and Ki67 in the presence of STIL knockdown in the tumors which also exhibited smaller size and lighter weight.

Conclusion

In summary, our study demonstrated that SF3A3 acts as a prognostic biomarker and oncogene in HCC by repressing the p53 activation. Importantly, we identified the STIL-FOXM1 axis supported the growth of HCC cells through activating SF3A3 expression. These findings provide a critical role of SF3A3 in HCC development and convincing evidence to support SF3A3 as a prognostic marker and therapeutic target for HCC.

Materials and methods

Patient and tissue collection

Tumors and corresponding adjacent tissues from 30 patients with HCC were collected from the First Hospital of Qiqihar between February 2021 and June 2023. All collected tissues were examined histopathologically and stored in liquid nitrogen. The research protocol was approved by the Ethics Committee of the First Hospital of Qiqihar, and each patient signed a written informed consent.

Cell culture and transfection

Human HCC cell lines (Hep3B and Huh7) were procured from Procell (Wuhan, Hubei, China), and transformed human liver epithelial-2 (THLE-2) was obtained from the ATCC (Manassas, VA, USA). All cell lines were cultured in DMEM containing 10% FBS and penicillin/streptomycin in a humid environment of 37 °C and 5% CO2. Cell lines were identified with short tandem repeat and tested negative for mycoplasma contamination.

Hep3B and Huh7 cells were subjected to 2 µg/mL Puromycin Dihydrochloride (ST551-10 mg, Beyotime Biotechnology Co., Ltd., Shanghai, China) treatment for 72 h. Cells stably infected with shRNA plasmids for SF3A3, STIL, FOXM1, and overexpressing SF3A3 plasmid were generated using lentivirus. The transfection efficacy was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). PFT-β (T3637, TargetMol, Boston, MA, USA) was used to treat cells at a concentration of 23 µM (EC50) for 24 h. DMSO-treated cells served as controls.

RNA isolation and RT‑qPCR

Total RNA in Hep3B, Huh7, and THLE-2 cells was isolated using TRIzol reagent (R0016, Beyotime) and reverse transcribed into cDNA using the RevertAid RT Reverse Transcription Kit (K1691, Thermo Fisher Scientific Inc., Waltham, MA, USA). cDNA amplification was performed using BeyoFast SYBR Green qPCR Mix (2X) premix (D7260-1 ml, Beyotime) to measure the expression of SF3A3, STIL, and FOXM1 in cells. All genes were normalized to the expression of β-actin. Relative expression was calculated using the 2−ΔΔCt method. Primer sequences are shown in Table 1.

Table 1 Primer sequences for RT-qPCR
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Cell proliferation analyses

The proliferation of Hep3B and Huh7 cells was evaluated using the EdU Cell Proliferation Assay Kit (E607204, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). EdU solution was added at a ratio of 500:1 in a 24-well plate to make 2X EdU solution, which was added to the original medium of the cells to obtain 1X EdU solution. The cells in each well were incubated with 300 µL of EdU-containing medium for 2 h, fixed with 4% paraformaldehyde, and permeabilized for 10 min at room temperature. After being incubated with 100 µL of configured assay mix and with 300 µL of 1X Hoechst staining solution in the dark (both at room temperature for 30 min), the cells were viewed under a fluorescence microscope.

For colony formation assays, stably transfected Hep3B and Huh7 cells (600 cells/well) were grown in 6-well plates and cultured for two weeks till the cells formed visible colonies. The medium was renewed at an interval of 4 to 5 days during cell growth. Cell colonies were fixed, stained with 0.5% (w/v) crystal violet for 20 min, imaged using a scanner, and quantitatively analyzed using ImageJ software.

Flow cytometry

Staining was performed using the ANNEXIN V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (CA1020, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China). First, 2 × 106 Hep3B and Huh7 cells (200 µL) were centrifuged at 1000 g for 5 min at room temperature and suspended in 100 µL of binding buffer. The cells were treated with 5 µL of ANNEXIN V-FITC for 15 min at 37 °C in the dark, followed by incubation with 10 µL of PI for 10 min with gentle shaking and loading into flow cytometry.

Wound healing and transwell invasion assays

For the wound healing assay, the cells were seeded into 6-well plates to obtain complete monolayers. Wounds were created on the cell monolayer using a small pipette tip. After washing with PBS, the cells were incubated with serum-free DMEM medium for 24 h. Each wound was photographed at 0 h and 24 h, and cell migration was thus measured.

To determine changes in cell invasion, Transwell 24-well plates (Merck Millipore, Darmstadt, Germany) were selected, and 106 Hep3B and Huh7 cells (200 µL) grown in serum-free DMEM were seeded in the apical chamber pre-coated with Matrigel. Then, 600 µL of medium containing 10% FBS was supplemented to the basolateral chamber. After 24 h, invaded cells were fixed with 4% formaldehyde and stained with 0.1% (w/v) crystal violet for 20 min. Randomly selected areas were counted microscopically.

ChIP-qPCR

Hep3B and Huh7 cells were cross-linked with 1% (v/v) formaldehyde. After sonication of the cells, lysates were pre-cleared in BSA-sealed protein A/G beads and incubated overnight at 4 °C with specific antibody to FOXM1 (2 µg/mg of lysate, ab245309, Abcam, Cambridge, MA, USA) and rabbit IgG isotype control (1 µg/mL, GTX35035, GeneTex, Inc., Alton Pkwy Irvine, CA, USA). The DNA was eluted and de-crosslinked overnight at 4 °C. The eluted DNA was used as a template for qPCR, and the input control was the supernatant before precipitation (5%). Input Dilution factor = 0.05− 1 = 20. Fold enrichment (relative to IgG) calculations of SF3A3 promoter fragments were performed using the 2−∆∆Ct method. ∆Ct (normalized ChIP) = Ct(ChIP)-[Ct(Input)-Log220], ∆∆Ct = ∆Ct (normalized ChIP) - ∆Ct (IgG).

Co‑immunoprecipitation (Co-IP) assay

The collected cells were lysed using RIPA buffer to release intracellular proteins and protease inhibitors were added to prevent protein degradation. The cells were probed with antibodies to STIL (2 µg/mg of lysate, ab89314, Abcam), FOXM1 (2 µg/mg of lysate, ab245309, Abcam), rabbit IgG isotype control (1 µg/mL, GTX35035, GeneTex) for 1 h at 4 °C to form antigen-antibody complexes, which were bound to Protein A/G agarose overnight to capture immunoprecipitated complexes. The immunoprecipitation complexes were washed with PBS to remove nonspecifically bound proteins. The precipitated proteins were separated by SDS-PAGE and transferred to a membrane to detect the protein expression using western blot assays.

Promoter reporters and dual-luciferase assay

The amplified SF3A3 promoter was inserted into the pGL4.2-basic-Luc reporter plasmid (Promega Corporation, Madison, WI, USA) using restriction endonuclease. pGL4.2-basic-Luc reporter plasmid and the internal control plasmid, pRL-TK (D2760-1 µg, Beyotime) were delivered into Hep3B and Huh7 cells reaching 70% confluence in 24-well plates with KD-STIL, KD-FOXM1 plasmids or empty vectors. After 48 h of transfection, reporter gene activity was analyzed using the Dual-Luciferase Assay Kit (D0011, Solarbio) according to the manufacturer’s instructions. Comparison of results between Hep3B and Huh7 cells transfected with KD-FOXM1, KD-STIL plasmid, or empty vector by normalizing pGL4.2-basic-Luc-SF3A3 promoter luciferase-reported activity to pRL-TK Rluc-reported activity.

Western blot analysis

Hep3B and Huh7 cells were lysed using RIPA lysis buffer containing protease inhibitors. Protein concentrations were determined using a BCA protein assay kit (PC0020, Solarbio). Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in PBS/Tween-20 containing 5% BSA and primarily blotted with primary antibodies against FOXM1 (1:1000, ab207298, Abcam), STIL (1:2000, ab89314, Abcam), p53 (1:5000, 10442-1-AP, ProteinTech Group, Chicago, IL, USA), Cleaved-caspase3 (1:1000, #9661, Cell Signaling Technologies, Beverly, MA, USA), Bax (1:2000, 50599-2-Ig, ProteinTech Group), Ki67 (1:5000, ab209897, Abcam), Glypican-3 (GPC3, 1:1000, ab95363, Abcam), and GAPDH (1:1000, 3683 S, Cell Signaling Technologies) overnight at 4℃. The goat anti-rabbit immunoglobulin G-horseradish peroxidase (1:2000, ab205718, Abcam) was used as a secondary antibody. Western blots were processed by enhanced chemiluminescence and analyzed using ImageJ Software.

Lentiviral infection and xenograft mouse models

For lentiviral infection, 4 × 105 Huh7 cells were incubated with 1 × 108 IU virus, and 5 µg/mL polybrene (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 24 h. The cells were treated with 2 µg/mL of puromycin and cultured continuously for 72 h. SF3A3, STIL, FOXM1 shRNA plasmid, and overexpression of SF3A3 plasmid were stably introduced into cells. The protocol for the xenograft tumor model experiments was approved by the Institutional Animal Care and Use Committee of the First Hospital of Qiqihar.

In the in vivo experiments, 4-week-old BALB/c nude mice (n = 40) procured from Vital River (Beijing, China) were randomly divided into the following 8 groups: the control (Huh7 cells without infection), KD-NC, KD-SF3A3, KD-STIL, KD-STIL + OE-NC, KD-STIL + OE-SF3A3, KD-STIL + KD-NC, KD-STIL + KD-FOXM1 groups. Huh7 cells were injected into the flanks of nude mice, and subcutaneous tumor formation was observed starting 5 days after injection. The tumor size was measured every 5 days using vernier calipers. Tumor volume was calculated as (length × width2)/2. At 30 days after injection, mice were euthanized under deep anesthesia, and tumors were collected for weighing immunohistochemistry, and mRNA expression analysis.

Immunohistochemistry

Paraffin-embedded and 4% paraformaldehyde-fixed xenograft tumor tissues were made into 4-µm-thick sections, deparaffinized with xylene and ethanol, and heated by microwave for 15 min in 0.1 mol/L citrate buffer (pH = 6.0). After cooling down to room temperature, the sections were incubated in 3% hydrogen peroxide for 20 min to block endogenous peroxidase and sealed with 10% BSA to inhibit nonspecific binding. The sections were probed with primary antibodies to Ki67 (1:200, 9027 S, Cell Signaling Technologies), GPC3 (1:100, ab95363, Abcam), and p53 (1:1000, 10442-1-AP, ProteinTech Group) at 4 °C overnight and with rabbit anti-IgG H&L (HRP) (1:1000, ab6721, Abcam) for 1 h. All sections were labeled with DAB substrate, counter-stained with hematoxylin, and observed. The intensity of positive staining was analyzed.

Quantification and statistical analyses

Graph Prism 8.0.2 (GraphPad, San Diego, CA, USA) was used for statistical analyses, and the data are presented as the mean ± SD from three experiments. The paired t-test was used to compare the significant difference between the 2 groups, and a one-way or two-way analysis of variance (ANOVA) test was performed to compare the differences among groups with Tukey’s correction for multiple testing. P values less than 0.05 were considered to be statistically significant.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

HCC:

Hepatocellular carcinoma

SF3A3:

Splicing factor 3 A subunit 3

STIL:

SCL-interrupting locus protein

FOXM1:

Forkhead box protein M1

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

FITC:

Fluorescein isothiocyanate

PI:

Propidium iodide

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Authors and Affiliations

  1. Second Department of General Surgery, the First Hospital of Qiqihar, No. 700, Pukui avenue, Long sha District, Qiqihar, Heilongjiang, 161000, P. R. China

    Haijun Zhang & Ziqi Wu

  2. Department of Pharmacy, the Second Affiliated Hospital of Qiqihar Medical College, Qiqihar, Heilongjiang, 161006, P. R. China

    Lin Zhang

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Contributions

LZ conceived the study and conducted the experiments; ZQW is responsible for data collection, analyzed and interpreted the data; HJZ wrote the manuscript and revised the manuscript and important intellectual content. All authors read and approved the final manuscript.

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Correspondence to Haijun Zhang.

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Zhang, H., Zhang, L. & Wu, Z. Interaction of STIL with FOXM1 regulates SF3A3 transcription in the hepatocellular carcinoma development. Cell Div 20, 1 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13008-025-00142-4

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Cell Division

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