Clinical samples
Between January 2018 and June 2020, surgical tissues were collected at the first affiliated hospital of Shandong first Medical University from 132 patients with hepatocellular carcinoma confirmed by fast pathology biopsy during the operation, after signed informed consent was obtained from the patients. All fresh tissues were immediately stored in a refrigerator at -80 ℃ and anonymized before transfer to the laboratory for further processing. Patient demographic data, including age, sex, clinical classification, survival time, and relative follow-up visits were collected. All control subjects, who were free of liver malignancy, were followed up for two and a half years.
Primary TC culture
Fresh liver para-cancer and hepatic hemangioma (as the control group) tissues, obtained after surgery, were cut into smaller pieces, and incubated with 5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Then, samples were washed twice with calcium- and magnesium-free PBS (pH 7.4, G0002, Servicebio, USA) and centrifuged at 10000 r.p.m. for 5 min, and then resuspended in DMEM (Gibco-8120217, NY, USA) supplemented with 10% fetal calf serum. The BJ-40 capillary glass tube (1.0 mm outer diameter, 0.8 mm inner diameter; Hengtong Technology Company, Beijing, China) was soaked in 1 mol/L hydrochloric acid for 24 h, rinsed continuously with ultrapure water, dried at 65 °C, and autoclaved. Under 200x magnification of the microscope and selected cells to 0.2 mL centrifuge tube containing 2 μL of lysate according to the morphology of TCs.26,27
Liver TC isolation and identification
Following the sacrifice of mature C57BL/6 mice (No. 4432, Weitonglihua animal company, Beijing, China) using an anesthetic, mouse hepatic tissue was collected under sterile conditions and transferred into sterile tubes containing DMEM supplemented with 100 UI/ml penicillin and 0.1 mg/ml streptomycin (20201013, Kaisu biology Co Ltd., Jiangsu, China), and transported to the cell culture laboratory. Dispersed cells were separated by filtration through a 40 m-diameter cell strainer (CLS431751,Falcon, NJ, Germany), collected by centrifugation at 1 000 rpm for 5 min, and resuspended in DMEM supplemented with 10% fetal calf serum, 100 UI/ml penicillin, and 0.1 mg/ml streptomycin (Sigma-Aldrich). Cells were distributed in 25 cm2 plastic culture flasks at a density of 1×105 cells/cm2 and maintained in a 37 ℃ and 5% CO2 atmosphere until they became semi-confluent. Typical TCs were photographed by auto-microscopy every 12 h. After adhesion of cells to the plate, Telocytes were selected, purified and further multiplied for the next experiment. TCs were identified according to their morphology and immunofluorescent staining assays.28
Lentivirus production and transfection
To build recombinant lentiviruses, 293T cells were co-transfected with progresses of package, envelop, and expression. The virus-containing supernatant was collected and concentrated by ultracentrifugation. The viral stock was supplemented with 8 mg/mL polybrene for infection.
Cell Transfection
To obtain stable experimental cell lines, primary TCs from mouse hepatic tissues were transfected with SV40 large and small T antigens to obtain TCSV40. TCSV40 cells were cultured in Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% fetal calf serum (Gibco-8120330, NY, USA). HepG2, SNU182, and SK-HEP-1 cell-Lines were cultured in DMEM (GIBCO,Beijing, China) supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml streptomycin. These cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. When cells reached 60-80% confluence, positive and stable transfectants were selected for the subsequent experiments.
RNA extraction and qRT-PCR analysis
To determine MMP2, MMP3, MMP9, MMP11, and MMP14 mRNA expression levels in HCC and para-cancer tissues, qRT-PCR analysis was performed. Total RNA was extracted from tissues using the Trizol reagent (CW0581, Kangweishiji company, China) according to the manufacturer’s protocol.29 Reverse transcription and cDNA amplification were performed using the SYBR master mix (CW0957, Kangweishiji company, China) according to the manufacturer’s guidelines. β-actin genes were used as internal controls. The primary sequences were shown in the Supplementary Table 1. The computational format of mRNA expression= 2-△△CT.
RNA interference
For the miR-942-3p test, complete miRNA sequences were obtained from the miRBase database; shRNAs and mimics of the indicated miRNA was purchased from RiboBio Company (Shanghai and Wuhan, China). Transfection with shRNAs and miRNAs was performed using riboFECT™ CP (RiboBio, Wuhan, China) according to the manufacturer’s instructions.
Luciferase assay
For the 3’-UTR analysis, cells were co-transfected with a psiCHECK-2-based construct and pre-miR-942-3p or a negative control. The Luciferase assay was conducted using the Luciferase Reporter Assay System (Promega, Madison, WI, USA). The Luciferase miRNA target expression vector (Promega, Madison, WI, USA) was used to construct the reporter vectors, MMP9 wild type (WT), MMP9 mutant (MUT), and negative vector-mimics (NC).
Western blot analysis
For western blot analysis, samples were thawed and resuspended using a lysis buffer (20% Glycerol, 4% SDS in 100 mM Tris Buffer, pH 6.8). Cell extracts were boiled for 10 min in loading buffer and then centrifuged at 12 000 rpm for 10 min at 4 °C using a microcentrifuge. The immune-reactive bands were colored using an ECL-PLUS/TM (Amersham company, UK). The antibodies are listed in Supplementary Table 2.
Immunohistochemistry (IHC) staining
Formalin-fixed paraffin-embedded primary tumor and para-cancer tissues were used for IHC analysis. For heat-induced antigen retrieval, slides were soaked in a citric acid buffer and maintained at a sub-boiling temperature for 8 min. Sections were observed using a light microscope (XSP-C204, CIC, China), and were scanned using a laser scanning confocal microscope (Eclipse Ti-E, Nikon, Japan) at a 40× magnification. Datums were quantified in immunohistochemistry digital slides using a Leica Aperio positive pixel count algorithm through whole slide analysis (PANNORAMIC DESK/MIDI/250/1000, 3DHISTECH, Hungary). Antibodies are shown in Supplementary Table 2.
Immunofluorescence (IF) staining
All sections were incubated twice in xylene for 15 min. The sections were dehydrated in 2 changes of pure ethanol using a dehydrator and were immersed in an EDTA antigen retrieval buffer (pH 8.0, G1206/G1203, Servicebio, USA). After the antibody reaction, photos were taken using a fluorescence microscope (NIKON ECLIPSE C1, Tokyo, Japan) with an imaging system (NIKON DS-U3, Tokyo, Japan). Images were captured at a magnification of 1 to 400 (Microscope Camera XSP-C204, Olympus Europa GmbH, Hamburg, Germany). Antibodies are listed in supplementary Table 2.
Transwell assay
For the invasion assays, transwell migration chambers and Matrigel coated chambers (Becton Dickinson, Waltham, MA) were used. In summary, 5 × 104 HCC cells were seeded into the upper chamber in a serum-free culture medium. The lower chamber was filled with 5 × 104 TCs in DME medium(Thermo Fisher, HyClone, UT, USA) with 10% FBS(Thermo Fisher Scientific, MA, USA). After 48 h of incubation, cells that crossed the membrane were stained with 1% crystal violet and quantified using a microscope (CKX-51, OLYMPUS company, Japan).
CCK-8 cell counting assay
TCs in logarithmic phase were digested and made into cell suspensions. After uniform spreading, the cells were incubated and then 10 μ L of 5 mg/mL cell counting kit-8(CCK-8)(HY K0301, MCE, Shanghai, China) was added to the wells starting from the second day after spreading and 4 h before the termination of the incubation. The OD value was measured at 450 nm by enzyme marker after 4 hours.
Wound healing assay
Using a ball marker, horizontal lines were uniformly drawn on the back of a 6-well plate. Approximately 2.5 × 105 HCC cells were added to the wells and incubated overnight to reach a fusion rate of 100%. A TCSV40 supernatant was prepared after 24 h of culturing and added to the chambers of the plate. MMP9 inhibitor group was a concentration of 3 µM, adding each group of co-culture chambers into the corresponding wells. Each chamber was incubated at 37 ℃ in a 5% CO2 atmosphere for 48 h. The area was estimated using the Image J software.
In vivo models
To establish a metastatic lung cancer model, 100 μl of PBS containing 1 × 107 HepG2 cells was injected into the tail veins of BALB/c-nu mice (No. 4272, Weitonglihua animal company, Beijing, China). After that, 50 μl with 0.9% normal saline containing 6 × 104 TCs was injected weekly. The mice were sacrificed on day 42, and their lung tissues were resected, photographed, fixed with 4% paraformaldehyde, and strained with hematoxylin and eosin (HE). For sub-axillary transplantation, HepG2 cells were injected into the right axilla of nude mice and 50 μl of 0.9% normal saline containing 6 × 104 TCs was injected around local tumors after every 3 days, with the group that received TCs and the MMP9 inhibitor serving as the comparison group. After 28 days, these mice were sacrificed to obtain sub-axillary transplanted tumors, the weights and volumes of which were measured (maximum axis × minimum axis2 × 1/2).
Statistical analyses
SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. Quantitative variables were represented as the mean ± standard deviation (SD). Two-tailed Student’s t-tests, paired t-tests, chi-square tests, and multivariate analyses were used to evaluate the differences between groups. Pearson correlation analysis was used to determine the relationship between MMP9 expression and TC number in HCC tissues. Survival curves were constructed through Kaplan–Meier analyses, and values of P < 0.05 were considered statistically significant.