Clinical sample collection

A total of 16 cases of invasive HCC tissues and adjacent non-cancerous tissues were collected immediately after surgical resection from Zhongshan Hospital, Fudan University. All cases were pathologically confirmed as HCC and none of the patients received either preoperative radiotherapy or chemotherapy. Informed consent was signed and obtained from each patient. All the clinical sampling was performed with approval of the Ethics Committee of the Zhongshan Hospital, Fudan University. Immediately after sample harvest, tissues were stored at -80 °C for further use.

Isolation and characterization of CAFs and NFs from HCC samples

Fresh HCC specimens and corresponding adjacent non-cancerous tissues were washed with serum-free DMEM/F-12 medium, cut into 0.2 × 0.2 mm fragments, and cultured in fresh medium for 24 h for attachment. At the end of the incubation period, the unattached cells were removed and the attached cells were grown in the culture dishes for 2–3 weeks. The culture medium was replenished every two or three days until fibroblasts began to grow out. To verify isolated CAFs and normal fibroblasts (NFs), expression of the fibroblast markers α-SMA and FAP was examined by immunofluorescence staining [13]. CAFs and NFs should be α-SMA- and FAP-positive but CD31-, AFP- and pan-cytokeratin-negative. CAFs and NFs of less than ten generations are used for the experiments.

Real-time quantitative polymerase chain reaction (RT-qPCR)

According to the kit protocols, total RNA was extracted from cells and tissues (TOYOBO, Tokyo, Japan). PCR-based analyses were performed following methods as previously described [13] using a universal SYBR Green Master System (Roche, Basel, Switzerland). Relative expression levels were calculated using the 2−ΔΔCt method. The primers used are listed in Table S1.

HCC cell lines

A metastatic HCC cell line, MHCC-97H, was established and provided by the Liver Cancer Institute, Fudan University (Shanghai, China) [31] and cultured following the methods described previously [13]. A metastatic HCC cell line, Huh-7 (JCRB0403), was obtained from the Japanese Cancer Research Resources Bank (JCRB; Osaka, Japan) and cultured following the methods described previously [13]. All cells were cultured at 37 °C in 5% CO2.

Cell transfection assay

For exogenous CXCL11 and LINC00152 overexpression, CAFs were transfected with plasmid-overexpressing CXCL11 or plasmid-overexpressing LINC00152 (CXCL11; LINC00152; GenePharma, Shanghai, China). For LINC00152 knockdown, a vector containing short hairpin RNA for LINC00152 (sh-LINC00152 #1/2; sh-NC as a negative control) was synthesized and obtained from GenePharma. For CXCL11 knockdown, a vector containing short hairpin RNA for CXCL11 (sh-CXCL11) was synthesized and obtained from GenePharma. All transfections were conducted using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for plasmid construction are listed in Table S1.

Preparation of conditioned medium (CM)

CM was obtained from CAFs following the methods described previously [13]. Cells and cell debris were removed by passing the collected CM through a 0.2 μm membrane syringe filter.

Transwell cell migration assay

Transwells without Matrigel gel were used for the migration assays following the methods described previously [13]. Transfected cells were seeded in the upper chambers and DMEM medium containing 10% FBS was added to the lower chambers. At the end of the migration assay, cells that stayed in the upper chambers were discarded, and cells that migrated to the lower chambers were fixed. Next, the cells were stained and counted under an optical microscope.

Fluorescence in situ hybridization (FISH) assay

A specific Biotin-labeled LINC00152 probe and a DIG-labeled miR-205-5p probe were purchased from General Bio. Ltd. Co, China. After being permeabilized with permeabilizer and digested with proteinase K, cells were prehybridized with a hybridization solution and then incubated with the LINC00152 probe in hybridization buffer overnight at 42 °C. After washing the cells with SSC reagent, they were incubated with HRP-streptavidin for 30 min at 37 °C. Then, the cells were incubated with TSA-520 reagent for 20 min at 37 °C in the dark. Next, the cells were incubated with the miR-205-5p probe, mouse anti-DIG-Biotin and TSA-570 reagent following the above procedures. Cell nuclei were stained with DAPI for 5 min at room temperature. LINC00152 is shown in green florescence and miR-205-5p is shown in red florescence. The cells were imaged by fluorescence microscopy (Olympus).

Argonaute2 (AGO2) assay

AGO2 was conducted using a Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, USA) according to the manufacturer’s guidelines. In short, approximately 1 × 107 cells were lysed and mixed with AGO2 antibody (ab32381, Abcam) or IgG-coated beads on a rotator at 4 °C overnight. Then, the beads were washed, and co-immunoprecipitated RNA was extracted using RNAiso Plus (TaKaRa, Japan), after which the levels of LINC00152, miR-205-5p and the 3’UTR of CXCL11 were detected by RT-qPCR.

RNA pull-down assay

Biotin-labeled LINC00152 or negative control (NC) oligo probes (General Bio) were pre-incubated with Streptavidin-Dyna beads M-280 (Invitrogen, USA). Next, the cells were crosslinked, lysed and incubated with the beads at 4 °C overnight. Then, the beads were washed, and the crosslinking was reversed. RNA was extracted using RNAiso Plus (TaKaRa, Japan), after which the level of miR-205-5p was measured by RT-qPCR.

ELISA

Cells, transfected and/or treated, were cultured for 48 h. Then, supernatants were collected, centrifuged (1500 rpm, 5 min), and examined for CXCL11 levels using a human CXCL11 ELISA kit (Invitrogen, USA).

Immunoblotting assay

Immunoblotting was performed using anti-CXCL11 (CSB-PA06119A0Rb; Cusabio), anti-PCNA (ab29, Abcam, UK) and anti-β-actin (66009-1-Ig; Proteintech, Wuhan, China) antibodies and secondary antibodies (HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG) following the methods described previously [13] to detect the protein levels of CXCL11 and PCNA. The ECL chemiluminescence method was used to visualize and detect the signals.

Establishment of a subcutaneous xenotransplanted HCC tumor model in mice

All animal experiments complied with the Guidelines for the Care and Use of Experimental Animals and were approved by the Experimental Committee of the Zhongshan Hospital, Fudan University. All experimental procedures were conducted following the methods described previously [13].

CAFs were infected with CXCL11 overexpression lentivirus (CXCL11) or LINC00152 knockdown lentivirus (sh-LINC00152) for 48 h followed by 4 days of selection with 2 µg/ml puromycin (Beyotime). BALB/c nude mice (SJA Laboratory Animal Co., Ltd, Shanghai, China) were divided into four groups: those recieving MHCC-97H cells + CAFs (NC + sh-NC-infected; n = 6), MHCC-97H cells + CAFs (CXCL11 + sh-NC infected; n = 6), MHCC-97H cells + CAFs (NC + sh-LINC00152 infected; n = 6) and MHCC-97H cells + CAFs (CXCL11 + sh-LINC00152 infected; n = 6). A total of 5 × 105 MHCC-97H cells mixed with 1.5 × 106 infected CAFs were suspended in 100 µl PBS and injected subcutaneously to the left armpit of the mice. After 4 weeks, anesthetized mice were sacrificed, and their tumor volumes and weights were examined. Tumor tissues were collected and subjected to RT-qPCR and immunoblotting.

Statistics analysis

GraphPad (San Diego, California, USA) was used to process the experimental results and express them as mean ± standard deviation (S.D.). Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test were used to assess statistical significance. P value < 0.05 is considered statistically significant.