UALCAN (http://ualcan.path.uab.edu) is a comprehensive and interactive web resource for the analysis of cancer omics data (19). The protein levels of MCM2-7 in human LIHC were assessed using UALCAN, and Student's t-test was used to generate P-values.

Plasmids containing RNF125 and MCM2-7 were purchased from Shanghai Cell Researcher Biotech Co., Ltd. and inserted into empty vectors, which were purchased from Cell Researcher Biotech Co., Ltd.), such as pCDH, pCDNA3.0-Myc, pET22b or pGEX4T-1. Briefly, pCDH-MCM2/MCM3/MCM6/MCM7/RNF125, pCDNA3.0-RNF126-Myc, pET22b-MCM2/MCM3/MCM6/MCM7/RNF125/UBA1/UBCH5A/catalytic core of human ubiquitin specific peptidase 2 (Usp2cc) and pGEX4T-1-MCM6/RNF125 were generated. Mutations were generated in plasmids containing RNF125 or MCM6 by site-directed mutagenesis as previously described (20). Short hairpin RNAs (shRNAs) inserted into the Plko.1 vector targeting RNF125 and MCM2, MCM3, MCM6 and MCM7 were also purchased from Shanghai Cell Researcher Biotech Co., Ltd., and the specific sequences of these shRNAs are shown in Table I.

The human HCC cell lines Huh7 and Hep3B were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. These cells were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/l penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The plasmids were transfected into the cells using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The ratio of the mass of nucleic acid (plasmid) to the volume of Lipofectamine 2000 was 1:2 (2 µg:4 µl), which was mixed together at room temperature for 20 min, and then added to cells for continued culture at 37°C. Stably expressed cell lines were generated using puromycin selection (2 µg/ml; Beyotime Institute of Biotechnology) for ≥7 days after 48 h transfection. Cycloheximide (CHX; Selleck Chemicals) was dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) at a concentration of 100 mM and stored at −40°C. Huh7 and Hep3B cells were treated with 100 µM CHX for 6 h, or at different time periods, at 37°C. For protein degradation pathway analysis, Huh7 cells were treated with 1 µM proteasomal inhibitor bortezomib (BTZ; Selleck Chemicals) or 20 nM autophagy inhibitor bafilomycin (BAF; Selleck Chemicals) for 6 h at 37°C prior to western blotting.

Huh7 and Hep3B cells stably expressing plasmids containing pCDH-MCMs, pCDH-RNF125 or pLKO.1-shMCMs, were seeded in 96-well plates at 5,000 cells/well. These cells were then incubated with Cell Counting Kit-8 (CCK-8) solution (Beyotime Institute of Biotechnology) for 4 h at different time points (0, 24, 48 or 72 h). The 0 h time point was 6 h after the cells were seeded in the plates. The product was then quantified by spectrophotometry at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). These experiments were performed with six replicates and repeated three times.

Cells stably expressing plasmids containing pCDH-MCMs, pCDH-RNF125 or pLKO.1-shMCMs were seeded into 6-well plates (1,000 cells/well). After 7 days culturing at 37°C, the cells were fixed with 4% paraformaldehyde (Merck KGaA) for 10 min at room temperature and then stained with 0.2% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 15 min. Images were captured using an iPhone 11 camera (Apple, Inc.) and the number of colonies (≥50 cells) was manually counted using a light-field microscope (CKX53; Olympus, Inc.).

The yeast two-hybrid (Y2H) screen was performed as described previously (21). Briefly, human MCM6 was used as bait, and its potential E3 ligase was screened from a library containing ~400 open reading frames (ORFs) of human E3 ubiquitin ligases. The positive colony was able to survive in SD-4 medium (deficient in uracil, histidine, leucine and tryptophan) and could be stained with X-Gal (Sangon Biotech Co., Ltd.).

Hexahistidine (His6)- and glutathione S-transferase (GST)-tagged proteins were purified from the BL21 E. coli system as described previously (22). Briefly, after induction with isopropyl-β-d-mercapto-galactopyranoside (Sigma-Aldrich), the cells transfected with protein-encoding plasmids were centrifuged, lysed in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄ and 2 mM KH₂PO₄, pH 7.4), incubated with Ni²⁺ or glutathione affinity gels, and eluted with 500 mM imidazole or 25 mM reduced L-glutathione solution (pH 8.0). The eluate was then dialyzed in PBS buffer containing 10% glycerol for 6 h at 4°C and stored at −70°C.

Purified GST-tagged protein (10 µg), His6-tagged protein (10 µg) and 25 µl Glutathione Sepharose 4B (Sangon Biotech Co., Ltd.) were incubated for 4 h at 4°C in 800 µl GST pull-down buffer [20 mM Tris-Cl, 5 mM MgCl₂, 100 mM NaCl, 1 mM EDTA, 1% NP-40 and fresh 1 mM dithiothreitol (DTT), pH 7.6] supplemented with fresh 10 mg/ml BSA. The samples were pelleted via centrifugation at 500 × g for 3 min at 4°C and washed five times with GST pull-down buffer. The immunoprecipitates were then denatured in 50 µl of 2X SDS protein loading buffer at 100°C for 10 min before immunoblotting (IB).

In vitro ubiquitination was performed as described previously (23). Briefly, 50 ng recombinant His6-UBA1 (an E1 or ubiquitin-activating enzyme), 100 ng His6-UBCH5A (an E2 or ubiquitin-conjugating enzyme), 200 ng GST-RNF125 (E3), 200 ng MCM6-His6 and 50 ng His6-ubiquitin were added to in vitro ubiquitination buffer (25 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl₂, pH 7.8, supplemented with 1 mM fresh ATP and 0.5 mM DTT) to a final volume of 50 µl, and incubated at 37°C for 1.5 h. Subsequently, 50 ng His6-tagged Usp2cc was added to the tube containing E1, E2, E3 and MCM6 and incubated at 37°C for a further 30 min. The level of MCM6 ubiquitination was detected by IB analysis using anti-MCM6 antibody.

For the Co-IP assay, transfected Huh7 and Hep3B cells were lysed in 800 µl Co-IP buffer (50 mM Tris-HCl, 1% NP-40, 150 mM NaCl and 5 mM EDTA, pH 7.6) supplemented with fresh protease inhibitor cocktail (Roche Diagnostics). The cell lysates were then incubated with anti-MCM6 antibody (1:100 dilution; 13347-2-AP; Wuhan Sanying Biotechnology) and 25 µl Protein G magnetic beads (L-1002; Biolinkedin) overnight at 4°C. For the IP assay, Huh7 and Hep3B cells were lysed in 800 µl RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% NP-40 and 0.1% SDS, pH 7.6) containing fresh protease inhibitor cocktail. Then, the cell lysates were incubated with the anti-MCM6 antibody (1:100 dilution) and 25 µl Protein G magnetic beads overnight at 4°C. The next day, the immunoprecipitates were pelleted via centrifugation at 500 × g for 3 min at 4°C and washed five times with Co-IP/IP buffer. Then, the immunoprecipitates were denaturated for 15 min at 100°C in 50 µl 2X SDS protein loading buffer. The immunoprecipitates, inputs and other lysates (10 µl) were subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 10% non-fat milk at room temperature for 45 min before incubation with the following antibodies: Anti-MCM2 (1:1,000 dilution; 10513-1-AP), anti-MCM3 (1:1,000 dilution; 15597-1-AP), anti-MCM6 (1:1,000 dilution), anti-MCM7 (1:1,000 dilution; 11225-1-AP), anti-GAPDH (1:6,000 dilution; 60004-1-Ig), anti-RNF125 (1:2,000 dilution; 13290-1-AP), anti-His (1:2,000 dilution; 66005-1-Ig), anti-GST (1:10,000 dilution; HRP-66001) or anti-ubiquitin (1:2,000, 10201-2-AP), all from Wuhan Sanying. The membranes were incubated with primary antibodies overnight at 4°C, and washed three times with TBST (50 mM Tris-HCl, 150 mM NaCl and 0.2% Tween-20; pH 8.0). Subsequently the membranes were incubated with the following horseradish peroxidase-labeled secondary antibodies: Goat anti-mouse IgG (1:10,000 dilution; SA00001-1) or goat anti-rabbit IgG (1:10,000 dilution; SA00001-2), both from Wuhan Sanying, at room temperature for 2 h and washed three times with TBST. The signals were visualized using high-signal ECL western blotting substrate (cat. no. 180-5001; Tanon Science and Technology Co., Ltd.) and a Tanon 5200 imaging system (Tanon Science and Technology Co., Ltd.).

Data are expressed as the mean ± SD and were analyzed by Student's t-test or one-way ANOVA with Tukey's post hoc test using GraphPad Prism 5 (GraphPad Software; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of genes encoding the MCM2-7 complex in human LIHC were analyzed using the GEPIA2 and UALCAN databases, respectively, and found to be significantly upregulated in LIHC compared with the corresponding normal tissues at the mRNA and protein levels (Fig. 1A and B).

The associations between different MCMs and the clinical outcomes of LIHC were analyzed using GEPIA2 (Fig. 1C). Stronger associations were observed for MCM2, MCM3, MCM6 and MCM7 than for MCM4 and 5. The associations between the mRNA expression of certain MCMs and the pathological stage of LIHC were also assessed using GEPIA2. The mRNA expression levels in the MCM2, MCM3, MCM6 and MCM7 groups were highly variable, and gradually increased with the progression of LIHC in the first three stages, but then markedly decreased in stage IV (Fig. S1A). In addition, patients with LIHC who had high mRNA expression levels of MCM2, MCM3, MCM6 and MCM7 showed poor overall and disease-free survival rates (Figs. 1D and S1B). Therefore, these four genes were selected for further study.

shRNAs targeting MCM2, MCM3, MCM6 and MCM7 were constructed and stably transfected into human Huh7 and Hep3B cells. The knockdown efficiencies were detected by IB analysis (Fig. 2A). On the basis of these results, shMCM2-1 and shMCM2-4 for MCM2, shMCM3-2 and shMCM3-4 for MCM3, shMCM6-1 and shMCM6-2 for MCM6, and shMCM7-1 and shMCM7-2 for MCM7 were selected for further study. The viability of Huh7 and Hep3B cells was determined by CCK-8 assay at different time points (0, 24, 48 and 72 h), and cell growth inhibition was observed in the MCM2/3/6/7-knockdown cells compared with that of the negative control (scramble) groups (Fig. 2B). Colony formation assays were also performed, the results of which were highly consistent with those of the CCK-8 assay (Fig. 2C). In addition, wound healing assays were performed and MCM2/3/6/7-knockdown exhibited no evident effect on the migration of Huh7 cells (data not shown).

Huh7 and Hep3B cells were stably transfected with pCDH or pCDH-MCM2/3/6/7 and the expression of the MDMs was detected by IB analysis (Fig. 3A). The viability of the transfected Huh7 and Hep3B cells was then determined by CCK-8 assay at various time points, and cell growth promotion was observed in the pCDH-MCM groups compared with the pCDH control groups (Fig. 3B). The results of colony formation assays were consistent with the results of the CCK-8 assay (Fig. 3C).

The pathways of MCM2, MCM3, MCM6 and MCM7 protein degradation were explored. Huh7 cells were treated with the proteasomal inhibitor BTZ or the autophagy inhibitor BAF for 6 h before being subjected to IB analysis. The results shown in Fig. 4A indicate that endogenous MCM6 protein is degraded mainly by the proteasome pathway, and that neither of these degradation pathways affect the other three MCMs. To investigate the regulation of MCM6 protein homeostasis, MCM6 was used as a bait to screen for its interacting partners using the Y2H prey library which contains ~400 ORFs of human E3 ubiquitin ligases. The E3 ligase RNF125 was identified as an interacting partner for MCM6 and verified in yeast cells (Fig. 4B). A direct interaction between RNF125 and MCM6 was detected by GST pull-down assay (Fig. 4C), whereas no interaction of RNF125 with MCM2, MCM3 or MCM7 was found (Fig. 4D). Protein-protein interaction domain analysis revealed that the MCM domain of MCM6 directly interacted with the N-terminal region (1–135 amino acids) of RNF125 containing the RING domain (Fig. 4E). Further Co-IP assays showed that endogenous MCM6 formed a complex with RNF125 in both Huh7 and Hep3B cells (Fig. 4F). These data indicate that RNF125 does indeed interact with MCM6.

To investigate whether RNF125 affects the stability of the MCM6 protein, Huh7 and Hep3B cells were transfected to express different amounts of RNF125, and the protein levels of MCM6 were detected by IB analysis. The results shown in Fig. 5A show that RNF125 reduced the protein levels of MCM6 in a dose-dependent manner. An in vitro ubiquitination assay was carried out, as shown in Fig. 5B. In the presence of E1 (UBA1), E2 (UBCH5A) and E3 (RNF125), the ubiquitination of MCM6 was detectable by IB analysis, and this modification was efficiently attenuated by Usp2cc. Huh7 and Hep3B cells were stably transfected with empty vector (pCDH) or pCDH-RNF125 and the upregulation of RNF125 by pCDH-RNF125 was confirmed by IB analysis (Fig. 5C). The lysates of the transfected Huh7 and Hep3B cells were immunoprecipitated with anti-MCM6 antibody, and the results revealed that the ubiquitination of MCM6 was markedly increased in cells stably expressing pCDH-RNF125 compared with the control cells (Fig. 5D). Three shRNAs targeting RNF125 were designed and tested in human Huh7 and Hep3B cells. shRNF125-2 and shRNF125-3 performed well in the knockdown of RNF125 and were selected for further study (Fig. 5E). The protein levels of endogenous MCM6 were increased in the RNF125-knockdown cells compared with the control cells in the presence of the protein synthesis inhibitor CHX (Fig. 5F). In addition, RNF125 knockdown reduced the ubiquitination of MCM6 and concurrently increased MCM6 protein levels in Huh7 and Hep3B cells (Fig. 5G). These data suggest that RNF125 is an E3 ligase for MCM6 and promotes its degradation.

pCDH-RNF125 or empty vector (pCDH) was stably transfected into Huh7 and Hep3B cells that also stably expressed scramble shRNA, shMCM6-1 or shMCM6-2, and the protein levels of RNF125 and MCM6 were detected by IB analysis (Fig. 6A). The results of CCK-8 and colony formation assays performed using these cells revealed that RNF125 inhibited the proliferation and colony formation of Huh7 and Hep3B cells with intact MCM6, but not of MCM6 knockdown cells (Fig. 6B and C). These data suggest that RNF125 promotes the proliferation of HCC cells mainly through MCM6. The expression of RNF125 in human LIHC was analyzed using the GEPIA2 database. As shown in Fig. 6D, the expression of RNF125 was downregulated in LIHC compared with normal tissues at the mRNA level. The association between RNF125 and the clinical outcome of LIHC was also analyzed, and the results showed that patients with LIHC and high mRNA expression of RNF125 had a higher overall survival rate (Fig. 6E). These findings suggest that RNF125 is a potential therapeutic target for human HCC.