2.1 Cell line information and cell transfection

The hepatic carcinoma cell line HCCLM3, obtained from Shanghai FuHeng Biotechnology Co., Ltd. (Catalog #FH0096), was utilized in this study. This cell line exhibits high metastatic potential and adherent growth characteristics. Cultivation was carried out in DMEM culture medium with high glucose (Catalog #C3113-0500, VivaCell, Shanghai, China), supplemented with 10% fetal bovine serum (Catalog #04-001-1ACS, VivaCell) and 1% penicillin–streptomycin (Catalog #BL505A, Biosharp, Hefei, China).

Based on the gene sequence of CXCR7 (NM_020311.3), an overexpression sequence and a small interfering RNA (siRNA) targeting CXCR7 (5′-CGCUCUCCUUCAUUUACAUUU-3′) were designed and synthesized by Shanghai Genechem Co., Ltd. The coding sequence (CDS) of CXCR7 was cloned into the pcDNA3.1 vector (pCAMBIA1301, Catalog #HZB800277, HZbscience, Hangzhou, China) to generate the recombinant overexpression vector pcDNA-CXCR7. As corresponding controls, si-NC (sense: 5′-ACGUGACACGUUCGGAGAAUU-3′ and antisense: 5′-UUCUCCGAACGUGUCACGU-3′) and an empty vector were used. HCCLM3 cells were resuspended and adjusted to a concentration of 8 × 105 cells/well before being seeded into a 6-well plate. For transfection, the overexpression plasmid or si-CXCR7 was mixed with PEI reagent (Catalog #23966-2, YEASEN, Shanghai, China) in Opti-MEM™ medium (Invitrogen, USA) and pre-incubated at room temperature for 20 min to form DNA-PEI complexes. The transfection complexes were then added to the cells in serum-free medium. After a 6-h incubation, the medium was replaced with complete growth medium, and the cells were further incubated at 37 °C for 24 h. Transfection efficiency was subsequently assessed.

2.2 CCK-8 activity assay and IC50 determination of verteporfin

Cells in the logarithmic growth phase were seeded into a 96-well plate at a density of 3000 cells/well, with each group replicated three times, and incubated at 37 °C. At predetermined time points (0, 24, 48, 72, and 96 h), CCK-8 solution (Catalog #C0037, Beyotime, Shanghai, China) was added, and the plates were returned to the incubator for an additional hour of incubation. Wells containing only the cell culture medium and CCK-8 solution, without cells, served as blank controls. Absorbance was measured at 450 nm, and the data were recorded.

Verteporfin (VP; Catalog #HY-B0146, ECM, USA) was dissolved in DMSO and further diluted with PBS. HCCLM3 cells were seeded into a 96-well plate at a density of 5,000 cells/well, with each condition replicated in triplicate. Cells were then treated with varying concentrations of Verteporfin (0, 2, 4, 6, 10, 14 μM) and incubated at 37 °C for 72 h. The cytotoxicity of Verteporfin was evaluated using the CCK-8 assay, measuring absorbance at 450 nm to determine the IC50 value. A Verteporfin concentration of 16 μM (median for 16 and 18 μM) was selected for subsequent experiments according to its calculated IC50 (about 17.90 μM).

2.3 Bioinformatics analysis of CXCR7

Gene expression data specific to CXCR7 in LIHC cases, along with corresponding clinical data, were retrieved from the TCGA database (The Cancer Genome Atlas, https://portal.gdc.cancer.gov). Patients were divided into high and low CXCR7 expression cohorts using the median expression level as a cutoff. Survival times and statuses were used to construct Kaplan–Meier survival curves, with a log-rank test assessing the statistical significance of survival differences between the two groups. Differential expression analysis was conducted using the Wilcoxon rank-sum test, applying criteria of |log2 Fold Change|> 1 and a p-value < 0.05 to identify significantly altered genes. The volcano plot of the differentially expressed genes (DEGs), including key DEGs, was visualized using the R package “ggplot2.” Enrichment analysis of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Set Enrichment Analysis (GSEA) were conducted using the R packages clusterProfiler and org.Hs.eg.db. Genes highly correlated with CXCR7 were identified using GEPIA (http://gepia.cancer-pku.cn/), while immune infiltration was assessed using Timer 2.0 (http://timer.cistrome.org/).

2.4 Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells using Trizol Reagent (Catalog #15596026, Invitrogen) and purified by phenol–chloroform extraction, following the manufacturer's instructions. The obtained RNA was converted into cDNA using a reverse transcription system (Catalog #R333-01, Vazyme, Nanjing, China). Quantitative PCR analysis of CXCR7 was performed on a Bio-Rad CFX96 system using ChamQ Universal SYBR qPCR Master Mix (Catalog #R711-03, Vazyme) with the following primer sequences: GAPDH forward (F): 5′-GTTCGTCATGGGTGTGAACC-3′, reverse (R): 5′-CATCCACAGTCTTCTGGGTG-3′; CXCR7 F: 5′-TGTCCTCACCATCCCAGTCT-3′, R: 5′-CTGCCGAAGAGGTTGATGGA-3′. The 2−ΔΔCt method was used to assess the relative expression levels of CXCR7 compared to the control group, with GAPDH as the internal reference.

2.5 Western blotting

Antibodies used in this study include: CXCR7 (1:3000, 60 kDa, Catalog #60216-1-Ig, Proteintech, Shanghai, China), Gα q/11 (1:500, 41 kDa, Catalog #sc-365906, Santa Cruz, USA), Gα s (1:500, 49 kDa, Catalog #sc-135914, Santa Cruz), phosphorylated (p)-LATS (1:1000, 140 kDa, Catalog #8654S, CST, USA), LATS (1:1000, 140 kDa, Catalog #17049-1-AP, Proteintech), p-YAP (1:1000, 72 kDa, Catalog #13008T, CST), YAP (1:1000, 72 kDa, Catalog #abs134112, absin), and β-actin (1:150,000, 42 kDa, Catalog #T0022, Affinity, USA). Protein concentration was determined (Catalog #P0010, Beyotime), and the samples were separated on a 12% SDS-PAGE gel, followed by semidry transfer. Membranes (0.22 µm) were blocked with a solution containing 5% non-fat milk and 0.1% Tween. Membranes were then incubated with primary antibodies overnight at 4 °C. The following day, membranes were exposed to the corresponding secondary antibody at room temperature for one hour. Immunoblots were visualized using an enhanced chemiluminescence system (Catalog #G2014, Servicebio, Wuhan, China), and images were captured on GenoSens1800 autoradiography film (Shanghai Qinxiang Scientific Instrument Co., Ltd., China). β-actin was used for normalization.

2.6 Colony formation assay

Cells were resuspended and adjusted to a concentration of 1 × 104 cells/mL. Cells were then seeded at a density of 300 cells per well in a 6-well plate, with 2 mL of fresh culture medium added. Plates were incubated at 37 °C in a 5% CO2 atmosphere. The colony size was periodically monitored, and the medium was refreshed regularly to maintain optimal growth conditions. After approximately two weeks, cells were fixed with tissue fixative (Catalog #P0099, Beyotime) for 10 min to preserve colony morphology and then stained with a 0.2% crystal violet solution (Catalog #C02121, Beyotime) for 15 min to visualize the cell clusters. Images of the colonies were captured for analysis.

2.7 Transwell assay

Cells were resuspended and seeded into a 96-well plate at a density of 5 × 104 cells onto the upper chamber of a Transwell insert, while the lower chamber was filled with culture medium supplemented with 10% serum. The Transwell plate was then incubated for 24 h. For the invasion assay, the membrane was precoated with Matrigel. Briefly, Matrigel (Catalog #111005, GENOM Bio, Hangzhou, China) was diluted with serum-free medium to a final concentration of 1 mg/mL. The diluted Matrigel was added to the center of the chamber and incubated at 37 °C for 4 h, followed by incubation at 4 °C overnight to allow for proper gelation. Migratory or invasive cells were fixed and stained using a 0.1% crystal violet solution for 10 min. Images were captured under identical parameters.

2.8 Statistical analysis

Protein gray scale analysis was performed using ImageJ software (1.48v, NIH, USA). Results are expressed as mean ± SD. The significance between two groups was evaluated using an unpaired two-tailed Student's t-test, while significance among multiple groups was assessed using one-way analysis of variance followed by Tukey's post hoc test. A p-value < 0.05 was considered significant.