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Intrahepatic cholangiocarcinoma (ICC) is a rare but highly aggressive malignant tumor arising within the liver, with a 5-year survival rate of only 20–40% after surgery. The role of

interleukin-8 (IL-8) in ICC progression remains elusive. A transcriptomic approach based on IL-8 stimulation first revealed significant upregulation of the prometastatic gene CD97 and key

epithelial–mesenchymal transition (EMT) factors E-cadherin and vimentin. Immunohistochemistry of 125 ICC tissues confirmed the positive correlation between IL-8 and CD97. Multivariable Cox

regression indicated that they are both independent predictors of ICC prognosis. Mechanistically, IL-8 treatment induced CD97 expression at 50 and 100 ng/ml in QBC-939 and QBE cells,

respectively. Moreover, the induction of cell migration and invasion upon IL-8 treatment was attenuated by CD97 RNA interference, and the expression of EMT-associated genes was dramatically

inhibited. To determine whether CXCR1 or CXCR2 are downstream effectors of IL-8, siCXCR2 was applied and shown to significantly attenuate the oncogenic effects of IL-8 by inhibiting the

phosphorylation of PI3K/AKT. Finally, the induction of CD97 expression by the PI3K pathway was verified by treatment with the inhibitor LY294002. In vivo, the significant tumor growth and

lung metastasis effects induced by intraperitoneal injection of IL-8 were greatly inhibited by silencing CD97 in nude mice. Collectively, the study presents a novel mechanism of the

IL-8-CXCR2-PI3K/AKT axis in regulating CD97 expression, which leads to ICC metastasis mainly through EMT. The study may provide alternatives for targeting the tumor microenvironment in

metastatic ICC.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor occurring in epithelial cells of secondary bile ducts and above branches. It ranks as the second most common primary liver

malignancy, accounting for 5–30% of all hepatic malignancies1. ICC is clinically rare in developed countries but is more common in East Asia, with an incidence of 10/100,000 persons in

China2. Systemic chemotherapy has shown limited success for treating ICC in clinical trials, and curative-intent resection (R0 resection) is the best treatment3. However, ICC patients

frequently do not present symptoms until the disease is in an advanced stage. The 5-year survival rates for patients with regional or distant metastasis are 8% and 2%, respectively, while

the rate is 25% in those with local disease4. Given the increasing incidence of advanced stage ICC and the limited efficacy of current treatment strategies, it is urgent to decipher the

biological events in metastatic types of ICC. Chronic inflammation, such as primary sclerosing cholangitis and primary biliary cirrhosis, is recognized as a major risk factor for ICC

progression5. IL-8 is a pro-inflammatory/chemokine and plays an important role in cancer-related inflammation. Studies have shown that the expression of IL-8 is significantly correlated with

the progression and prognosis of various cancers, such as ovarian cancer6, breast cancer7, bladder epithelial cancer8, small cell lung cancer9, etc. As the primary cytokine, IL-8 is

implicated in tumorigenesis, but its exact impact on ICC is not clear.

In the present study, high-throughput RNA sequencing of QBC-939 cells under IL-8 treatment was utilized to identify the potential target genes of IL-8 in ICC. Among the 7282 differentially

expressed genes (DEGs), CD97 and a cluster of EMT-related genes were significantly upregulated. CD97 is a prometastatic epidermal growth factor-seven-transmembrane (EGF-TM7) receptor of G

protein-coupled receptor (GPCRs), and its presence in scattered tumor cells at the invading front of several carcinomas has clinical significance. CD97 contributes to an invasive phenotype,

correlating with tumor grade, lymph node invasion, metastatic spread, and overall prognosis10,11,12,13,14. Aust et al. illustrated the presence of CD97 in gastric, pancreatic, and esophageal

tumors but not in normal tissues15. We previously demonstrated that CD97 is a prognostic marker for ICC; however, the functional consequence remains elusive16. Here, the oncogenic role of

CD97 in promoting cell invasion, tumor growth and eventually lung metastasis was validated by in vitro and in vivo ICC models.

At present, the mechanism of IL-8 induced CD97 expression in EMT metastasis of ICC cells is not clear. IL-8 plays its biological role mainly through CXCR1 and/or CXCR2 receptors17 and can

activate PI3K/AKT signaling in prostate cancer, nasopharyngeal cancer and thyroid cancer cell lines18,19,20. Other studies have shown that wortmannin (a specific inhibitor of PI3K/AKT) can

significantly down-regulate CD97 expression in colorectal adenocarcinoma cell lines21. Combined with RNA-seq results, we speculated that IL-8 induced CD97 expression through CXCR2/PI3K/AKT

signaling pathway, which may play an important role in EMT metastasis of ICC cells.

This study was approved by the Ethics Committee of the Affiliated Union Hospital of Fujian Medical University, Fuzhou, People’s Republic of China (Patient protocol 2018KY067). Written

informed consent was obtained from all patients before surgery and all specimens anonymized. We confirmed that all methods were carried out in accordance with relevant guidelines and

regulations. A total of 125 clinical specimens were collected from ICC patients, and the relevant clinicopathological factors are shown in Table 1. All of the radical resections (R0

resection) were performed by the same hepatobiliary surgical team. These ICC patients had not received preoperative radiotherapy or chemotherapy, and patients who had distant metastasis or

died due to an accident or other causes unrelated to ICC were excluded from the study. From January 2009 to January 2019, the above patients underwent hepatic radical resection. The

diagnoses corresponding to all tumor specimens were histologically confirmed as adenocarcinoma from intrahepatic ducts. The follow-up began from the date of surgery and lasted until patient

death, and the follow-up lasted up to 60 months (4–60 months). Additional surgical intrahepatic bile duct tissue was obtained for comparative analysis from 10 patients who underwent live

resection for hepatolithiasis.

The animal experiments were approved by the Institutional Animal Care and Use Committee of the Affiliated Union Hospital of Fujian Medical University, Fuzhou, People’s Republic of China

(Animal Protocol IACUC FJMU 2022-0039). The care and use protocols of the animals were performed in conformity with the Regulations for the Administration of Affairs Concerning Experimental

Animals approved by the State Council of People’s Republic of China.

The ICC cell lines QBC-939 and RBE, which were purchased from the National Center for Certified Cell Culture (Shanghai, China), were maintained in RPMI-1640 medium (HyClone) with 10% fetal

bovine serum (HyClone). QBC-939/shCD97, QBC-939/small interfering RNA (si) CXCR1, QBC-939/siCXCR2, QBC-939/negative control (NC), RBE/shCD97, RBE/siCXCR1, RBE/siCXCR2, and RBE/NC cell lines

were established by infection with shRNA or siRNA according to the manufacturer’s instructions (System Bioscience). The cells were supplemented with 1% penicillin–streptomycin (Biological

Industries, Israel) and cultured at 37 °C under 5% CO2.

The shRNA sequences targeting CD97 were as follows: sh-CD97#1: 5′-GCCGAACTGGAGGAGATATAT-3′; sh-CD97#2: 5′-GCACGCATGAAGCTGAATTGG-3′; sh-CD97#3: 5′-CTCAAACCTTGAAGATATC-3′; sh-NC:

5′-TTCTCCGAACGTGTCACGT-3′. The siRNA sequences targeting CXCR1 and CXCR2 were as follows: siRNA-CXCR2: sense 5′-GGUCAAGUUUGUUUGUCUUTT-3′, antisense 5′-AAGACAAACAAACUUGACCTT-3′; siRNA-CXCR2:

sense 5′-CCCUGGAAAUCAACAAGUATT-3′, antisense 5′-UACUUGUUGAUUUCCAGGGTT-3′; si-negative control (NC): sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′.

The primer sequences are summarized as follows: CD97: (forward) 5′-GATACTGCTGGTTGGACTTTGAG-3′ and (reverse) 5′-CCCTCGCCTTCTTTAATTTCTTCA-3′. CXCR1: (forward) 5′-TTCTCCATAGCTGCCTCAACC-3′,

(reverse) 5′-TGTAGGAGGTAACACGATGACG-3′. CXCR2: (forward) 5′-TACTGGCCTGCATCAGTGTG-3′, (reverse) 5′-CAGGCTGGGCTAACATTGGA-3′. GAPDH: (forward) 5′-GGTGTGAACCATGAGAAGTATGA-3′, (reverse)

5′-GAGTCCTTCCACGATACCAAAG-3′.

The following antibodies and reagents were used: IL-8 (200-08, PeproTech, USA), anti-IL-8 (A2541, ABclonal, USA), anti-CD97 (A3780, ABclonal, USA), anti-CXCR1 (A16386, ABclonal, USA),

anti-CXCR2 (A3301, ABclonal, USA), anti-PI3K (4249, Cell Signaling Technology, USA), anti-P-PI3K (17,366, Cell Signaling Technology, USA), anti-AKT (4691, Cell Signaling Technology, USA),

anti-P-AKT (13,038, Cell Signaling Technology, USA), anti-E-cadherin (GB11082, Servicebio, China), anti-N-cadherin (GB111273, Servicebio, China), anti-Vimentin (GB11192, Servicebio, China);

LY294002 (9901S, Cell Signaling Technology, USA), GP-Transfect-Mate reagent (Shanghai GenePharmaCo., LTD), reverse transcription kit (Life Technologies, USA), Fast Start Universal SYBR Green

Master Mix (Roche Group, Swiss), and Transwell kit 8.0 µm (Falcon, Becton Dickison, USA).

Total RNA was extracted from QBC-939 cell lines treated with IL-8 or control solvent, and three biological replicates were assessed. According to the manufacturer's instructions, cDNA was

synthesized in an M-MuLV reverse transcriptase system. cDNA fragments of approximately 370–420 bp were screened with AMPure XP Beads for PCR amplification, and PCR products were purified

again with AMPure XP Beads to obtain the library. Illumina NovaSeq 6000 sequencing was performed after different libraries were combined according to the requirements of effective

concentration and target offline data volume. DESeq2 software (1.20.0) was used to analyze the differential expression between the two comparison combinations. Genes were considered DEGs if

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