Clinical specimens and ethical approval
Human HCC and para-cancer tissues were obtained from HCC patients who underwent the surgical operation at Shandong Cancer Hospital and Institute between January 2020 and January 2022 (78 cases). The patients never received chemo- or radiotherapy before surgery. Before and after the operation, peripheral venous blood(10ml) was collected to separate exosomes of serum from HCC patients (10 cases). All pathological diagnoses should be confirmed according to the guidelines of the American Joint Committee on Cancer. The study was approved by the Ethics Committee of Shandong Cancer Hospital and Institute(SHTHEC: 2022003015), and informed consent was gained from all patients and healthy volunteers.
Cell lines and co-culture
Human-derived HCCLM3 cell line, MHCC97 cell line and 293T-cell line were purchased from Zhongqiaoxinzhou company of China (www.zqxzbio.com) and cultured by DMEM or RPMI-1640 media at 37℃ in a humidified 5% CO2 incubator, respectively. Primary TCs were separated from the fresh para-cancer tissues and incubated for 72 hours, and then generated into enough cells. Dual-containers were used while TCs incubated with HCC cell lines, making TCs in the upper space and cancer cells in the nether space but the supernate was interlinked.
Telocytes identification
The protocol of TCs identification in the liver tissue could be referenced in our previous study(Suppl Fig 1-A)[14].
Exosomes isolation and identification
Serum and cancer cell supernate were certificated at 2000×g for 15 min, 10000×g for 30 min, and then filtered through a 0.22um filter to remove debris. The pellet was under ultracentrifugation at 1200000×g for 70 min(BECKMAN: Optima XPN-100), and then resuspended into PBS(E607008, Sangon biotech). CD81, HSP70 and TSG were used to identify exosomes by western blot assay, and the three-dimensions of exosomes were observed by transmitted electron microscope(TEM, HITACHI: HT7800). The volume of exosomes was detected by nanometre particle analysis(Particle Metrix, ZetaView PMX110).
Exosomes fluorescence tracking assay
After collecting exosomes from HCC cells-line suspension, put 5ul EvLINK505(CL12100220, TINGO, USA) dye into 150ul exosomes, lucifuge incubated at room temperature 30 min. Then 15ug/ml stained exosomes co-cultured with TCs for 24 hours in a 37℃ 5% CO2 incubator. Washing cells by PBS buffer three times and fixed by 4% paraformaldehyde for 30 min and then put 5ul CellLINK555 dye(1:100 dilution; EL012100200, TINGO, USA) into cells for 30 min in the dark condition. All images were photographed by confocal laser scanning microscope(3869000207, LSM800 with Airyscan2, Zeiss, Germany).
Quantitative real-time PCR(qRT-PCR)
Total RNA was extracted by Trizol reagent (Invitrogen) and reverse transcription was 2ug of total RNA. The mRNA or miRNA expression levels were conducted and calculated by the Bio-Rad CFX96 system. Expression levels were normalized to GAPDH (for mRNA) or U6 (for miRNA) and data was assessed by 2-ΔΔCt values. All primers were purchased from Jinweizhi company of China(Suppl Tab 7).
Overexpression and short hairpin RNAs
MHCC97 cells and TCs were instantaneously transfected with shRNA or scrambled shRNA negative control (sh-NC). All individual shRNAs were designed and synthesized by ABCAM company. Constructing all sequences of miR-942-3p and LncSNHG16, combined with a pWSLV-05 carrier, and then cells were transfected according to the manufacturer’s instructions for over-expression. The cells were harvested for 48 hours. MiR-942-3p or LncSNHG16 with pWSLV-05 was transfected into TCs or MHCC97 cells using Lipofectamine 2000 (Invitrogen; Suppl Fig 1-B, C, D).
Divergent LncRNAs bioinformatics analysis
The distinct expression of LncRNAs in exosomes between HCC patients and healthy volunteers was detected by Novogen. Company. LDT of China. Comparing those different LncRNAs by Genbank database, the functional enrichment analysis of LncRNAs was performed using the Genetic Ontology(GO) database. After screening interested miRNAs, their targets were selected by Starbase, TargetScan database and JEFFERSON and Venn Diagram Web Tools helped to focus finally targets. The co-expression and survival analysis of LncSNHG16 and MMP9 in HCC were analyzed by StarBase v3.0.
Western Blot
Cells or exosomes were dissolved in RIPA lysis buffer and 30 µg protein was run on SDS/PAGE gels and transferred to PDVF membranes. After blocking for 1 hour and rinsing with TBST three times, the membranes were incubated in relevant primary antibodies at 4℃ overnight. Thereafter, the membranes were incubated with secondary antibodies, and finally imaged by the ECL system (Thermo Fisher Scientific). All primary antibodies were listed in Suppl Tab 1.
Immunohistochemistry(IHC)
Formalin-fixed paraffin-embedded primary tumour and paracancerous tissues were used for IHC analysis(Suppl Tab 1). For heat-induced antigen retrieval, slides were soaked in 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 scanned using a laser scanning confocal microscope (Eclipse Ti-E, Nikon, Japan) at 40× magnification. Datums were quantified on digital immunohistochemical slides using a Leica Aperio positive pixel count algorithm through whole slide analysis (PANNORAMIC DESK/MIDI/250/1000, 3DHISTECH, Hungary).
Cell invasion and migration assays
Transwell assay for cancer cells invasive detection used 8 μm pore size chamber in 24-well plats, and the upper was filled with 100 μl of diluted Matrigel (1:20) and then 5×104 MHCC97 cells in 150μl serum-free media were added into the upper chamber, while 750μl of media containing 10% FBS or experimental supernate from TCs was added to the lower chambers. After 48 hours, the invaded cancer cells were fixed with methanol and stained by DAPI for 30min. At least five random fields were calculated under fluorescence microscope(Thermo.Com, USA). Each sample was run into /and repeated three times. Cell wound healing assay for cancer cell migration detection utilized different stimuli according to experimental groups.
Dual-Luciferase Gene Report
The 358 bp wild-type or mutant 3′UTR region of LncSNHG16 was inserted into the psiCHECK2-basic vector (Promega) and transduced into TCs with miR-942-3p mimics or MMP9 mutative type and 293T-cells using Lipofectamine 3000 (Invitrogen). Luciferase activities were measured 48 hours after transfection by a Dual-Luciferase Assay (Promega).
RNA immunoprecipitation (RIP) assay
RIP was conducted using the Magna Nuclear RIP™ (Cross-Linked) Nuclear RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. IgG antibodies (ab150077) were used for the RIP assay. Subsequent sequencing after RIP was performed and Tophat(v1.4.0) was used to map the RIP-seq raw reads to the human reference genome(hg19/GRCh37). GAPDH was considered as an internal reference[15].
In vivo model construction
To construct the subaxillary transplantation nude mice model, 100ul of PBS containing 1×106 MHCC97-luciferase cells with distinct groups were injected into the right axilla of nude mice, and sacrificed on day 18 according to the volume of tumors, from them, we randomly selected one mouse of the control group and resected the tumor in an aseptic condition. Then putting the same weight tumor into liver tissues of nude mice to construct the orthotopic liver transplantation tumor model, and observe the lung metastasis(Suppl Fig 2-A). As successfully established, the mice will be detected by IVIS Spectrum CT In Vivo Imaging System per 7 days. The interference of GW4869 and MMP9 inhibitor(ab142180, ABCAM company, China) groups were constructed by the intraperitoneal injection of mice with 100ul every two days. The mice were sacrificed by the high carbon dioxide after 21 days, and their tissues were fixed with 4% paraformaldehyde. We assured that all animals received humane care.
Statistical analysis
SPSS 26.0 version software(Chicago, IL, UAS) and Prism 8(San Diego, CA, USA) analyzed all the data in the study and each experiment was repeated at least 3 times. A p value < 0.05 was considered to represent significantly. The Student’s test was utilized to compare microarray data and two groups of data, while the mean ± standard deviation was used for all numerical results. Kaplan-Meier analysis and log-rank test were used to evaluate the OS of patients, as well that the Spearman’s rank correlation was calculated. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.000)