Establishment of the mouse model of spleen deficiency and classification criteria for the spleen deficiency index
All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University, Guangzhou, China. First, 1 mg of reserpine powder (Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, China; CAS code: 50-55-5) was completely dissolved in 25 mL of glacial acetic acid and stored at 4°C as a stock solution. Each mouse was injected subcutaneously with approximately 100 µL of the solution once a day (i.e., 0.1 mg∙kg–1∙d–1) for 14 consecutive days to establish the SD model. The control group was injected subcutaneously with 100 µL of a sodium chloride solution(0.9%) for the same number of days. The body weight and feed amount of all mice were measured daily. Simultaneously, the smell, mental state, body temperature and heat, breathing state, hair color, food intake, and stool of all mice, described in the SD rating scale, were observed and noted.The classification criteria[26–28] for determining the SD index were divided into four grades, each corresponding to a relevant score as follows: a score less than or equal to 7 = no SD symptoms; 8–14 = mild SD; 15–21 = typical SD; and 22–28 = severe SD (Table 1).
Table 1
Classification criteria for determining the spleen deficiency index
Index/Score
|
Grading standards
|
1
|
2
|
3
|
4
|
Body odor
|
Odor-free
|
Mild odor
|
Medium odor
|
Severe odor
|
Mental state
|
Stable
|
Irritable
|
Fatigued
|
Somnolent
|
Fever & chills
|
Normal
|
Cowered
|
Chills
|
Arched back & trembling
|
Respiration
|
Normal
|
Panting
|
Tachypnea
|
Faint
|
Fur
|
Glossy
|
Matted
|
Fluffy & erect
|
Brown & erect
|
Stool
|
Normal
|
Wet
|
Wet & rotten
|
Mucous texture
|
Appetite
|
Normal
|
Reduced to 50%
|
Reduced to 25%
|
None at all
|
Note: A total score of 7 represents no spleen deficiency, 8–14 represents mild spleen deficiency, 15–21 represents typical spleen deficiency, and 22–28 represents severe spleen deficiency.
Cell lines and culture
Human-derived hepatoma cells (MHCC97H, HCCLM3, and HepG2), mouse-derived hepatoma cells (Hepa1-6), and human embryonic kidney 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, St. Peters, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) at 37°C in a 5% CO2 atmosphere.
Extraction of exosomes
The mice were enucleated, and blood was collected in a 1.5 mL anticoagulant tube. The exosomes were then extracted from the plasma using the Wayen Exosome Isolation Kit (Cat# EIQ3-02001, H-Wayen Biotechnologies, Shanghai, China). In brief, 4 µL of reagent C that had been completely thawed on ice was added to 200 µL of mouse plasma, with up and down pipetting and vortex mixing performed until a homogeneous mixture was obtained. Then, 50 µL of extraction reagent A and 50 µL of reagent B were added to the suspension, and exosomes were extracted according to the steps outlined in the manufacturer’s instruction manual. Finally, the obtained pellet was resuspended in 50–120 µL of sterilized 1× phosphate-buffered saline (PBS), following which the exosome-containing suspension was aliquoted and stored at − 80°C for further use and analysis.
Transmission electron microscopy
The morphology of the exosomes was examined using a transmission electron microscope (JEM-1200EX, JEOL, Tokyo, Japan). In brief, the purified exosomes were first incubated with 4% osmium tetroxide at 4°C for 30 min, then transferred to copper grids with carbon-coated membranes, and subsequently stained with 2% phosphotungstic acid for 3 min. The filter paper on which the sample was absorbed was dried for 5 min and then imaged under the electron microscope at 10 kV.
Luciferase reporter gene assay
We predicted the targeting relationship between miR-29a-3p and the FAM167A gene using the bioinformatics databases TargetScan (https://www.targetscan.org/vert_80/) and miRDB (http://mirdb.org/). The 3'-untranslated region (3'-UTR) of the FAM167A promoter region constituted the miR-29a-3p-binding site for construction of the wild-type (WT) plasmid of FAM167A 3'-UTR (FAM167A-WT).
Based on this plasmid, a site mutation kit (Takara, Dalian, China) was used to mutate the miR-29a-3p-binding site on FAM167A-WT to construct the FAM167A 3'-UTR mutant (MUT) plasmid (FAM167A-MUT). In the meantime, HCCLM3 cells were seeded into the wells of 24-well plates and grown to 70% confluence. Using Lipofectamine™ 3000 reagent, the correctly sequenced FAM167A-WT or FAM167A-MUT plasmids were then co-transfected into the HCCLM3 cells together with mock NC or mock miR-29a-3p plasmids. At 48 h after transfection, the cells were lysed, and their luciferase activity was detected using a luciferase assay kit (Cat: 11402ES60,Yeasen,Guangzhou, China).
ELISA detection
We used enzyme-linked immunosorbent assay (ELISA) kits (Cat: MM-0163M1, MM-0040M1, and MM-0123M1) to detect target proteins in cell culture supernatants collected in sterile tubes. In brief, after centrifugation of the cell culture at 2–8°C for approximately 20 min (2,000–3,000 rpm), the supernatant was carefully collected and stored. If a precipitate had formed during storage, the supernatant was centrifuged again. The sample was then tested with the ELISA kit according to the manufacturer’s instructions. Finally, the optical density at 450 nm of the solution in each well was measured within 15 min of adding the stop solution.
RNA extraction and qRT-PCR
Total RNA was extracted from the cells using the MolPure® Cell/Tissue Total RNA Kit (Cat: 19221ES50, Yeasen, Shanghai, China) and reverse transcribed using SuperScript™ III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific,Shanghai, China) and specific primers. The real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the SYBR Green PCR Master Mix (Cat: 11184ES08, Yeasen), and detection was performed. The sequences of all the indicated primers are listed in Supplementary Table S1.
Wound-healing and Transwell assay
The mouse HCC cell line Hepa1-6 was co-cultured with approximately 100ug/ml exosomes for 48 h, following which the cells were washed with 2 mL of PBS. Then, 1 mL of 0.25% trypsin was added to dissociate the cells and a pipette was used to disperse them into single cells. Subsequently, 2–3 mL of DMEM containing 10% fetal bovine serum and trypsin was added and the thoroughly mixed cell suspension was transferred to a 15 mL centrifuge tube. The sample was centrifuged at 1,000 rpm for 5 min, after which the liquid was discarded and 1 mL of the medium was added. After thorough mixing, the cells were counted and 200,000–400,000 were seeded into 2 mL of serum-free DMEM in 6-well plates and cultured at 37°C. After overnight incubation, the medium was removed, and the cell layer in each well of the 6-well plate was scratched with a 200 µL pipette tip. The cells were then washed with PBS, and 1 mL of the wash solution was used for imaging analysis (adding PBS before photography to avoid a medium-colored background). After scratching of the cell layer, a time gradient was used, where images were taken at 12, 24, and 48 h.
For the cell migration assay, 800 µL of DMEM containing 10% fetal bovine serum was added to the lower chambers of a 24-well Transwell plate, and 200 µL of a serum-free cell suspension was added to the cell culture inserts in the upper chambers. The cells were cultured in an incubator for 20–24 h (no more than 24 h to avoid cell proliferation affecting the migration experiment). Then, the cell culture inserts were carefully removed from the chambers with tweezers, and the upper chambers and lower chambers were blotted to remove liquids and then washed with PBS. The cell culture inserts were then returned to the wells and 800 µL of methanol was added to the lower chamber and 200 µL to the upper chamber. After the cells had been fixed at ambient temperature (25℃) for 15 min, the methanol was removed from the chambers. The upper and lower chambers were again blotted and the fixatives were dried, after which 800 µL of crystal violet was added to the lower chamber and 200 µL to the upper chamber. The cell culture inserts were incubated for 30 min at ambient temperature, following which they were gently rinsed, then soaked several times with water, and finally removed from the chamber and blotted dry. The upper chamber liquid was blotted, and the cells were carefully wiped from the membrane surface on the bottom of the upper chamber with a damp cotton swab. The plates were inverted and dried thoroughly overnight in the oven. Pictures were taken, and samples were obtained.
Nude mouse tumor xenograft models
To generate tumor xenograft models, 0.1 mL of a precultured Hepa1-6 cell suspension (1 × 107/mL) was injected subcutaneously into the armpit of BALB/C nude mice. When the subcutaneous tumors were approximately 1 cm in diameter (after ~ 2 weeks), the mice were anesthetized with a 1% sodium pentobarbital solution (50 mg/kg) and then euthanized by cervical dislocation. The tumor was rapidly excised under sterile conditions, and the meat-like tissue was cut into 1 mm3 small pieces in PBS solution. The mouse model of liver cancer was then established as follows. First, the skin of C57 mice was incised at the xiphoid process under anesthesia, and the left liver lobe was removed from the abdominal cavity. A 1 mm3 piece of the tumor tissue was placed in a cannula (5 mm from the tip), which was then inserted into the liver surface at a 10° angle. Then, the tumor tissue was implanted under the liver capsule, an absorbable gelatin sponge was applied to the bleeding area, and the liver lobes were returned to the abdominal cavity, which was subsequently flushed with penicillin solution and closed with absorbable sutures. Blood and tissue samples were collected 28 d after the establishment of the liver cancer model.
Western blot analysis
The liver cancer tissues and HCC cells of the two groups of mice were lysed using RIPA lysis buffer, following which the total proteins were extracted using the corresponding extraction kit. The manufacturer instructions, “gel preparation–electrophoresis–transfer membrane–blocking and incubation antibody–incubate secondary antibody–ECL luminescence,” were then followed step by step, and finally data statistics and images were obtained. Primary antibodies used in this study were FAM167 Ab (1:1000),α1-integrin Ab (1:1000), Alix Ab (1:1000), HSP70 Ab (1:1000), CD81 Ab (1:1000), NF-κB Ab (1:1000), phosphorylated-NF-κB Ab (1:1000), and GAPDH mAb (1:1000). Secondary antibodies were anti-rabbit (1:2000) and anti-mouse (1:2000).
Survival analysis
The Kaplan–Meier plotter (http://kmplot.com/), which is an interactive online tool for investigating survival correlations, can assess the effects of 54,000 genes on survival in 21 cancer types [29]. To determine the clinical significance of the α1-integrin and FAM167A genes, patients with HCC were divided into high- and low-expression groups. The overall survival (OS) and recurrence/relapse-free survival (RFS) rates of the two groups were assessed using Kaplan–Meier plots and the log-rank p-value (p < 0.05). The past 10 years of follow-up statistics of 70 liver cancer patients of the Department of Traditional Chinese Medicine of the First Affiliated Hospital of Sun Yat-sen University were used for the OS and RFS analyses with the R language.
RNA interference and plasmids
Small interfering RNAs (siRNAs) (viz., siNC and siRNAs targeting FAM167A or α1-integrin) and mimics of the indicated miRNAs were obtained from Kinco Co., Ltd. (Beijing, China). The sequences of the siRNAs and miRNA mimics are listed in Supplementary Table S2. Lentiviral vectors containing the miR-29a-3p inhibitor and control sequence were constructed and generated by GeneCopoeia (Rockville, MD, USA). The selection of lentiviral-transfected cells using puromycin was performed by Guangzhou Weijia Technology Co., Ltd. (Guangzhou, China).
Immunohistochemistry and immunofluorescence staining
The 2-Step Plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution) (E-IR-R217-6 mL, Elabscience, Wuhan, China) and immunofluorescence staining kit (Cat: E-IR-R323-100T, Elabscience) were used for detecting target proteins by immunohistochemistry and immunofluorescence staining, respectively. First, the mouse liver cancer tissue was fixed with 4% cold paraformaldehyde for 15 min and then washed three times with PBS. This was followed by membrane perforation, blocking, primary antibody incubation, and secondary antibody incubation steps, and finally 0.5 µg/mL 4′,6-diamidino-2-phenylindole was added for nuclear staining. Immunohistochemistry was performed using semi-quantitative methods, and the percentage of positive cells and staining intensity were scored under a microscope.
Establishment of the lung metastasis model via tail vein injection
Twenty immunodeficiency mice were randomly divided into HCC and SD-HCC groups. Then, the HCC group was injected with MHCC97H-luc liver cancer cells and exosomes extracted from the plasma of mice with liver cancer only, whereas the SD-HCC group was injected with MHCC97H-luc liver cancer cells and exosomes extracted from the plasma of mice with liver cancer and SD symptoms. The exosomes were mixed and injected into the immunodeficiency mice via the tail vein. The number of cells was 106/100 µL per mouse, and the exosome dose was 10 µg∙mouse–1∙week–1. After 6 weeks, each mouse was administered an intraperitoneal injection of 150 mg/kg fluorescein sodium salt (Cat. No: 4090ES03, Yeasen). After subjecting the nude mice to gas anesthesia, tumor growth in each mouse was detected using the AniView100 multimodal animal in vivo imaging system. Thereafter, the nude mice were euthanized, and lung tissue was harvested for hematoxylin and eosin (HE) staining.
Hematoxylin and eosin staining
Tissue sections were dewaxed with xylene for 10 min and then rehydrated using an ethanol gradient (100%, 95%, 80%, 70%, and 60%) for 5 min at each concentration. Thereafter, the sections were stained with hematoxylin for 2 min, differentiated in ethanolic hydrochloric acid for 20 s, stained with eosin for 10 min, and then rinsed with tap water. Finally, the sections were sealed with neutral resin and examined under a microscope.
Flow cytometry
The spleens of the two groups of mice were removed, stored in RPMI-1640 medium, and immediately crushed within 4 h to prepare single-cell suspensions. The immune cells were subsequently detected through staining with antibodies against the following mouse antigens: anti-CD3, anti-CD45, anti-CD4, and anti-CD8. CD45 MicroBeads (Miltenyi, Bergisch Gladbach, Germany) were used to enrich the tumor-infiltrating immune cells, which were then analyzed using FlowJo v10.0 or FACS Diva v8.0 software, according to the manufacturer’s instructions. The difference in immune cell proportions between the two groups of mice was calculated.
Statistical analysis
GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used to perform all statistical analyses. Each experiment was conducted in at least triplicates, with all results presented as the mean ± standard deviation. The χ2 and Student’s t-test were used to assess the statistical significance of differences between the various mouse groups. Analysis of variance with Tukey’s multiple comparisons post-hoc test and Pearson’s correlation analysis were performed for statistical comparisons. Kaplan–Meier analysis and log-rank tests were applied for the survival analyses. A p-value of less than 0.05 was considered statistically significant.