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ZSCAN16 expedites hepatocellular carcinoma progression via activating TBC1D31

Cell Division volume 19, Article number: 31 (2024) Cite this article

Abstract

Objective

Hepatocellular carcinoma (HCC) is fatal and poses great challenges to early diagnosis and effective treatment. This paper sought to expound the function of Zinc finger and SCAN domain-containing protein 16 (ZSCAN16) and TBC1 domain family member 31 (TBC1D31) in HCC progression.

Methods

ZSCAN16 and TBC1D31 levels were detected by RT-qPCR, Western blot, and immunohistochemistry. The transcriptional regulation of TBC1D31 by ZSCAN16 was demonstrated by ChIP-qPCR and dual-luciferase assay. Colony formation assay, migration and invasion assays, TUNEL staining, CCK-8 assay, flow cytometry, and western blot analysis were adopted to evaluate the biological activity of HCC cells. The role of the ZSCAN16/TBC1D31 axis in HCC was demonstrated by lentiviral gene intervention combined with functional rescue experiments. Hep3B cells were used to establish a nude mouse xenograft tumor model to study the role of the ZSCAN16/TBC1D31 axis in vivo.

Results

ZSCAN16 and TBC1D31 were highly expressed in HCC. Downregulation of ZSCAN16 repressed the proliferation, migration, and invasion of HCC cells while promoting apoptosis, as well as curbing tumor growth in vivo. Mechanistic studies showed that ZSCAN16 mediated the transcriptional activation of TBC1D31, which in turn led to tumor development. TBC1D31 overexpression reversed the inhibitory effect of ZSCAN16 knockdown on the malignant behavior and tumor growth of HCC cells and accelerated tumor development.

Conclusion

ZSCAN16 mediates transcriptional activation of TBC1D31 and promotes HCC progression.

Background

Hepatocellular carcinoma (HCC) is a prevalent and fatal cancer worldwide and a major challenge in global healthcare [1]. The incidence of HCC has been on the rise for the past 30 years, and more than 1 million people will be affected annually by 2025 [2]. Moreover, HCC is typically detected at advanced stages when it is unresectable in many parts of the world, making these treatments ineffective [3]. Effective systemic and local treatment prolongs survival in patients with advanced HCC and enables them to undergo surgery [4]. Therefore, a proper understanding of the molecular mechanisms of HCC is the key to the search for effective targeted therapies [5].

Zinc finger and SCAN domain-containing (ZSCAN) transcription factors (TFs), which comprise the smallest and most recently defined subgroup of the Zinc finger (ZNF) family, may promote or inhibit angiogenesis, apoptosis, cell migration, and invasion, and stem cell characteristics in diverse tumors [6]. Through dataset analysis and intersection, we focused on a new TF, ZSCAN16. ZSCAN16, also known as ZNF392 or ZNF435, is expressed in all tested tissues and is capable of homoassociation [7]. Even though ZSCAN16 antisense RNA 1, the long noncoding RNA transcribed from the opposite strand of the ZSCAN16 gene has been reported to promote the malignant properties of HCC [8], the expression pattern and possible roles of ZSCAN16 in HCC progression are rarely studied. Dissecting the function and molecular mechanism of ZSCAN16 might provide more insights into the known oncogenic role of the ZNF family members in the progression of HCC [9, 10].

For the downstream factor of ZSCAN16, database analysis directed our focus to TBC1D31, a member of the TBC1 domain family, also called WDR67. TBC domain family members are highly homologous and highly conserved [11]. The TBC domain is often strung together with other domains associated with cell membrane function, indicating that TBC domain proteins may mediate the occurrence and development of cancer or other diseases [12]. For example, TBC1D23 is widely expressed in human tissues and correlated with tumor size, differentiation, metastasis, and unfavorable prognosis in lung cancer [13]. TBC1D3 is expressed in human tissues and overexpressed in prostate, breast, pancreatic, and bladder cancers [14]. WDR6, a member of the WDR superfamily, promotes HCC progression [15]. However, there is little information about TBC1D31, let alone its role in HCC. Therefore, this paper was to investigate the potential expression and mechanism of ZSCAN16 and TBC1D31 in HCC, hoping to provide novel biomarkers for HCC diagnosis and treatment.

Results

ZSCAN16 is significantly elevated in HCC patients and correlates with poor prognosis

Differentially expressed genes (DEGs) in HCC were analyzed in the GEO database (p. adj. < 0.01), where dataset GSE57957 was used to analyze transcriptome differences between 39 tumors and 39 adjacent non-tumorous samples and dataset GSE46408 was used to analyze transcriptome differences between 6 primary HCCs and their corresponding nontumorous liver parenchyma (Fig. 1A). We also downloaded human TFs from Human TFDB (http://bioinfo.life.hust.edu.cn/HumanTFDB#!/) and obtained seven intersecting genes of the DEGs in GSE57957 and GSE46408 datasets and downloaded human TFs via Jvenn: ZGPAT, CSRNP1, ZSCAN16, ATF5, CCDC88A, EBF3, and TCF3 (Fig. 1B). Of these, only ZSCAN16 has not been studied and reported in HCC. Analysis by UALCAN (https://ualcan.path.uab.edu/index.html) showed that ZSCAN16 was markedly highly expressed in HCC (Fig. 1C). It was also highly expressed in datasets GSE57957 (LogFC = 0.60545552) and GSE46408 (LogFC = 1.6864497). In addition, Kaplan-Meier Plotter (http://kmplot.com/analysis/index.php?p=background) analysis found that HCC patients with high ZSCAN16 expression had lower recurrence-free survival (RFS) (Fig. 1D). However, whether ZSCAN16 also acts as an oncogene in HCC remains to be further revealed.

Fig. 1
figure 1

ZSCAN16 was significantly upregulated in human HCC cells and tissues. A: The volcano maps of DEGs in the GSE57957 and GSE46408 datasets. B: Intersection genes in human TFs and DEGs (p. adj. < 0.01) in GSE57957 and GSE46408 datasets obtained from Jvenn. C: Expression of ZSCAN16 in HCC was analyzed on UALCAN. D: RFS of HCC patients with high or low expression of ZSCAN16 was analyzed on Kaplan-Meier Plotter. E: mRNA expression of ZSCAN16 in human HCC tissues and paracancerous tissues was detected by RT-qPCR (n = 12). F: Comparison of ZSCAN16 protein expression in human HCC and paracancerous tissues by IHC scoring (n = 12). G, H: RT-qPCR and WB for detection of transcriptional and translational levels of ZSCAN16 in THLE-2 and HCC cell lines. All cell experiments were repeated three times independently. Comparisons between two groups were analyzed by paired t-test (EF), and comparisons between multiple groups were analyzed by ANOVA (GH). * p < 0.05 compared with adjacent tissue or THLE-2 cells

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To investigate the potential role of ZSCAN16 in HCC, we first detected the expression of ZSCAN16 in HCC tissues and paracancerous liver tissue samples. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) demonstrated that ZSCAN16 was greatly upregulated in HCC tissues compared with that in paracancerous liver tissues (Fig. 1E, F). In addition, in two human HCC cell lines (Hep3B and PLC/PRF/5), the mRNA and protein levels of ZSCAN16 were significantly upregulated compared with those in THLE-2 cells (Fig. 1G, H).

Knockdown of ZSCAN16 represses malignant growth of HCC cells

To elucidate the functional role of ZSCAN16 in HCC, we used sh-ZSCAN16 or sh-Scramble to infect HCC cell lines (Hep3B and PLC/PRF/5). The mRNA expression of ZSCAN16 was significantly reduced in HCC cells following sh-ZSCAN16 infection (Fig. 2A). Colony formation assay showed that ZSCAN16 knockdown curbed the colony formation ability of Hep3B and PLC/PRF/5 cells (Fig. 2B). ZSCAN16 knockdown also significantly hindered cell migration and invasion (Fig. 2C, D). TUNEL and flow cytometry revealed that apoptotic cells were visibly upregulated in HCC cells with stable knockdown of ZSCAN16 (Fig. 2E, F). Western blot assay for the expression of EMT-related markers N-cadherin and Snail and apoptosis-associated protein Cleaved Caspase-3 showed that knockdown of ZSCAN16 downregulated the expression of N-cadherin, Snail, and upregulated Cleaved Caspase-3 expression in Hep3B and PLC/PRF/5 cells (Fig. 2G).

Fig. 2
figure 2

ZSCAN16 knockdown represses malignant biological behaviors in HCC cells. A: The infection effect of sh-ZSCAN16 in HCC cells was examined using RT-qPCR. B: The proliferation of HCC cell lines treated with knockdown ZSCAN16 was examined using a colony formation assay. C: Migration assay of HCC cell lines with knockdown ZSCAN16. D: Analysis of the invasive ability of HCC cell lines with knockdown ZSCAN16. E: Apoptosis in HCC cell lines with knockdown ZSCAN16 treatment was examined using TUNEL assay. F: Apoptosis in HCC cell lines with knockdown ZSCAN16 treatment was examined using flow cytometry. G: The protein expression of N-cadherin, Snail, and Cleaved Caspase-3 in HCC cell lines with knockdown ZSCAN16 treatment was examined using WB. All cell experiments were independently repeated three times. Comparisons between two groups were performed using the unpaired t-test (ABCDEF), and comparisons between multiple groups were analyzed by ANOVA (G). * p < 0.05 compared with the sh-Scramble group

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In addition, we overexpressed ZSCAN16 in THLE-2 cells using lentiviral vectors as well. After verifying the successful overexpression of ZSCAN16 using RT-qPCR (Fig S1A), we conducted colony formation, wound healing, and Transwell assays. As expected, the normal THLE-2 cells showed enhanced potential to grow (Fig S1B), migrate (Fig S1C), and invade (Fig S1D) in the presence of ZSCAN16 upregulation. In addition, the TUNEL assay (Fig S1E) and flow cytometry (Fig S1F) showed a significant decrease in apoptosis in THLE-2 cells stably overexpressing ZSCAN16.

ZSCAN16 knockdown inhibits malignant progression of HCC in vivo

To further elucidate the effect of ZSCAN16 on HCC tumors, we set up animal experiments. We established an HCC xenograft tumor model by subcutaneously injecting sh-ZSCAN16-treated Hep3B cells into nude mice (Hep3B cells were chosen because of their more prominent pro-carcinogenic effect in the above cell experiments). RT-qPCR found that the mRNA level of ZSCAN16 was decreased in tissues after sh-ZSCAN16 treatment (Fig. 3A). ZSCAN16 knockdown inhibited HCC growth during the 32-d monitoring (Fig. 3B). At the end of the experiment, the tumor weight was reduced compared with the control group (Fig. 3C). In tumor tissues formed by Hep3B cells with sh-ZSCAN16, the protein level of Ki67, N-cadherin, and Snail was reduced (Fig. 3D), while the expression of Cleaved Caspase-3 was enhanced. Consistently, the apoptosis of subcutaneous tumor cells was elevated as well (Fig. 3E). Altogether, ZSCAN16 knockdown inhibited HCC progression in vivo.

Fig. 3
figure 3

ZSCAN16 knockdown inhibits HCC progression in vivo. A: mRNA expression of ZSCAN16 in tumor tissues formed by Hep3B cells with sh-ZSCAN16 was detected by RT-qPCR. B: Subcutaneous tumor volume was measured every four days during the subcutaneous tumorigenicity assay in nude mice. C: After the tumorigenicity assay and nude mice were euthanized, subcutaneous tumors were collected and weighed. D: Expression of Ki67, N-cadherin, Snail, and Cleaved Caspase-3 in mouse tumor tissues was detected by IHC. E: TUNEL to detect apoptosis of subcutaneous tumor tissues in mice. All animal experiments were independently repeated five times. Comparisons between two groups were analyzed by unpaired t-test (A, C, D, E), and comparisons between multiple groups were analyzed by ANOVA (B). * p < 0.05 compared with the sh-Scramble group

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ZSCAN16 knockdown inhibits transcriptional activation of TBC1D31

To investigate the downstream molecular mechanisms of ZSCAN16, we downloaded the first 1500 downstream targets of ZSCAN16 from hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/), which were intersected with DEGs (p.adj. < 0.01) in the datasets GSE57957 and GSE46408 on Jvenn, and 12 intersecting genes were obtained: FAM49B, CPD, CKAP2L, C1RL, CENPE, ACTG2, KPNA2, KIF20A, TBC1D31, INCENP, ORC6, OLFML2B (Fig. 4A). Only TBC1D31 had not been reported in HCC. Therefore, it was selected for further analysis. UALCAN prediction revealed that TBC1D31 (WDR67) was significantly highly expressed in HCC (Fig. 4B). Similarly, TBC1D31 was greatly highly expressed in GSE57957 (LogFC = 0.7929022) and GSE46408 (LogFC = 1.8427321) datasets. Kaplan-Meier Plotter analysis noted that HCC patients with high expression of TBC1D31 (WDR67) had worse RFS (Fig. 4C). In the LIHC samples in the TCGA Tumor database of GEPIA (http://gepia.cancer-pku.cn/index.html) and liver samples in the GTEx database, the analysis revealed that ZSCAN16 was significantly positively correlated with TBC1D31 expression (Fig. 4D). Finally, analysis in the ChIP-seq database on UCSC (https://genome.ucsc.edu/cgi-bin/hgGateway) revealed that ZSCAN16 had a significant binding peak in the TBC1D31 promoter region (Fig. 4E). Thus far, we hypothesized that ZSCAN16 mediates transcriptional activation of TBC1D31 to promote HCC progression.

Fig. 4
figure 4

TBC1D31 is a target of ZSCAN16 in HCC cells. A: The first 1500 downstream targets of ZSCAN16 downloaded from hTFtarget were intersected with DEGs (p. adj. < 0.01) in the GSE57957 and GSE46408 datasets obtained on Jvenn; B: UALCAN predicted the expression of TBC1D31 (WDR67) in HCC. C: Kaplan-Meier Plotter to analyze the prognostic significance of TBC1D31 (WDR67) in HCC. D: Correlation between ZSCAN16 and TBC1D31 in LIHC samples in the TCGA Tumor database and in liver samples in the GTEx database was analyzed on GEPIA. E: ChIP-seq database in the UCSC to analyze the binding of ZSCAN16 in the promoter region of TBC1D31. F: RT-qPCR to detect the transcription level of TBC1D31 in HCC tissues and paracancerous liver tissues. G: IHC to analyze the protein expression of TBC1D31 in HCC tissues. H: RT-qPCR to detect the expression of TBC1D31 in THLE-2 and HCC cell lines. I: WB detection of protein levels of TBC1D31 in THLE-2 and HCC cell lines. J: RT-qPCR detection of mRNA expression of TBC1D31 in HCC cell lines with ZSCAN16 knockdown. K: ChIP-qPCR detection of DNA fragments enriched by ZSCAN16 antibody. L: Dual-luciferase assay detected the relationship between ZSCAN16 and TBC1D31. All cellular experiments were independently repeated three times. The comparison between two groups was performed using the paired t-test (FG) or unpaired t-test (JL), and ANOVA analysis was used for comparisons among multiple groups (HIK). * p < 0.05 compared with adjacent tissue, THLE-2 cells, or the sh-Scramble group

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To further determine whether TBC1D31 plays a role in the biological behavior of HCC cells, we evaluated the levels of TBC1D31 in HCC tissues and paracancerous liver tissues by RT-qPCR and IHC analysis. The mRNA and protein levels of TBC1D31 in HCC tissues were substantially higher than those in paracancerous tissues (Fig. 4F-G). Meanwhile, RT-qPCR and WB results showed that the transcriptional and translational levels of TBC1D31 were markedly higher in HCC cell lines than in THLE-2 cells (Fig. 4H-I).

To further explore whether TBC1D31 is a target of ZSCAN16, we analyzed the mRNA expression of TBC1D31 in HCC cell lines with stable knockdown of ZSCAN16 using RT-qPCR. TBC1D31 transcriptional levels were similarly significantly downregulated after ZSCAN16 silencing (Fig. 4J). ChIP-qPCR and dual-luciferase assays further confirmed that ZSCAN16 bound to the TBC1D31 promoter in HCC cells and that there was an enrichment of the TBC1D31 promoter fragment in DNA fragments enriched by the ZSCAN16 antibody (Fig. 4K-L). Taken together, TBC1D31 is a transcriptional target of ZSCAN16.

TBC1D31 is an important factor in ZSCAN16-mediated malignant progression of HCC

Therefore, we treated HCC cells with knockdown lentivirus of ZSCAN16 combined with overexpression lentivirus of TBC1D31. RT-qPCR revealed that the downregulated TBC1D31 mRNA expression by knockdown of ZSCAN16 alone was completely reversed by TBC1D31 overexpression, whereas the combined treatment of TBC1D31 overexpression did not affect ZSCAN16 expression (Fig. 5A). CCK-8 analysis showed that TBC1D31 overexpression reversed the proliferation of HCC cells inhibited by knockdown of ZSCAN16 alone (Fig. 5B). Cell migration and invasion assays displayed that TBC1D31 overexpression in HCC cells rescued the migratory and invasive abilities of HCC cells inhibited by ZSCAN16 knockdown (Fig. 5C-D). Flow cytometry and TUNEL assays revealed that the apoptotic rate of HCC cells upregulated by ZSCAN16 knockdown was reversed by TBC1D31 overexpression (Fig. 5E-F). Meanwhile, we found that upregulated N-cadherin and Snail, and downregulated Cleaved Caspase-3 expression were all reversed by TBC1D31 overexpression in the presence of sh-ZSCAN16 (Fig. 5G). Overall, the pro-carcinogenic effect of ZSCAN16 in HCC is mediated by TBC1D31.

Fig. 5
figure 5

TBC1D31 overexpression enhances the biological activities of HCC cells in the presence of sh-ZSCAN16. A: The expression of ZSCAN16 and TBC1D31 in HCC cells infected with sh-ZSCAN16 + oe-NC or sh-ZSCAN16 + oe-TBC1D31 by RT-qPCR. B: CCK-8 to analyze the viability of HCC cell lines. C: Migration ability of HCC cells detected by the migration assay. D: Analysis of the invasive ability of HCC cell lines by the migration assay. E: Apoptosis in HCC cell lines was examined using the TUNEL assay. F: Apoptosis of HCC cells was examined using flow cytometry. G: The protein expression of N-cadherin, Snail, and Cleaved Caspase-3 in HCC cell lines was examined using WB. All cell experiments were independently repeated three times. Comparisons between groups were analyzed by unpaired t-test (CDEF), and comparisons between multiple groups were analyzed by ANOVA (ABG). * p < 0.05 compared with the sh-ZSCAN16 + oe-NC group

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ZSCAN16/TBC1D31 axis is essential for HCC malignant progression in vivo

To further validate the above results in vivo, we established an HCC xenograft tumor model. Hep3B cells co-treated with sh-ZSCAN16 + oe-TBC1D31 were injected subcutaneously into nude mice. TBC1D31 overexpression reversed the inhibitory effect of ZSCAN16 knockdown on HCC growth (Fig. 6A). Tumor weight and subcutaneous tumor volume growth trend had the same result, i.e., overexpression of TBC1D31 reversed the tumor weight growth inhibited by ZSCAN16 knockdown alone (Fig. 6B). IHC revealed that the protein expression of Ki67, N-cadherin, and Snail was also increased, while that of Cleaved Caspase-3 was decreased in the tumor tissues formed by Hep3B cells overexpressing TBC1D31 (Fig. 6C). TUNEL staining revealed a significant decrease in TUNEL-labeled cells after co-transfection (Fig. 6D). Lastly, we found that TBC1D31 overexpression had no significant effect on the protein level of ZSCAN16, while only the protein level of TBC1D31 was significantly upregulated in the tumor tissues (Fig. 6E).

Fig. 6
figure 6

Overexpression of TBC1D31 induces tumor growth in nude mice in the presence of ZSCAN16 knockdown. A: The subcutaneous tumor volume was measured every four days during the subcutaneous tumorigenic assay in nude mice. B: After the tumorigenic assay and the nude mice were euthanized, the subcutaneous tumors were collected and weighed. C: IHC detected the expression of Ki67, N-cadherin, Snail, and Cleaved Caspase-3 in the tumor tissues of nude mice. D: TUNEL detected the apoptosis of the subcutaneous tumor cells in nude mice. E: IHC analysis of protein expression of ZSCAN16 and TBC1D31 in tumor tissues. All animal experiments were independently repeated five times. Comparisons between groups were analyzed by unpaired t-test (BCDE), and comparisons among multiple groups were analyzed by ANOVA (A), * p < 0.05 compared with the sh-ZSCAN16 + oe-NC group

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Discussion

Despite the notable progress achieved, the overall outcomes of HCC patients are still not satisfactory [16]. Further investigations are required to enhance the management and outcomes of HCC. Hence, we investigated the expression and potential mechanisms of ZSCAN16 and TBC1D31 in HCC and illustrated the high expression of ZSCAN16 and TBC1D31. In mechanism, ZSCAN16 facilitated TBC1D31 transcriptional activation and promoted malignant biological behaviors in HCC cells.

In this study, Kaplan-Meier analysis revealed that HCC patients with high ZSCAN16 expression had a lower RFS, indicating that ZSCAN16 may be linked with the prognosis of HCC. Likewise, ZSCAN16 was upregulated and notably affected the overall survival of patients with late-onset ovarian serous cystadenocarcinoma [17]. Additionally, our further cellular and animal experimental findings noted the repressing effect of ZSCAN16 knockdown on HCC cell growth in vitro and tumor progression in vivo. There was a dearth of research on the oncogenic function of ZSCAN16. Only several studies displayed the anti-HCC effect of a new lncRNA ZSCAN16‑AS1 silencing. Patients with high ZSCAN16‑AS1 levels had worse prognoses, and ZSCAN16‑AS1 knockdown curbed HCC cell viability in vitro and restricted tumor growth in vivo [8]. ZSCAN16-AS1 silencing inhibited HCC cell proliferation and invasion, while accelerating apoptosis [18]. Besides, other ZNF family members have been implicated in HCC progression. Elevated ZNF384 levels in HCC tissues were markedly correlated with tumor recurrence and poor overall survival, while ZNF384 downregulation hindered HCC cell proliferation by inhibiting Cyclin D1 levels [19]. ZNF233 was notably upregulated in HCC tissues and tightly linked to tumor grade, pathologic T stage, and poorer overall survival [20]. Altogether, this paper initially revealed the significant implication of ZSCAN16 in HCC. In addition, another member of the ZNF family, ZNF281 has been identified as an EMT driver in HCC [21]. Therefore, we hypothesized that ZSCAN16 exerted a similar function here. The induction of EMT is orchestrated by several core EMT TFs, including Snail, and EMT plays pleiotropic roles to promote the expression of mesenchymal genes, such as N-cadherin [22]. Here, we found the expression of these two markers was downregulated by ZSCAN16 knockdown not only in HCC cells in vitro but also in tumor tissues formed by HCC cells, which preliminarily evidenced our hypothesis.

During the further downstream investigation, sample analysis in the database revealed a positive correlation between the expression of ZSCAN16 and TBC1D31, and ZSCAN16 exhibited a significant binding peak in the TBC1D31 promoter region, indicating TBC1D31 was a transcriptional target of ZSCAN16. Furthermore, database analysis revealed the high expression of TBC1D31 in HCC, consistent with the expression patterns in cells and tissue samples. Additionally, HCC patients with high TBC1D31 (WDR67) expression had a lower RFS, indicating that TBC1D31 may be linked with the development and prognosis of HCC. Similarly, WDR48 was substantially overexpressed in HCC, and high WDR48 was correlated with poor RFS [23]. Upregulated WDR12 was associated with shorter overall survival and recurrence in HCC patients [24]. The subsequent loss-of-function assays unveiled that TBC1D31 overexpression nullified the protective effects of ZSCAN16 knockdown. TBC1D16 appeared to be a driver for melanoma, and its overexpression stimulates cell growth, drug resistance, and melanoma progression [25]. TBC1D3 promoted clear cell renal cell carcinoma proliferation and was positively linked with CD4+ T cell infiltrating levels [14]. More relevantly, enhanced WDR6 levels were greatly related to increased tumor size and distant metastasis and promoted HCC progression possibly by reprogramming the immune microenvironment [15]. In addition to promoting HCC cell proliferation and inhibiting apoptosis, WDR4, which was activated by the TF MYC, enhanced metastasis and sorafenib resistance [26]. WDR74 was markedly elevated in HCC tissues, and WDR74 deficiency prevented HCC cell proliferation by inhibiting G1/S cell cycle turnover and inducing apoptosis [27].

There are several limitations. First, although cell and animal experiments confirmed the expression, association, and mechanism of ZSCAN16 and TBC1D3D, their diagnostic and prognostic value in HCC has not been explored in our enrolled patients. Second, there were many downstream targets of ZSCAN16, but we only studied TBC1D3D. Finally, the downstream pathways of TBC1D3D were not investigated. Other members of the TBC1 domain family mainly function through the JNK/p38 MAPK cascade [28] and ERK pathway [13], which are also implicated in HCC progression [29, 30]. Therefore, more research should be performed to confirm the diagnostic and prognostic value of ZSCAN16 and TBC1D3D, other downstream targets of ZSCAN16, and the possible downstream pathways.

In conclusion, our data highlighted the high levels of ZSCAN16 and TBC1D3D and their important oncogenic roles in HCC. Gene-targeted therapy has emerged as a new avenue for cancer medicine. Targeting oncogenic ZSCAN16 and TBC1D3D in HCC may have profound clinical benefits.

Materials and methods

Patients and tissue samples

Tumor and paraneoplastic non-tumor liver tissues were collected from 12 HCC patients who underwent hepatectomy from June 2020 to December 2022 at Pingxiang People’s Hospital (none of these patients had other malignancies and had not received radiotherapy). All patients signed a written informed consent, and the procedure was performed in strict accordance with the Declaration of Helsinki. In addition, all samples were used for RT-qPCR and IHC.

Cell culture and reagents

HCC cell lines Hep3B (CL-0102, Procell, Wuhan, China) and PLC/PRF/5 (CRL-8024, ATCC, USA) were cultured in minimum essential medium (MEM) (PM150410, Procell) (Hep3B) or Eagle’s MEM (30-2003, ATCC) (PLC/PRF/5) supplemented with 10% FBS and 1% P/S. Transformed human liver epithelial-2 (THLE-2, CRL-2706, ATCC) was cultured in BEGM (CC-3170, Lonza, Fair Lawn, NJ, USA) supplemented with 10% FBS, 5 ng/mL EGF (SRP3027, Merck KGaA, Darmstadt, Germany), and 70 ng/mL phosphoethanolamine (P3275, Merck KGaA). The cultures were all placed in a humidified incubator with 5% CO2 at 37 °C.

Gene expression intervention

Lentiviruses encoding human ZSCAN16 knockdown, TBC1D31 knockdown, and TBC1D31 overexpression were obtained from Origene (Beijing, China), and the corresponding scramble lentiviruses were used as control treatments. Specifically, HCC cells were seeded in the corresponding medium at 106 cells the day before lentiviral infection. After 24 h, when confluence reached 70%, lentivirus with a titer of 3 × 108 TU/mL was added with a multiplicity of infection of 15. Stably infected cells were screened using puromycin after 24 h of infection, and finally, the effect of genetic intervention in infected cells was verified by RT-qPCR.

RT-qPCR

Total RNA was extracted from cells or tissues using Biozol Total RNA Extraction Reagent (BN20537, Biorigin, Beijing, China) and reverse transcribed to cDNA using Random Hexamer and FastKing cDNA First Strand Synthesis Kit (KR116-01, Tiangen, Beijing, China). SYBR Green Master Mix (A46012, Thermo Fisher Scientific, Rockford, IL, USA) was employed for qPCR according to the manufacturer’s recommendations. The primers designed were as follows: ZSCAN16, forward: 5’-TGCACACCATCCAGAGACTGGA-3’, reverse: 5’-CTTGTCCATGAGTATGTCCCGTC-3’; TBC1D31, forward: 5’-ATTGCCAGCTCCACCTGAAAGC-3’, reverse: 5’-AAGAGCTGCCTAGCTTCCAAGC-3’; GAPDH, forward: 5’-GTCTCCTCTGACTTCAACAGCG-3’, reverse: 5’- ACCACCCTGTTGCTGTAGCCAA-3’. The results were analyzed by the 2−ΔΔCT method with GAPDH as a control.

IHC

HCC tissue or paracancerous tissue sections were deparaffinized in xylene, rehydrated with ethanol, and added with 3% H2O2 for blocking endogenous peroxidase activity, followed by the addition of 0.01 M sodium citrate buffer and microwave heating for antigen retrieval. The slides were subsequently pre-incubated in 10% goat serum overnight at 4 °C with the primary antibodies against ZSCAN16 (1:50, NBP1-85184, Novus Biologicals, Littleton, CO, USA), N-cadherin (1:500, ab76011, Abcam, Cambridge, UK), Snail (1:100, A5243, ABclonal, Wuhan, Hubei, China), Cleaved Caspase-3 (1:100, PAF5-114687, Thermo Fisher), Ki67 (1:100, 701198, Thermo Fisher) and TBC1D31 (1:200, NBP1-81818, Novus Biologicals). Then, goat anti-rabbit IgG H&L (HRP) (1:20,000, ab205718, Abcam) was added for 2-h incubation. Afterward, the DAB substrate was added to develop the color, and sections were counterstained with hematoxylin and observed.

The IHC intensity was recorded as 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). Stained area scores were 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%) and 4 (> 75%). The IHC score was calculated by multiplying the intensity score by the stained area score [31].

Western blot (WB)

Total protein was extracted from cells using RIPA lysis buffer (AR0105-100, Boster, Wuhan, Hubei, China). Protein concentration was then measured using the BCA kit (P0012, Beyotime, Shanghai, China). The extracted proteins were separated via SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies ZSCAN16 (1:250, NBP1-85184, Novus Biologicals), TBC1D31 (1:500, NBP1-81818, Novus Biologicals), N-cadherin (1:5000, ab76011), Snail (1:1000, A5243, ABclonal, Wuhan, Hubei, China), and Cleaved Caspase-3 (1:1000, PAF5-114687, Thermo Fisher) overnight, with GAPDH antibody (1:2500, ab9485, Abcam) as a control. Secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:10000, ab205718, Abcam) was added for 1-h incubation. A super-sensitive ECL luminescence reagent (MA0186, Meilunbio, Dalian, Liaoning, China) was added to develop the color.

Colony formation assay

HCC and THLE-2 cells from different treatments were seeded into 6-well plates containing fresh medium at approximately 1000 cells/well and then cultured in MEM at 37 °C for 2 weeks. The cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% (w/v) crystal violet for 30 min, then counted, and imaged using a microscope. ImageJ software was used for counting the number of colonies [32].

Cell migration assay

HCC and THLE-2 cells were seeded in 6-well plates with fresh DMEM. When the cells grew to a tight cell monolayer, they were scratched and recorded using a 200 µL sterile plastic pipette tip (set to 0 h), and the cell surface layer was washed with PBS to remove cell debris. After the medium was removed, serum-free DMEM was added to each well. After 48 h of incubation, wound photographs were recorded using a microscope and analyzed by ImageJ software.

Cell invasion assay

Transwell chambers were previously coated with 100 µL of 1:8 diluted Matrigel (M8370, Solarbio, Beijing, China) and incubated for 5 h. Subsequently, 5 × 104 differently treated HCC and THLE-2 cells in 200 µL of serum-free medium were placed in the upper chamber, and 600 µL of DMEM containing 10% FBS was loaded into the lower chamber. After incubation at 37 °C for 36 h, the Matrigel in the upper chamber was gently removed with cotton swabs. Cells invading the lower chamber were then fixed with 4% paraformaldehyde and stained with crystal violet. Cells were photographed using a fluorescence microscope and counted using a microscope [33].

TUNEL

Cell apoptosis was examined using the one-step TUNEL Apoptosis Assay Kit (C1090, Beyotime). Briefly, HCC and THLE-2 cells with different treatments were added with PBS to resuspend the cell precipitates, fixed with 4% paraformaldehyde at room temperature for 15 min, centrifuged at 600 × g for 5 min, and resuspended in PBS. Next, 25 µL of cell suspensions were smeared on slides, dried, immersed, and permeabilized with 100 µL of 0.1% Trixon X-100 at 37 °C for 10 min. After rinsing with PBS, 100 µL of TUNEL assay solution was added dropwise to each sample at 37 °C for 25 min. Subsequently, the cell smears were incubated with DAPI for 5 min in the dark, and the nuclei were counterstained. The staining results were analyzed under a fluorescence microscope and photographed.

Cell viability assay

Cell Counting Kit-8 (CCK-8, C0005, TargetMol Chemicals, Shanghai, China) was used for analysis. HCC cells with different treatments were seeded in 96-well plates at 3,000 cells and pre-incubated with 100 µL of MEM containing 10% FBS in an incubator (37 °C, 5% CO2) for 24 h. The incubation was then continued for 0, 24, 48, 72, and 96 h. At each moment, 10 µL of CCK-8 was added for another 2 h of culture. The 96-well plates were shaken on a shaker for 1 min to ensure that the color of the plate was uniform. Finally, optical density (OD) values were measured at 450 nm using a microplate reader, and cell viability was calculated.

Flow cytometry

Apoptosis analysis was performed using the APC-Annexin V/PI Apoptosis Kit (A6030S, UElandy Biotechnology, Suzhou, Jiangsu, China). Specifically, when HCC and THLE-2 cells were cultured to 85% confluence, an appropriate amount of trypsin (without EDTA) was added to digest the cells, and the blown-down cells were transferred to a centrifuge tube and centrifuged at 10,00×g for 5 min. The supernatant was discarded, and the cells were gently resuspended with PBS and counted. About 5 × 104 resuspended cells were mixed with 5 µL of Annexin V-APC and then incubated with 5 µL of PI at 25 °C for 15 min in dark. Afterward, 400 µL of 1× Annexin V binding buffer was supplemented to resuspend the cells, and then apoptosis was assessed immediately.

Subcutaneous xenograft tumor

The animal experimental protocol was approved by the Ethical Review Committee of Pingxiang People’s Hospital and was conducted in strict compliance with the Guidelines for the Care and Use of Laboratory Animals. Six-week-old female BALB/c nude mice (401, Charles River) were placed in a 12-h light/dark cycle, with free access to food and water. After one week of acclimatization, nude mice were assigned into four groups (sh-NC, sh-ZSCAN16, sh-ZSCAN16 + oe-NC, sh-ZSCAN16 + oe-TBC1D31) with five mice randomized to each group. Mice were subcutaneously injected with 2 × 106 Hep3B cells. When the tumor reached 50 mm3, it was set as the starting point of the experiment. The subcutaneous tumor volume was measured every four days, and the volume calculation formula was V = 0.5 × L × W2 (V, volume; L, length; W, width [34]. After 32 days (the end point of the experiment), the nude mice were euthanized by intraperitoneal injection of 150 mg/kg sodium pentobarbital, and the tumors were collected, weighed, and made into paraffin sections for IHC [35].

Chromatin immunoprecipitation (ChIP)-qPCR

HCC cells were cross-linked for protein-DNA interactions with formaldehyde, followed by the addition of glycine to quench unreacted formaldehyde. Cells from each dish were scraped into 1× PBS, centrifuged at 800× g for 5 min, and resuspended in SDS lysis buffer on ice. After centrifugation again, the cells were resuspended in a nuclear lysis buffer. The resulting suspension was subjected to fragmentation by sonication and shaking, and probed with anti-ZSCAN16 (1:20, RAB-S238, Absolute Antibody), Mouse IgG2a Isotype Control-ChIP Grade (1:50, ab18413, Abcam), and Pierce Protein A beads (88846, Thermo Scientific) overnight at 4 °C, followed by reverse cross-linking. Purified DNA fragments were obtained for qPCR analysis.

Dual luciferase assay

HCC cells were plated in 96-well plates for 24 h. After that, cells were co-transfected with pGL3-basic (E1751, Promega Corporation, Madison, WI, USA) inserted with TBC1D31 promoter sequence using Lipofectamine 2000 Transfection Reagent (11668019, Invitrogen, with the Renilla luciferase vector pRL-SV40-N reporter gene plasmid (D2762, Beyotime) as an internal control. After 48 h of incubation, the luciferase activity of the cells was evaluated using a dual-luciferase assay system (Promega) as per the manufacturer’s protocol.

Statistical analysis

Prism 8.0.2 (GraphPad, San Diego, CA, USA) was used for all statistical analyses and graph plotting. Continuous values were recorded as mean ± SEM. Depending on data distribution, a t-test or ANOVA (followed by Tukey’s post hoc test) was used to analyze the statistical p-values between different groups. p < 0.05 was considered statistically significant and was represented by *.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

HCC:

Hepatocellular carcinoma

TFs:

Transcription factors

RT-qPCR:

Reverse transcription-quantitative polymerase chain reaction

IHC:

Immunohistochemistry

MEM:

Minimum essential medium

WB:

Western blot

ECL:

Enhanced chemiluminescence

ChIP:

Chromatin immunoprecipitation

CCK-8:

Cell Counting Kit-8

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Authors and Affiliations

  1. Department of Hepatobiliary Surgery, Pingxiang People’s Hospital, Pingxiang, Jiangxi, 337000, P.R. China

    Xiaofang Wang, Fuping Zhong, Yong Zhou, Qibo Wang & Jihao Jiang

  2. Department of Imaging, Pingxiang People’s Hospital, NO.88, Wugongshan Middle Avenue, Pingxiang, Jiangxi, 337000, P.R. China

    Bo Xiao

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Contributions

XFW designed the study and drafted the manuscript. FPZ and YZ were primarily responsible for collecting data, performing data analysis, and revising the manuscript. QBW, JHJ, and BX performed the experiments. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Bo Xiao.

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Ethics approval and consent to participate

Tumor and paraneoplastic non-tumor liver tissues were collected from 12 HCC patients who underwent hepatectomy from June 2020 to December 2022 at Pingxiang People’s Hospital (none of these patients had other malignancies and had not received radiotherapy). All patients signed a written informed consent, and the procedure was performed in strict accordance with the Declaration of Helsinki. The animal experimental protocol was approved by the Ethical Review Committee of Pingxiang People’s Hospital and was conducted in strict compliance with the Guidelines for the Care and Use of Laboratory Animals.

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Wang, X., Xiao, B., Zhong, F. et al. ZSCAN16 expedites hepatocellular carcinoma progression via activating TBC1D31. Cell Div 19, 31 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13008-024-00135-9

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Keywords

Cell Division

ISSN: 1747-1028

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