Targeting B4GALT7 suppresses the proliferation, migration and invasion of hepatocellular carcinoma through the Cdc2/CyclinB1 and miR-338-3p/MMP2 pathway

Cell lines and tumor tissues

Human HCC cell lines Huh-7, HepG2, SMMC-7721 and SK-Hep-1 were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Human hepatocyte cell line HL-7702 and human HCC cell line SNU-423 were purchased from American Type Culture Collection (ATCC, USA). They were maintained in DMEM (Huh-7, HepG2 and SK-Hep-1) or RPMI-1640 (SMMC-7721, SNU-423 and HL-7702) medium with 10% fetal bovine serum. All cell lines used in the study were tested and authenticated using short tandem repeat (STR) matching analysis. 10 pairs of HCC tissues and corresponding para-tumor specimens were collected from the Affiliated Tumor Hospital of Shanxi Medical University (Shanxi, China). Written informed consents were obtained from all participants before surgery. Collections and use of tissue samples were approved by the ethics committee of the Affiliated Tumor Hospital of Shanxi Medical University (approval number: KY2023017) and were in accordance with the Declaration of Helsinki.

Transfection

The short hairpin RNA (shRNA)-B4GALT7 and an empty vector were designed by GenePharma (Shanghai, China). The lentiviral vector plasmid used was LV3 (H1/GFP&Puro). Puromycin (4 µg/ml) was applied to select stable cell lines using shRNA vector to mediate B4GALT7 suppression. The three shRNA sequences targeting B4GALT7 were as follows:

shRNA-B4GALT7-1: 5′-GCAACAGCACGGACTACATTG- 3′;

shRNA-B4GALT7-2: 5′-GCCTGAACACTGTGAAGTACC- 3′;

shRNA-B4GALT7-3: 5′-GCACTGTCCTCAACATCATGT- 3′.

LV3 NC: 5′-TTCTCCGAACGTGTCACGT- 3′ (NC: negative control).

The miR-338-3p inhibitor and mimics were purchased from GenePharma, and were transfected into SNU-423 and SK-Hep-1 cells using siRNA-mate (GenePharma, Shanghai, China). The sequence information is shown in Table 1. Plasmid DNA (pEX-3/B4GALT7) with the restriction enzyme cutting site XhoI/EcoRI was obtained from GenePharma. Transfection was conducted according to the manufacturer’s instructions.

Cell proliferation, colony formation, migration, and invasion assays

MTT (Solarbio, Beijing, China) was conducted to assay cell proliferation. Absorbance at 492 nm was examined on consecutive four days using a BioTek microplate reader (Winooski, VT, USA). For the colony formation assay, the colonies were stained with 0.5% crystal violet and photographed. Absorbance at 595 nm (OD₅₉₅) was determined with a BioTek microplate reader. Each experiment was carried out three times. For the wound healing assay, a scratch was made to the monolayer formed by indicated cells in 6-well plates. Cells were further maintained without FBS for 48 h. The wound was photographed using light microscope and the wound healing area was calculated by Image J software. For the migration and invasion assay, the upper chambers were coated with or without 100 µL of Matrigel (1:8 mixed with FBS-free medium; Corning, New York, USA). 5–8 × 10⁴ indicated HCC cells were seeded in the upper chamber of transwell plates (8 µm pore size; Corning) without serum. Medium with 10% FBS was filled into the lower chamber. Cells on the bottom chamber were fixed, stained, and counted in five randomly selected fields using light microscope after 48 h.

Dual-luciferase reporter assay

The GP-miRGLO-B4GALT7 WT (wild-type) plasmids and its corresponding mutant-type (mut) plasmids were designed by GenePharma. The above luciferase vectors, miR-338-3p mimics or miR-338-3p NC was co-transfected into HEK-293T or SNU-423 cells using Lipofectamine 2000 (Invitrogen). The dual luciferase reporter gene assay kit (GenePharma) was conducted to detect the renilla and firefly luciferase activities after incubation for 48 h.

RNA extraction and real-time PCR

TRIzol reagent (Takara, Beijing, China) or miRNA Isolation Kit (Omega Bio-Tek, Guangzhou, China) was performed to extract total RNA. PrimeScript™ RT reagent Kit with gDNA Eraser (Takara) or Mir-X miRNA First-Strand Synthesis Kit (Takara) was performed to reversely transcribe cDNA from mRNA and miRNA. qRT-PCR was performed to calculate the mRNA levels by the 2^−ΔΔCtmethod using TB Green® Premix Ex Taq™ II (Takara). mRNA and miRNA expression levels were normalized to β-actin and small nucleolar RNA U6, respectively. The qRT-PCR primer sequences are shown in Table 2.

Flow cytometry analysis of cell apoptosis and cell cycle

For analysis of cell apoptosis, the indicated HCC cells (1 × 10⁶ cells/mL) were incubated with 10 µL 7-AAD, 500 µL binding buffer and 5 µL Annexin V-APC for 15 min at 37 °C in the dark. For analysis of cell cycle, the indicated HCC cells were harvested, fixed in 70% ethanol, and incubated with RNase A and propidium iodide (PI) for 1 h at room temperature. The apoptosis rate and cell cycle were examined with an Agilent NovoCyte flow cytometer (Agilent, Santa Clara, USA). Each experiment was carried out three times.

Western blot analysis

Total proteins were extracted from the indicated HCC cells in RIPA buffer (Beyotime, Shanghai, China) and quantified using the BCA protein quantitation kit (Boster Biotechnology, Wuhan, China). Proteins in 60 µg samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The primary antibodies included B4GALT7 (1:500; NBP1-88652) from Novus Biologicals (Shanghai, China), MMP-2 (1:1,000; ab92536) from abcam (Cambridge, UK), and p44/42 MAPK (Erk1/2) (137F5) (1:1,000; #4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (1:1,000; #4370), Akt (pan) (C67E7) (1:1,000; #4691), Phospho-Akt (Ser473) (D9E) (1:1,000; #4060), E-Cadherin (24E10) (1:1,000; #3195), N-Cadherin (D4R1H) (1:1,000; #13116), Vimentin (D21H3) (1:1,000; #5741), Phospho-Chk2 (Thr68) (1:1,000; #2661), Phospho-Wee1 (Ser642) (D47G5) (1:1,000; #4910), Phospho-cdc2 (Tyr15) (10A11) (1:1,000; #4539), Cyclin B1 (D5C10) (1:1,000; #12231), phosphor-ATM (Ser1981) (D6H9) (1:1,000; #5883), Phospho-Histone H2A.X (Ser139) (20E3) (1:1,000; #9718) from Cell Signaling Technology (Danvers, MA), and β-actin (1:2,500; TA-09) from ZSGB-Biotechnology (Beijing, China). The membranes were visualized using an enhanced chemiluminescent (ECL) blot detection system (Transgene, Beijing, China) after the primary antibodies were incubated by anti-mouse or anti-rabbit secondary antibodies.

Statistical analysis

Statistical analyses were performed using Student’s t-test or one-way ANOVA by SPSS 19.0 statistical software. P < 0.05 was set as statistically significant.

Clinical relevance of B4GALT7 expression in HCC cancer patients

B4GALT7 was upregulated in HCC tissues compared to para-tumor specimens in three GEO datasets (GSE14520, GSE25097, GSE84402) (Fig. 1A, P < 0.001) and the TCGA database using the UALCAN portal (P < 0.001, Fig. 1B) (Chandrashekar et al., 2017). Consistently, upregulated B4GALT7 expression (n = 90) correlated with shorter survival probability in HCC patients (P = 0.0032, Fig. 1C). We further validated B4GALT7 expression levels in 10 paired human HCC tissues by western blotting (Fig. 1D) and qPCR (Fig. 1E). B4GALT7 was overexpressed in seven (70%) HCC tissues compared with paired para-tumor specimens (Figs. 1D and 1E). B4GALT7 was mainly located in the cytoplasm and HCC tissues demonstrated stronger B4GALT7 staining than the paired para-tumor specimens (Fig. 1F), as revealed in the Human Protein Atlas database (https://www.proteinatlas.org/). We further applied the TIMER2.0 database (http://timer.comp-genomics.org/) to identify the expression landscape of B4GALT7. B4GALT7 was highly expressed in a large number of cancer tissues compared to para-tumor specimens (Fig. 1G).

B4GALT7 suppression reduces HCC cell proliferation in vitro

We then examined the endogenous expression levels of B4GALT7 in five HCC cell lines by qPCR (Fig. 2A) and western blotting (Fig. 2B). B4GALT7 was highly expressed in SNU-423, SMMC-7721, SK-Hep-1, HepG2 and Huh-7 cells compared with normal liver cell HL-7702 (Figs. 2A and 2B). SNU-423 and SK-Hep-1, with the highest B4GALT7 expression levels, were chosen for further investigation. To examine the molecular mechanism by which B4GALT7 is associated with HCC, the SNU-423 and SK-Hep-1 cells were transfected with shRNA vectors to mediate B4GALT7 suppression. The green fluorescence intensity in both SNU-423 and SK-Hep-1 cells was above 80% (Fig. 2C). B4GALT7 was significantly downregulated in the above two cell lines by qPCR (Fig. 2D) and western blotting (Fig. 2E). ShRNA mediated B4GALT7 suppression in SNU-423 and SK-Hep-1 cells reduced cell proliferation rates (Figs. 2F–2G). However, no significant cell apoptosis was observed (Fig. 2H). Collectively, down-regulation of B4GALT7 reduces HCC cell proliferative abilities, but does not promote significant apoptosis in vitro.

Down-regulation of B4GALT7 arrests the cell cycle at the G2/M phase

Then, we examined whether DNA was damaged after shRNA mediated B4GALT7 suppression by measuring the expressions of DNA damage markers, including ataxia-telangiectasia mutated (ATM) and H2AX (Li et al., 2021b; Sharma & Almasan, 2021). The phosphorylation of ATM and H2A.X was increased after shRNA mediated B4GALT7 suppression (Fig. 3A) and was reduced after plasmid pEX-3/B4LGAT7 mediated B4GALT7 overexpression (Fig. 3B). B4GALT7 expression was further rescued using plasmid pEX-3/B4GALT7 in SNU-423 transfected with shB4GALT7 (Fig. 3B). More cells stayed in the G2 phase for both cell lines after shRNA mediated B4GALT7 suppression, suggesting that B4GALT7 regulated the progression from G2 to M phase (Fig. 3C). Chk2 phosphorylation at Thr68 was significantly elevated after shRNA mediated B4GALT7 suppression (Fig. 3D). ShRNA mediated B4GALT7 suppression markedly promoted phosphorylation of Cdc2 at Tyr15 and induced the levels of cyclin B1 (Fig. 3D), which was assumed to extend the time for cells to fix DNA damages. ShRNA mediated B4GALT7 suppression in SNU-423 and SK-Hep-1 cells markedly promoted the phosphorylation of Wee1 at Ser642 (Fig. 3D). B4GALT7 overexpression rescued the cell cycle arrest caused by B4GALT7 suppression (Fig. 3E). Collectively, these results indicated that the ATM-Chk2-Cdc2/cyclin B1 pathway was involved in the G2/M cell cycle arrest caused by B4GALT7 suppression.

B4GALT7 interacts with miR-338-3p in HCC cells

To examine the mechanism of B4GALT7 in modulating cell proliferative and invasive abilities, the online software TargetScan (http://www.targetscan.org/vert_72/) and miRPathDB v2.0 (https://mpd.bioinf.uni-sb.de/) were applied to screen for candidate miRNAs that might regulate B4GALT7. There is a potential 8mer binding site in the 3′ UTR of B4GALT7 for miR-338-3p (Fig. 4A). Low expression of miR-338-3p in SK-Hep-1 and SNU-423 cells was validated by qPCR analysis (Fig. 4B). MiR-338-3p suppressed the luciferase activity of the B4GALT7-WT vector in the HEK-293T and SNU-423 cells, but not the B4GALT7-MUT vector, confirming that miR-338-3p targeted the 3′ UTR of B4GALT7 (Fig. 4C). B4GALT7 overexpression reduced miR-338-3p level (Fig. 4D) and shRNA mediated B4GALT7 suppression elevated miR-338-3p level in HCC cells (Fig. 4E). Overexpression of miR-338-3p (Fig. 4F) suppressed both mRNA (Fig. 4G) and protein expression levels (Fig. 4H) of B4GALT7, and miR-338-3p inhibition elevated both mRNA (Fig. 4G) and protein expression levels (Fig. 4H) of B4GALT7, implying that miR-338-3p degrades B4GALT7 mRNA by targeting its 3′ UTR.

Previous reports have shown that miR-338-3p is involved in the EMT (epithelial-mesenchymal transition) in HCC and other malignant tumors (Li et al., 2021a; Lu et al., 2019; Song et al., 2020; Li et al., 2019). We found that miR-338-3p mimics reduced HCC cell invasive abilities (Fig. 5A); and suppressed the phosphorylation of Erk (Fig. 5B), MMP2 and the expression of mesenchymal markers (N-cadherin and vimentin), whereas elevated the expression of epithelial marker E-cadherin (Fig. 5B). However, the phosphorylation of Akt was not affected with miR-338-3p overexpression in the above two cell lines (Fig. 5B).

B4GALT7 suppression reduces HCC cell migration and invasion in vitro

ShRNA mediated B4GALT7 suppression reduced the phosphorylation of Erk, MMP2 and the expression of mesenchymal markers (N-cadherin and vimentin), whereas elevated the expression of epithelial marker E-cadherin (Fig. 6). However, the phosphorylation of Akt was not affected after shRNA mediated B4GALT7 suppression in the above two cell lines (Fig. 6). Then, SK-Hep-1 and SNU-423 cells were transfected with different shRNAs/miR-338-3p inhibitors as demonstrated in Fig. 7A. We found that shRNA mediated B4GALT7 suppression suppressed the migrative and invasive abilities of HCC cells, whereas miR-338-3p inhibitor significantly rescued these phenotypes (Figs. 7A–7B). The expression levels of MMP2, N-cadherin, vimentin and E-cadherin were rescued after miR-338-3p inhibitor was co-transfected (Fig. 7C). In contrast, B4GALT7 overexpression induced HCC cell invasive abilities (Fig. 8A); and elevated the expression of MMP2 and the mesenchymal markers (N-cadherin and vimentin), whereas reduced epithelial marker E-cadherin (Fig. 8B). The invasion stimulative phenotypes were rescued after miR-338-3p mimics were co-transfected (Fig. 8A), and the expression levels of MMP2, N-cadherin, vimentin and E-cadherin were reversed (Fig. 8B). The expression levels of MMP2 and EMT marker proteins were reversed after transfection with plasmid pEX-3/B4GALT7 in SNU-423 with shRNA mediated B4GALT7 suppression (Fig. 8C). Collectively, these results suggested that miR-338-3p rescued the tumor-promoting effect of B4GALT7 in HCC.