Cell culture

MGC803, Hs746T, and HEK-293 T cells were obtained from the American Type Culture Collection (ATCC). MGC803, Hs746T and HEK-293 T cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Cat: 11,965,092) containing 4.5 g/L glucose and 4 mM L-glutamine (DMEM, 41,965, Life Technologies) and supplemented with 10% foetal bovine serum (FBS, Biological Industries, BISH0744). Cultures were maintained in a humidified incubator at 37 °C with 5% CO2. The Power Plex 21 system applied short tandem repeat (STR) analysis to identify all cell lines described in the article. The STR data of MGC803, Hs746T and HEK-293 T cell lines were examined and matched with the STR data of ATCC.

Cell cryopreservation

MGC803, Hs746T and HEK-293 T cells in the logarithmic growth phase were detached using trypsin–EDTA (Thermo Fisher Scientific, Cat:25,200,072) and suspended in serum-free cell cryopreservation solution (CELLSAVING, cat: C40100). Cell suspensions were aliquoted into cryovials and stored in liquid nitrogen.

Cell maintenance and passage

Cells were passaged using a standard trypsin–EDTA dissociation protocol. Upon reaching 70–80% confluence, the cells were harvested, washed with prewarmed PBS, and resuspended in fresh culture medium. Subcultures were initiated at a seeding density of 1 × 105 cells/mL.

RNA extraction and qPCR analysis

Total cellular RNA was extracted using the RNeasy Plus Mini Kit (Tiangen, DP451) according to the specifications provided by the manufacturer. The total RNA obtained in the previous step was reverse transcribed using HiScript II Q RT SuperMix (Vazyme, R223-01). qRT‒PCR was performed using SYBR qPCR Master Mix (Vazyme, Q511-02) under a 7500 Fast Real-Time PCR System (Applied Biosystems, Singapore) machine, and 36B4 was used as an internal reference. The primer sequences designed for the qPCR assay are shown below: 36B4 F: GCA GCA TCT ACA ACC CTG AAG; R: CAC TGG CAA CAT TGC GGA C. CTGF F: ACC GAC TGG AAG ACA CGT TTG; R: CCA GGT CAG CTT CGC AAG G. CYR61 F: GGT CAA AGT TAC CGG GCA GT; R: GGA GGC ATC GAA TCC CAG C. CXCR7 F: CTA TGA CAC GCA CTG CTA CAT C; R: CTG CAC GAG ACT GAC CAC C. ANKRD1 F: CGC ATT TCG GCA AGT TGG AG; R: TCC TGC TTG AAT CAG CCA GAC; YAP F: CAA GAA AGC AGG CTC ACA GAA; R: GCT GGG TGT TAG GGC TTC G. The specifics of all primer pairs were assessed by melting curve analysis.

Plasmids and siRNA

The CXCR7 and LATS1 plasmids required for the experiments were obtained from HANBIO (https://www.hanbio.net). Plasmids were transiently transfected with Lipofectamine 2000 (1662298, Invitrogen). Specific target genes were thereby knocked down using small interfering RNA. The sequences of CXCR7 siRNA are as follows: (1) CGC ACU GCU ACA UCU UGA ATT and UUC AAG AUG UAG CAG UGC GTT (2) CCG UUC CCU UCU CCA UUA UTT and AUA AUG GAG AAG GGA ACG GTT. The sequences of YAP siRNA are as follows: (1) GUC AGA GAU ACU UCU UAA ATT and UUU AAG AAG UAU CUC UGA CTT (2) GUC UCA GGA AUU GAG AAC ATT and UGU UCU CAA UUC CUG AGA CTT. The sequences of GNA11 siRNA are as follows: ACU UGU AGA GGA UCU UGA GCG and CUC AAG AUC CUC UAC AAG UAC. The sequences of GNAQ were UUA UCU UCA UCA GAG UAU CCU and GAU ACU CUG AUG AAG AUA AAA. The negative control siRNA sequences were as follows: UUC UCC GAA CGU GUC ACG UTT and ACG UGA CAC GUU CGG AGA ATT. The reagent used for siRNA transduction was RNAiMAX reagent (13,778,150, Invitrogen). For lentivirus CXCR7 silencing, shCXCR7 was inserted into the insertion vector pLKO.1 and cotransfected with pMD2.G envelope plasmid and psPAX2, bound as a packaging plasmid in HEK293T cells and cotransfected to express the corresponding gene. Forty-eight hours later, the CXCR7 shRNA-expressing lentivirus solution was collected for subsequent experiments. The gene was expressed by adding 3 ml of antibiotic-free medium containing 300 µl of lentiviral suspension to MGC803 gastric cancer cells for coculture.

Reagents

The reagents used were XMU-MP-1 (MCE, Cat. No. HY-100526), doxycycline (Dox) (Sigma, Cat. No. 33429), ACT-1004–1239 (MCE, Cat. No. HY-142617), TC14012 (MCE, Cat. No. HY-P1102), verteporfin (MCE, Cat. No. HY-B0146), GSK429286A (MCE, Cat. No. HY-11000), Y27632 (MCE, Cat. No. HY-10071), and C3 (Cytoskeleton, Inc., Cat. No. CT03).

Immunoblot analysis

The method used to analyse relative protein expression in cells was the standard Western blotting technique. The catalogue of antibodies used for immunoblotting tests was as follows: anti-CXCR7 (ab117836, Abcam, 1:1000), anti-YAP (SC-101199, Santa Cruz, 1:1000), anti-tubulin (11,224–1-AP, Proteintech, 1:1000), anti-histone-H3 (17,168–1-AP, Proteintech, 1:1000), anti-pYAP (S127) (13,008, Cell Signaling Technology, 1:1000), anti-actin (3700, Cell Signaling Technology, 1:1000), anti-Gαq/11 (sc-392, Santa Cruz, 1:1000), anti-Gαs (sc-135914, Santa Cruz, 1:1000), anti-LATS1 (3477, Cell Signaling Technology, 1:1000), and anti-Myc (9E10, Santa Cruz, 1:1000). The protein signal was detected by an ECL kit (Millipore Co., Billerica, Massachusetts, USA).

In vitro kinase activity assay

The phos-tag reagent Phos-tag Acrylamide (Wako, 93521) was purchased from Wako Chemicals. Gels containing phos-tag and MnCl2 (Wako, 13446–34-9) were prepared according to the manufacturer's instructions. On the prepared phos-tag gels, anti-YAP (SC-101199, Santa Cruz, 1:1000) was used for primary antibody, the YAP protein can be separated into multiple visible bands by itself according to the phosphorylation effect to observe the change in phosphorylation status.

Quantifying cell viability

Gastric cancer cells MGC803 and Hs746T cells were treated in 12-well plates with siCXCR7 or siControl transfection or by drugs. Twenty-four hours after the cells were treated, the cells in the 12-well plates were counted, and 5000 cells were transferred to 96-well plates after counting the distillation. The number of cells was determined by measuring the relative viability of the cells at 450 nm using the CCK-8 Cell Proliferation Reagent at different time points to measure absorbance.

Wound healing assay

In the wound healing test, MGC803 and Hs746T gastric cancer cells were transfected with siCXCR7 or siControl or treated with the indicated drugs in a 6-well plate until the cells grew without gaps, and then, a straight line was drawn along a straight edge with a 200 µl yellow sterile tip gun. Scratches of the cells were assessed by microscopic photography at the indicated time points after the line was made. The separation between the two edges was measured and compared to the distance on the first day. The recovery rate of wound healing was calculated as follows: [1—(Woundwound width at a given time/wound width at t = 0)] × 100%.

Transwell assays

Transwell plates (8 μm pore size, Corning) were used for cell migration experiments and invasion experiments. The membranes in the upper chamber of the plates were coated with a special matrix gel (BD Biocoat, USA). After treatment with the appropriate siRNA or drugs, the cells were fixed on the membrane bottom with 4% paraformaldehyde for 10 min. After fixation, they were washed three times with water. Subsequently, the cells were stained with crystal violet for 10 min, followed by another three washes with water. Finally, cell counting was performed under a microscope using a 20 × objective. Each experiment was conducted in triplicate.

Immunofluorescence (IF) staining

MGC803 and Hs746T cells were added to 24-well plates, treated with small molecule reagents after they were plated, and transferred to small dialysis slides after 24 h. After they were plated, gastric cancer cells were fixed on bottom coverslips using 4% paraformaldehyde and stained with anti-CXCR7 (ab117836, Abcam, 1:200) and anti-YAP (14,074, Cell Signaling Technology, 1:200) primary antibodies for one hour at room temperature. After that, the cells were washed with PBS. Cells were then incubated again with fluorophore-conjugated secondary antibody (Invitrogen, Carlsbad, CA). After PBS washes, the cells were stained for nuclei with the cell nuclear dye DAPI (Life Technology). After the cells reacted and were washed, images of staining for cell-specific proteins were obtained with a confocal laser scanning microscope (Leica TCS SP8 STED) after dropping in anti-quenching reagent. The stained images taken were integrated into a single image using ImageJ software, and their fluorescence intensity was measured for analysis.

Immunoprecipitation

Western blotting was conducted as described in previously published studies [16, 31]. Total lysates of MGC803 and Hs746T gastric cancer cells were preincubated with rabbit IgG for 1 h. We used Cell lysis buffer for Western and IP (Beyotime, P0013).It contained 20 mM Tris(pH7.5), 150 mM NaCl, 1% Triton X-100. sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin with Protease inhibitor Cocktail (TargetMol, C0001). After removal of IgG as a negative control, immunoprecipitation was performed with anti-LATS1 (3477, Cell Signaling Technology, 1:1000), and antibodies were incubated with protein for 4 h at 4 °C, with rabbit IgG (Santa Cruz) used as a negative control. Afterwards, protein A was added for the corresponding binding of beads to antibodies, and finally, western blot loading buffer was added. After lysis, the cell solution was detected with anti-CXCR7 (ab117836, Abcam, 1:1000) or anti-YAP (SC-101199, Santa Cruz, 1:1000) for the presence or absence of bound proteins.

Colony formation assay

Gastric cancer cells MGC803 and Hs746T cells were spread in 12-well plates and treated with CXCR7 siRNA or siControl or the indicated concentrations of chemical reagents after they were allowed to grow against the wall. Twenty-four hours later, the cells were suspended in trypsin, and cell counting was performed (5,00 cells/well in six-well plates). The cells were cultured for two weeks after adding sufficient medium, and the medium was changed every three days with fresh medium containing serum. After they grew to the appropriate density, the cells were fixed with 4% paraformaldehyde for ten minutes, washed with PBS and then stained with crystal violet staining solution for the fixed colonies. Colonies were counted by counting specific areas of each condition, and the counts were compared.

Extreme limiting dilution assay and self-renewal capacity

Control or experimental cells were inoculated into ultralow adherence 96-well plates (Corning, CLS3474) at different cell doses and incubated for 10 days at 37 °C in standard pH under sphere-forming conditions. Decreasing numbers (200–100-50–20 cells per well) of cells were cultured in medium plus 20 ng/ml basic fibroblast growth factor (bFGF) (R&D Systems, 233-FB-010) and 20 ng/ml epidermal growth factor (EGF) (R&D Systems, 236-EG-200). Colony formation was assessed by visual observation. For each dilution series, we counted the wells that showed sphere formation on Day 11. Data were analysed and displayed using the Extreme Limiting Dilution Assay (ELDA) software, which can be downloaded at: https://bioinf.wehi.edu.au/software/elda/ [32].

Flow cytometry analysis

In cell cycle analysis experiments, MGC803 and Hs746T cells were synchronized at G0/G1 by serum starvation. MGC803 and HS746T cells were seeded in 12-well plates and transfected with siCXCR7 or siControl or the appropriate concentration of chemical reagents specified with 10% FBS and left to react for 24 h or treated with the appropriate chemical reagents for the specified time. The suspended cells were first collected with trypsin, followed by fixation treatment with 70% ethanol, and then propidium iodide was used to stain the cells. In addition, the percentage of cells in each phase of the cell cycle was analysed by Modfit software. In the apoptosis assay, the cells were treated with the appropriate reagents. After that, the cells were stained with propidium iodide and Annexin V. The proportion of each cell phase was determined.

Luciferase reporter assay

Gastric cancer MGC803 and Hs746T cells were cotransfected and plated with siCXCR7 or siControl or with appropriate chemistry and TEAD luciferase reporter vector, and the cells were collected after 24 h by digestion with trypsin and then assayed. A dual luciferase assay kit (Promega) was used for this experiment. The pRL-null vector (Promega) expressing Renilla luciferase was an internal control.

In vivo tumorigenesis assay

For the in vivo tumorigenesis assay in mice, MGC803 cells were inoculated with the corresponding lentiviral cells. After 48 h of infiltration, the cells were screened for resistant target cells by incubation with 0.5 μg/ml puromycin for three days. Gastric cancer cells MGC803 cells (2 × 106) of the screened stable transgenic strain were injected into the backs of 4-week-old female BALB/c nude mice. Alternatively, mice were treated with the drug after cell injection, and tumour formation in nude mice was monitored for 4 weeks. Tumour volume = 0.5 × length × width2. Mice were sacrificed 5 weeks after injection. The weight of their removed tumours was calculated, and photographs were taken. Mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. This experiment was conducted according to the protocol approved by the ethics commission of the Second Hospital of Shandong University. Approval Protocol Number KYLL-2023LW030.

Tissue microarray (TMA) and immunohistochemistry (IHC) analysis

Ninety paraffin-embedded gastric cancer samples were received from Shanghai Odo Biotech (http://www.superchip.com.cn). Approval Protocol Number: HStmA180Su11. The samples were inspected by pathologists. Pathological grade and lymph node metastasis conditions were acquired for each sample. The utilization of the samples was sanctioned by the company, and written informed consent was obtained from all patients. Samples were stained using antibodies specific for CXCR7 (Abcam, 72,100) and YAP (Cell Signaling Technology, 14,074) and assessed according to the Fromowitz criteria. The staining was graded in intensity as follows: no positive staining, 0; weak positive staining, 1; intermediate positive staining, 2; and high positive staining, 3. The percentage of positive cells was categorized into four categories: staining percentage of 0–25%, 1; staining percentage of 26–50%, 2; staining percentage of 51–75%, 3; and staining percentage of 76–100%, 4. Staining scores of 1–2 was regarded as indicating low expression, while staining scores of 3–4 was regarded as indicating high expression. At least three regions were counted per core region.

ChIP assay

The ChIP (chromatin immunoprecipitation) assay was conducted following to the methodology outlined in a previously published study [33]. In brief, cells were crosslinked by adding formaldehyde to a final concentration of 1% for 10 min only or 2 mM DSG crosslinker (CovaChem, Cat. No.13301) at room temperature for 1 h followed by secondary fixation with 1% formaldehyde (Pierce, Cat. No. 28908) for 10 min and quenched by adding glycine. Subsequently, cells were washed with PBS and subject to cell lysis. Cell lysis buffer contains SDS Lysis Buffer (Beyotime P2078-11), Phosphatase Inhibitor Cocktail (TargetMol, Cat: C0002) and Protease Inhibitor Cocktail (TargetMol, Cat: 0001). The cell extracts were subject to sonication. To achieve chromatin fragmentation, sonication was employed using a Bioruptor sonicator (Diagenode). ChIP assay involved sonication of chromatin fragments, resulting in fragments typically ranging from approximately 100 to 500 base pairs (bp) in size. The sonication parameters were set as follows: Power: Typically set 25%, adjust as needed depending on the sample type. Sonication Cycle: 30 s on, 30 s off (pulse mode), repeated 10–20 times. Amplitude: High setting. Temperature: Samples were maintained in an ice-water bath during sonication to prevent overheating. After centrifugation, the cell extracts were incubated with prepared YAP antibody (CST, Cat. No. 14074), washed in wash buffer for five times and decross-linked ChIP in elution buffer at 65 °C overnight. Quantitative PCR analysis was performed with a DNA extraction kit (Qiagen, Cat. No. 28106). ChIP-qPCR analysis was performed with the following primer sequences: CTGF F: TGT GCC AGC TTT TTC AGA CG, R: TGA GCT GAA TGG AGT CCT ACA CA; CYR61 F: AGC AAA CAG CTC ACT GCC TT, R: ATG GTA GTT GGA GGG TCG TG; CXCR7: F: CAG GAA CAG AGA GAG CCA GC, R: ATT GAG AGG CCC AGG GTT G.

RNA sequencing and data analysis

The RNA sequence data were stored in the Gene Expression Repository (GEO) (GSE233094). Genes with differential representation (P < 0.05 and fold change > 2) were analysed by Ingenuity Pathway Analysis (IPA). For genomic rich analysis of RNA-seq data, the conserved Hippo signature gene set was utilized and downloaded from Molecular Signatures Database v7.4. Volcano plots were created with the 'ggplot2' package in R.

TCGA data and analysis of survival data

Gene exposure databases of 376 gastric cancer patients were obtained from the TCGA database. The correlation between CXCR7 expression and patient survival in gastric cancer was generated using GEPIA online software; GSEA online software was used to generate the results using default parameters. Volcano plots were generated with the 'ggplot2' package in R (threshold P < 0.05, equivalent change > 1.5).

Statistical analysis

Data were analysed using GraphPad Prism 8 software or SPSS version 23. Data are presented as the mean ± s.e.m. values. Variations among each individual set were appraised with Student’s t test. Survival analysis was performed by the Kaplan‒Meier method and log-rank test. A difference was assumed to be statistically significant when *P < 0.05 (**P < 0.01; ***P < 0.001).