Exosomal miR-29a-3p in the immune microenvironment of spleen deficiency promotes hepatocellular carcinoma lung metastasis by activating FAM167A-α1-integrin-NF-κB signaling axis

Exosomal miR-29a-3p in the immune microenvironment of spleen deficiency promotes hepatocellular carcinoma lung metastasis by activating FAM167A-α1-integrin-NF-κB signaling axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosomal miR-29a-3p in the immune microenvironment of spleen deficiency promotes hepatocellular carcinoma lung metastasis by activating FAM167A-α1-integrin-NF-κB signaling axis Jin Luo, Qiu-Xia Chen, Pan Li, Zhi-Ming Yang, He Yu, Bao-Qi Liu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2266609/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Hepatocellular carcinoma (HCC), a common type of cancer, has a strong metastatic ability and poor prognosis. The tumor microenvironment is the “soil” for the occurrence and development of tumors, with exosomes playing an important role in these processes. In traditional Chinese medicine(TCM), the tumor microenvironment corresponds to the internal environment of the syndrome known as spleen deficiency (SD). Numerous studies have shown that exosomes contain high levels of miRNAs, which have been shown to contribute to tumor immune regulation and metastasis. The aim of this study was to explore the mechanisms underlying the changes in the tumor microenvironment under the condition of spleen deficiency in order to find better treatments for cancer. Methods The effects of exosomal miR-29a-3p on lung metastasis from hepatocellular carcinoma (HCC) were evaluated using the scratch test, migration test, mouse SD model, HCC model, and tail-vein injection model of lung metastasis. The western blot assay, ELISA, flow cytometry, luciferase reporter gene analysis, qRT-PCR and immunofluorescence staining were among the methods used to study the molecular mechanism of lung metastasis promotion under the SD internal environment. Results Compared with the mice with HCC only, the mice with HCC and SD symptoms secreted more miR-29a- 3p-enriched exosomes, and their tumor tissue expressed significantly higher levels of α1-integrin and lower levels of FAM167A. These changed the immune microenvironment of mice (Decreased infiltration of T cells (CD3 + CD4 + and CD3 + CD8 + ), activated α1-integrin-NF-κB signaling pathway, and secreted more interleukin inflammatory factors(IL-1β, IL-6, and IL-8), which promoted the invasion and infiltration of HCC and its lung metastasis both in vivo and in vitro . In a series of patients with liver cancer, SD was found to have affected their overall survival and relapse-free survival. Conclusion Our study showed that under conditions of SD, the body releases more miRNA-containing exosomes, changes the immune microenvironment of the body, and ultimately promotes tumor metastasis and growth. These results highlight potential therapeutic targets and methods for the prevention of cancer metastasis, which may help to screen possible anticachexia TCMs and elucidate its mechanism in the future. Exosomes hepatocellular carcinoma miR-29a-3p Spleen deficiency internal environment Lung metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality. Metastasis of tumors is one of the most common causes of mortality in HCC. Its etiology and exact molecular mechanisms remain unclear, and therapeutic strategies against it remain unsatisfactory. In patients with HCC, lung metastasis is the most common form of distant disease invasion and progression as well as a leading cause of death[ 1 , 2 ].Lung metastasis of HCC is affected by many factors, the most important of which is the tumor microenvironment. The liver tumor microenvironment is generally divided into cellular and non-cellular components, including fibroblasts, hepatic stellate cells, immune cells, endothelial cells, mesenchymal stem cells, growth factors, cytokines, extracellular matrix, hormones, and viruses[ 3 ]. The “seed–soil” theory holds that the growth[ 4 – 6 ], invasion, and metastasis of tumors are closely related to the tumor microenvironment—“soil”[ 7 ], and the overall body environment eventually reaches a level that is conducive to the survival of tumor cells but not of normal cells. When the body environment changes during development, the “big soil” of the internal environment of the organism plays a decisive role in many physiological processes. According to the theory of traditional Chinese medicine (TCM), spleen deficiency (SD) is a core syndrome underlying the pathogenic mechanisms of HCC, with the SD internal environment corresponding to the tumor microenvironment, which as mentioned above provides the “soil” for the occurrence and development of the malignant disease.In clinical practice, we have found that many patients with tumors also have SD symptoms, such as pale complexion, excessive sweating, loss of appetite, abdominal distension, cold body, fatigue, cold hands and feet, and diarrhea[ 8 ]. In recent years, therapeutic strategies targeting components of the tumor microenvironment have become a popular choice for combating tumor metastasis[ 9 – 12 ]. Exosomes, which are extracellular vesicles with a diameter of approximately 30–200 nm, are secreted by almost all cells. These vesicles carry a variety of substances, including DNA, RNA, lipids, proteins, and metabolites. Studies have shown that exosomes are involved in various physiological and pathological processes in the human body and play a role in material and information transfer between cells. Exosomal microRNAs (miRNAs) are closely associated with various human diseases[ 13 – 16 ].Specific proteins (e.g., TSG101, CD81, HSP70, CD63, and CD9) that are highly enriched in exosomes are often used to identify the vesicles[ 17 , 18 ]. Numerous studies have shown that exosomes can mediate communication between cells and tumor-related proteins in the tumor microenvironment and promote tumor metastasis[ 17 – 20 ]. miRNAs are small non-coding RNAs that inhibit the translation of messenger RNAs (mRNAs). It had been shown that exosomes contain high levels of miRNAs, which have been demonstrated to contribute to tumor immune regulation, metastasis, and chemotherapy resistance [ 21 – 25 ]. However, the relationship between plasma-derived exosomal miRNAs extracted from HCC mice with SD and lung metastasis remains unknown. In this study, we divided mice into two groups: those with HCC with SD symptoms (designated the SD-HCC group) and those with HCC alone (designated the HCC group). A C57 mouse model of SD was also successfully established and its exosomes were extracted from the plasma. The exosomes extracted from the SD-HCC mice and HCC mice were injected into immunodeficient mice via tail vein injection. In the lung metastasis model, we found that exosomal miRNAs in the SD-HCC mice promoted lung metastasis by secreting more pro-inflammatory cytokines. Our RNA sequencing results suggested that the gene that was differentially expressed between SD-HCC and HCC mice was FAM167A (encoding family with sequence similarity 167 member A). Therefore, we speculated that mice with liver cancer and SD could secrete exosomal miR-29a-3p, directly targeting FAM167A and changing the immune microenvironment of the body, thereby activating the α1-integrin–nuclear factor-kappa B (NF-κB) signaling pathway and ultimately promoting cancer metastasis. These results highlight potential therapeutic targets for the prevention and treatment of cancer metastasis. Materials And Methods 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% CO 2 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 × 10 7 /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 mm 3 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 mm 3 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 10 6 /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 χ 2 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. Results Spleen deficiency changed the body weight, appetite, Mental state and other indicators in mice Two groups of normal male specific-pathogen-free C57BL/6 mice were used: one group was injected with reserpine (0.1 mL/10 g, once a day for 14 d), and the other group was injected with an equal volume of normal saline. One week later, the pre-prepared HCC tumor from a nude mouse was cut into 1 mm 3 pieces and orthotopically transplanted into the livers of the above two groups of mice, thereby generating mice with HCC only (HCC group) and those with both SD and HCC (SD-HCC group). We analyzed the body weight and daily feed amount of the C57BL/6 mice before, during, and after SD modeling and found that there was no statistical difference between the two groups before modeling ( p > 0.05) (Fig. 1 a, d). However, statistically significant differences were found between the two groups during and after the SD modeling process ( p < 0.001) (Fig. 1 b, c, e, f). At the same time, the SD scores between the two groups were significantly different before and after modeling ( p < 0.001) (Fig. 1 g). Spleen deficiency promoted the secretion of more exosomes in mouse plasma, thereby promoting cell migration, invasion, and wound healing in vitro There is an abundance of evidence that tumor metastasis is closely related to the tumor microenvironment[ 30 – 32 ].In this study, exosomes were extracted from mouse plasma using an exosome isolation kit, and their cup-shaped structure, size, and number were determined using electron microscopy and Nanosight particle tracking analysis (Fig. 2 a, b). We found that the C57 mice that had been injected with reserpine for 14 d secreted more exosomes after successful SD modeling than the normal C57 mice did (Fig. 2 b, c). The exosome markers were all detected on the western blot, where their protein contents were significantly higher than those of the control group (Fig. 2 d). Next, we added exosomes extracted from the plasma of mice with SD to the liver cancer cell lines MHCC97H and Hepa1-6 for co-culture and tested their migratory activity using the Transwell assay. Surprisingly, the liver cancer cells in the exosome-treated group had stronger migration and invasion abilities than those in the blank control group. Moreover, the cancer cells treated with exosomes from the mice with SD had stronger migration and invasion abilities than the cells treated with exosomes from a normal mice (Fig. 2 e, f, g, h). Additionally, we added the plasma-derived exosomes from the SD-HCC and HCC groups of mice to Hepa1-6 cells to conduct scratch experiments and found that the extracellular vesicles from mice with SD symptoms could better promote cell wound healing (Fig. 2 i, j). MiR-29a-3p released by exosomes from mice with spleen deficiency affected FAM167A–α1-integrin activation of the NF-κB pathway and the promotion of inflammatory cytokine release We sent the two groups of exosomes for sequencing and obtained 15 miRNAs with statistically significant differences between the groups, among which the up-regulation of miR-29a-3p was the most obvious (Fig. 3 a). Then, we performed qRT-PCR analysis of the two groups of exosomes to obtain the multiple differences in miR-29a-3p between them on the RNA level (Fig. 3 b). We performed RNA sequencing analysis of the liver cancer tissues from the HCC and SD-HCC groups of C57 mice. The conditions for screening differentially expressed gene sequences were a fold change value of greater than 1 and a p -value of less than 0.05. As a result, the genes coding for FAM167A, α1-integrin, and serglycin were found to differ significantly between the two mouse groups (Fig. 3 c). (Serglycin has been studied in depth in another article and is not discussed herein.) A heatmap of the sequences was generated using TBtools software[ 33 ].Using miRDB and TargetScan, the target gene of miR-29a-3p was predicted to be FAM167A (Fig. 3 d). Alignment of the miR-29a-3p sequence with the full-length FAM167A sequence confirmed that the FAM167A coding sequence was a potential target of this miRNA (Fig. 3 e). Subsequently, we cloned the binding sites of WT and MUT miR-29a-3p into the luciferase vector for transfection into HCCLM3 cells. The luciferase assay results showed that in HCCLM3 cells, the vector containing the WT binding site caused a significant decrease in luciferase activity, whereas cells transfected with the MUT binding site did not show this trend (Fig. 3 f). Additionally, we transfected cells with the mimics and siRNAs of miR-29a-3p and α1-integrin for 48 h and then extracted the cellular proteins for analysis. The western blot results showed that the miR-29a-3p mimic or knockdown of FAM167A promoted the expression of α1-integrin, and miR-29a-3p high expression or FAM167A inhibition also promoted NF-κB phosphorylation (Fig. 3 g). More importantly, knockdown of α1-integrin attenuated the effect of miR-29a-3p on NF-κB phosphorylation (Fig. 3 h). To further investigate the effect of miR-29a-3p, HCC cells were stably transfected with an miR-29-3p inhibitor. As a result, the impact of miR-29a-3p on the cells was abolished by its specific inhibitor (Fig. 3 i, j). Additionally, we found that the miR-29a-3p mimic also contributed to the motility of HCC cells (Fig. 3 k, l). It is well known that interleukin (IL)-1β, IL-6, and IL-8 are targets of NF-κB. We found that the mRNA levels of IL-1β, IL-6, and IL-8 in cells co-cultured with exosomes from the mice with SD were higher than the levels in cells co-cultured with exosomes from mice without SD and in the blank control group (Fig. 3 m). The ELISA results confirmed that the levels of the IL-type factors in the supernatant of the cells were increased to varying degrees (Fig. 3 n). Collectively, these results suggest that exosomal miR-29a-3p from SD-HCC mice mediates α1-integrin and FAM167A gene expression and activates the NF-κB pathway. Specifically, it promotes NF-κB phosphorylation and increases the levels of the IL-type factors, thereby promoting cancer cell migration and invasion. Spleen deficiency changed the tumor immune microenvironment and promoted tumor growth in vivo To further explore how the internal environment of the body changes under SD conditions, we generated Hepa1-6 tumors in the armpits of BALB/C mice and then transplanted the tumor tissue into the liver of spleen-deficient C57 mice. We found that the tumor size in mice with SD was significantly larger than that in the mice without the syndrome (Fig. 4 a, b). We then used qRT-PCR to detect the α1-integrin expression level in the liver cancer tissues of the two groups of mice and found that the level was significantly higher in the SD-HCC group (Fig. 4 c). The same trend was obtained in the western blot analysis of this protein (Fig. 4 D). Immunohistochemical analysis of tissue sections of the tumors also verified that the α1-integrin expression level was higher in the SD-HCC group(The positive rate of tumor cells was higher in the SD-HCC group) (Fig. 4 e, f). We used immunofluorescence staining to detect the difference in FAM167A levels between the two groups and found that it was lower in the SD-HCC group (Fig. 4 g), which was opposite to the α1-integrin expression level (Fig. 4 h). HE staining of the two groups of tissues showed that the SD-HCC group had a higher cell density, a disordered arrangement, more multinucleated cells, destruction of the hepatic cord and sinusoids, and a higher degree of liver stasis and cirrhosis (Fig. 4 i). Secreted exosomes in SD-HCC mice accelerated the lung metastasis process According to previous studies, exosomes and integrins are closely associated with cancer metastasis[ 34 – 37 ]. In our study, mouse exosomes and integrins showed apparent changes under the SD internal environment. Therefore, to further clarify whether these changes were related to tumor metastasis, we established a lung metastasis model in immunodeficient mice. To this end, the HCC group was injected with MHCC97H-luc liver cancer cells and plasma-derived exosomes from mice with liver cancer only, whereas the SD-HCC group was injected with MHCC97H-luc liver cancer cells and plasma-derived exosomes from mice with liver cancer and SD. The exosomes were respectively mixed with the hepatoma cells and injected into immunodeficiency mice via the tail vein. After 6 weeks, fluorescein sodium salt was administered intraperitoneally into each mouse for in vivo imaging of the animals (Fig. 5 a). The metastatic lung nodules in the SD-HCC group had enhanced fluorescein intensity and were larger in area. We euthanized the mice and stained the lung tissue with HE (Fig. 5 c). Surprisingly, the SD-HCC group not only had an increased amount of lung metastases (Fig. 5 e) but also a higher degree of tissue differentiation and a higher cell density. The increased disorderly arrangement occurred mostly with the multinucleated cells. Immunohistochemical analysis of the differences in FAM167A, α1-integrin, and p-NF-κB gene expression between the two groups of tissues revealed consistency of their levels with the previous in vitro results, in that the α1-integrin and p-NF-κB levels were increased and the FAM167A level was decreased in SD-HCC lung metastasis (Fig. 5 f). We selected cases from the Gene Expression Omnibus and The Cancer Genome Atlas databases and analyzed the OS and RFS rates in relation to the FAM167A and α1-integrin genes. The results, which were calculated from the time the disease was first diagnosed, also revealed that low FAM167A and high α1-integrin expression levels were associated with shorter OS and RFS in liver cancer. (Fig. 5 b, d). Spleen deficiency changed the immune state of mice and affected the overall survival and relapse-free survival of patients with liver cancer In order to further explore the specific mechanism of action, we did a further experiment and follow-up of patients with liver cancer. We did flow cytometry to analyze the changes in the relevant immune cells. Flow cytometric analysis of the level of spleen cell infiltration in the two groups of mice showed decreased infiltration of T cells (CD3 + CD4 + and CD3 + CD8 + ) in the SD-HCC group (Fig. 6 a, b, c).We had followed up on 70 liver cancer patients at the Department of Traditional Chinese Medicine of the First Affiliated Hospital of Sun Yat-sen University in the past 10 years.We found that the patients without symptoms of SD (LC group) had significantly longer OS (Fig. 6 d) and RFS rates (Fig. 6 e) than the patients with symptoms of SD (SD-LC group). Discussion The tumor microenvironment, a dynamic system coordinated by intercellular communication, is closely related to tumor progression and metastasis [ 38 – 43 ].Clinically, we have found that patients with SD symptoms are more likely to develop cancer, and cancer patients with SD symptoms have higher rates of metastasis and mortality and a shorter tumor-free survival time after surgery than those without such symptoms. The malignant disease progresses rapidly in patients with SD. Because there is a paucity of data on the effects of an SD internal environment on HCC metastasis, we constructed animal models of SD-HCC and of orthotopically transplanted liver cancer (HCC), extracted plasma exosomes from the two groups of mice, and injected them into immunodeficient mice through the tail vein to observe the effects of lung metastasis. We found that the SD-HCC mouse-derived exosomes had a significantly enhanced ability in promoting lung metastasis compared with the HCC mouse-derived exosomes. We performed cell experiments with the extracted exosomes and also found that the SD-HCC group could better promote the migration and invasion abilities of liver cancer cells. RNA sequencing analysis of the liver cancer tissues revealed apparent differences between the two mouse groups. Finally, miRDB and TargetScan predicted FAM167A as the target gene of miR-29a-3p. FAM167A is found in some leukemias and lymphomas and is typically associated with a poor prognosis [ 44 ]. Subsequently, we added the mimic and siRNA of miR-29a-3p and α1-integrin to cancer cell cultures and extracted cellular proteins 48 h after transfection. Western blot analysis showed that knockdown of the miR-29a-3p mimic or FAM167A promoted the expression of α1-integrin, and miR-29a-3p expression or FAM167A inhibition also promoted NF-κB phosphorylation. More importantly, knockdown of α1-integrin attenuated the effect of miR-29a-3p on NF-κB phosphorylation. Integrins are cell surface receptors composed of α and β subunits. Studies have shown that integrins accelerate cell migration by promoting the nuclear transport of NF-κB and activating the NF-κB signaling pathway, which is closely related to tumor metastasis [ 45 ]. To further investigate the effects of miR-29a-3p, HCC cells were stably transfected with an miR-29-3p inhibitor, which abolished the effects of the miRNA on cells, as expected. Additionally, we found that the mRNA levels of IL-1β, IL-6, and IL-8 in cells exposed to SD-HCC mouse-derived exosomes for 72 h were higher than the levels in cells exposed to HCC mouse-derived exosomes and cells in the blank control group. These findings reveal that exosomal miR-29a-3p in the SD-HCC environment regulates the gene expression of α1-integrin and FAM167A , activates the NF-κB pathway, promotes phosphorylation, and increases inflammatory IL-type factor levels, thereby ultimately promoting cancer cell migration and invasion and eventually leading to lung metastasis ( Fig. 6 f ) . First invoked by Paget, the seed and soil hypothesis suggests that the successful growth of metastatic cells depends on the interactions and properties of cancer cells (seeds) and their potential target organs (soil). The “seed–soil” theory holds that cancer cells (seeds) must find their appropriate soil to growth, invasion, and metastasis. The “soil”, the body’s internal environment, which is also called as the body with SD syndrome in TCM theory. It is the core pathogenic mechanism for the occurrence and development of malignant disease. In previous studies, there have been many discussions about exosomes promoting tumor growth and metastasis [ 46 – 48 ]. Metastatic organogenesis has been one of the greatest mysteries since Stephen Paget’s 1889 “seed–soil” hypothesis was introduced. Exosomal proteomics has revealed distinct integrin expression patterns, and numerous studies have demonstrated that exosomal integrins can be used to predict organ-specific cancer metastasis [ 49 , 50 ]. There are new exciting strategies in the fight against cancer, with changing the tumor microenvironment and implementing immunotherapy methods being breakthroughs in cancer treatment development [ 51 , 52 ]. Many studies have shown that the human body is in a state of immunodeficiency when SD symptoms are present [ 53 – 55 ]. Therefore, it is of great significance to improve the symptoms of SD and change the human body environment for improving immunity. We also found that exosomal miR-29a-3p in the SD internal environment promotes the metastasis of HCC via activation of the FAM167A–α1-integrin–NF-κB signaling pathway. It has been reported [ 44 ], that the FAM167A gene can activate the NF-κB pathway and induce BCR–ABL-independent tyrosine kinase inhibitor resistance. The FAM167A protein activates the non-canonical NF-κB pathway by binding to desmoglein-1 (DSG1), a cell adhesion protein. Under the state of SD, exosomes can activate the NF-κB signaling pathway in liver cancer cells and significantly promote the expression of phosphorylated NF-κB. Studies have shown that exosomes and integrins are closely related to tumor metastasis [ 56 – 58 ],Our study showed that the levels of exosomes and integrins will increase significantly under the state of SD, leading to activation of the NF-κB pathway and the promotion of NF-κB phosphorylation, which also changes the tumor immune microenvironment and reduces the proportion of CD4 + and CD8 + immune cells. These findings have important guiding significance for our clinical practice. For patients with TCM symptoms of SD, we can apply Chinese herbs for strengthening the spleen and replenishing the “qi” for symptomatic treatment, starting on the basis of the “seed–soil” theory to change the microenvironment that tumors rely on for survival. Our follow-up project will be to study the therapeutic mechanisms of related TCMs. Such as Spleen strengthening medicine ingredient atractylodes enol, quercetin, etc. One particularly exciting candidate is quercetin, an active ingredient in the Chinese medicinal plants Lobelia and Scutellaria barbata . It is hoped that more effective ingredients for both SD and cancer treatment can be found in natural medicines to overcome the global problem of malignant diseases in humans. Conclusions In conclusion, our study showed that under conditions of SD, the body releases more miRNA-containing exosomes, which mediate the gene expression of α1-integrin and FAM167A , activate the NF-κB pathway, and promote the release of more IL-type inflammatory factors. At the same time, SD changes the immune microenvironment of the body and ultimately promotes tumor metastasis and growth. TCM strategies for strengthening the spleen and its active components can start from the “seed–soil” theory; that is, to change the immune microenvironment or “soil” wherein tumors survive. The mechanism of such therapeutic strategy needs to be further studied and explored before it can be applied clinically. Abbreviations ATCC: American Type Culture Collection DMEM: Dulbecco’s modified Eagle’s medium ELISA, enzyme-linked immunosorbent assay HCC: Hepatocellular carcinoma HE: hematoxylin and eosin LC: liver cancer miRNA: microRNA mRNA: messenger RNA MUT: mutant OS: overall survival PBS: phosphate-buffered saline qRT-PCR: real-time reverse transcription-polymerase chain reaction RFS: recurrence/relapse-free survival RIPA: Radio Immunoprecipitation Assay SD: spleen deficiency SD-HCC: Hepatocellular carcinoma in spleen deficiency syndrome siRNAs: small interfering RNAs TCM: traditional Chinese medicine UTR: untranslated region WT: wild type Declarations Ethics approval and consent to participate All animals received humane care throughout the experiments. The study protocols were approved by the Committee on the Use of Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University (Number: Ethical review [2020] No.402). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the National Natural Science Foundation of China (No. 81873248, 82174173, 81903967, 82104962, 82104647, 81972785, and 81773162) and the Postdoctoral Science Foundation of China (No. 2021M700964). The study was also supported by the Project of Inheriting Famous TCM Masters of Guangdong Provincial Administration of Traditional Chinese Medicine (No. [2020]1), China (No. 2017A030313866 and 2022A1515012298 to B.H.) and the Open Funds of State Key Laboratory of Oncology in South China (No. HN2021-09). Author contributions JL, QC, and PL designed the experiments, analyzed the data, and prepared the manuscript. SZ, BH, HH, LY, and CQ conducted the experiments. 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15:45:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2266609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2266609/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29789497,"identity":"bb68956f-00b3-4215-a3f7-1642c84e60fd","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":581281,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences before and after orthotopic liver transplantation in mice with spleen deficiency. \u003cstrong\u003e(a) \u003c/strong\u003eStatistical chart of body weight differences between the two mouse groups before modeling. \u003cstrong\u003e(b) \u003c/strong\u003eStatistical chart of body weight difference between the two groups during modeling. \u003cstrong\u003e(c) \u003c/strong\u003eStatistical chart of body weight difference between the two groups after modeling. \u003cstrong\u003e(d) \u003c/strong\u003eDifferences in daily diet weight between the two groups before modeling. \u003cstrong\u003e(e) \u003c/strong\u003eDifferences in daily diet weight between the two groups during modeling. \u003cstrong\u003e(f) \u003c/strong\u003eDifferences in daily diet weight between the two groups after modeling. \u003cstrong\u003e(g) \u003c/strong\u003eThe scores of spleen deficiency were different between the two groups of mice. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/7c462b15dcbd35794cf2cda3.png"},{"id":29789498,"identity":"ef59c2c5-5086-4e1d-ba1d-3e089d18b724","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4414338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e identification of and differences between exosomes from two mouse sources. \u003cstrong\u003e(a) \u003c/strong\u003eElectron microscopy images of exosomes from the plasma of different C57 mice (HCC and SD-HCC). Scale bars = 200 nm and 500 nm. \u003cstrong\u003e(b) \u003c/strong\u003eNanoparticle tracking assay detection of exosomes in plasma from HCC and SD-HCC mice. \u003cstrong\u003e(c) \u003c/strong\u003eStatistical chart of the exosome concentration in the two groups. \u003cstrong\u003e(d) \u003c/strong\u003eWestern blot analysis of surface markers (ALIX, HSP70, CD81) of exosomes from two groups of mice (samples are a mixture of exosomes from 4 mice in each group). \u003cstrong\u003e(e) \u003c/strong\u003eEffects of exosomes from different mouse sources and of the blank (BLK) control on the migration ability of HCC cells. \u003cstrong\u003e(f) \u003c/strong\u003eStatistical chart of the cell migration abilities for the three groups. \u003cstrong\u003e(g) \u003c/strong\u003eEffects of exosomes from different mouse sources and of the blank (BLK) control on the invasion ability of HCC cells. \u003cstrong\u003e(h) \u003c/strong\u003eStatistical chart of the cell invasion abilities for the three groups. \u003cstrong\u003e(i) \u003c/strong\u003eResults of the cell scratch experiment for the Control, HCC exosome, and SD-HCC exosome groups. \u003cstrong\u003e(j) \u003c/strong\u003eStatistical chart of the cell scratch experimental results. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. HCC, mice with hepatocellular carcinoma only; SD-HCC, mice with both spleen deficiency and hepatocellular carcinoma.\u003c/p\u003e","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/1a8aaa3dbcd3536c0f6381b6.png"},{"id":29790449,"identity":"c797ea1e-5527-4f26-a88e-e6d73a42f939","added_by":"auto","created_at":"2022-12-01 19:33:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3681292,"visible":true,"origin":"","legend":"\u003cp\u003eExosomal miR-29a-3p regulated FAM167A–α1-integrin to activate the NF-κB pathway and promote inflammatory factor release. \u003cstrong\u003e(a) \u003c/strong\u003eThe two groups of exosomes were sequenced to identify differentially expressed miRNAs. \u003cstrong\u003e(b) \u003c/strong\u003eqRT-PCR analysis of miRNA29a-3p expression differences between the two groups. \u003cstrong\u003e(c) \u003c/strong\u003eThe differentially expressed gene \u003cem\u003eFAM167A\u003c/em\u003ewas obtained by RNA sequencing of hepatocellular carcinoma tissues from SD-HCC and HCC C57 mice. \u003cstrong\u003e(d) \u003c/strong\u003eTarget gene prediction for miR-29a-3p using two bioinformatics tools. \u003cstrong\u003e(e) \u003c/strong\u003eWild-type and mutated type of binding sites between miR-29a-3p and FAM167A. \u003cstrong\u003e(f) \u003c/strong\u003eRelative luciferase activity in HCCLM3 cells subjected to the indicated treatments. \u003cstrong\u003e(g) \u003c/strong\u003eImmunoblots of the indicated proteins in HCCLM3 cells subjected to the indicated treatments. \u003cstrong\u003e(h) \u003c/strong\u003eEffect of\u003cstrong\u003e \u003c/strong\u003emiR-29a-3p on the expression of the indicated proteins in HCCLM3 cells with or without the suppression of α1-integrin expression. \u003cstrong\u003e(i, j) \u003c/strong\u003eComparison of the migration abilities of HepG2 and HCCLM3 cells treated with exosomes derived from SD-HCC C57 mice stably expressing the miR-29a-3p inhibitor or negative control. Migrated cells were counted, and representative images are shown. Scale bar = 150 μm.\u003cstrong\u003e (k, l) \u003c/strong\u003eEffect of the miR-29a-3p mimic on the migration abilities of Hepa1-6 and HepG2 cells. Scale bar = 150 μm. \u003cstrong\u003e(m) \u003c/strong\u003eqRT-PCR analysis of the expression of interleukin genes in MHCC97H and HCCLM3 cells treated with exosomes extracted from different C57 mouse plasmas or the blank control. \u003cstrong\u003e(n) \u003c/strong\u003eELISA analysis of the differences in interleukin levels after co-incubation of hepatoma cells with different exosomes. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. HCC, mice with hepatocellular carcinoma only; SD-HCC, mice with both spleen deficiency and hepatocellular carcinoma; ELISA, enzyme-linked immunosorbent assay; qRT-PCR, real-time reverse transcription-polymerase chain reaction.\u003c/p\u003e","description":"","filename":"Figure31.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/4d8c161974219fac60efa90d.png"},{"id":29789504,"identity":"91e02334-a3a9-4a17-b42a-60466790f005","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3606316,"visible":true,"origin":"","legend":"\u003cp\u003eSpleen deficiency changed the tumor immune microenvironment and promoted tumor growth in vivo. \u003cstrong\u003e(a) \u003c/strong\u003eDifferences in tumor size between the two groups of mice. \u003cstrong\u003e(b) \u003c/strong\u003eStatistical chart of the difference in tumor sizes between the two groups of mice.\u003cstrong\u003e (c) \u003c/strong\u003eqRT-PCR analysis of the changes in α1-integrin expression level in the liver tissue of the two groups of mice and normal mice. \u003cstrong\u003e(d) \u003c/strong\u003eWestern blot of the changes in protein levels in hepatocellular carcinoma tissues from the two groups of mice. \u003cstrong\u003e(e) \u003c/strong\u003eImmunohistochemical detection of the difference in α1-integrin levels between tumor tissues from the two groups of mice. \u003cstrong\u003e(f) \u003c/strong\u003eStatistical chart of the immunohistochemical results. \u003cstrong\u003e(g) \u003c/strong\u003eImmunofluorescence staining to detect the difference in \u003cem\u003eFAM167A\u003c/em\u003e gene expression in liver cancer tissues between the two groups of mice. Scale bar = 100 μm. \u003cstrong\u003e(h) \u003c/strong\u003eImmunofluorescence staining to detect the difference in α1-integrin gene expression in liver cancer tissues between the two groups of mice. Scale bar = 100 μm. \u003cstrong\u003e(i) \u003c/strong\u003eHE staining of the tumors from the two mouse groups. Data are presented as the means ± SD. Student’s \u003cem\u003et\u003c/em\u003e-test was used to analyze the data. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/f68c3db48774596b963530ad.png"},{"id":29789991,"identity":"445a9c95-5497-4fba-a990-f5cacd3b7502","added_by":"auto","created_at":"2022-12-01 19:25:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3851022,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of two groups of exosomes on lung metastasis in immunodeficient mice. \u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of lung metastases in immunodeficient mice treated with exosomes derived from two different sets of mouse plasma, as determined by luciferase-based bioluminescence imaging. \u003cstrong\u003e(b) \u003c/strong\u003eKaplan–Meier curves of overall survival (OS) for patients with low versus high expression of α1-integrin and FAM167A in the GEO and TCGA databases. \u003cstrong\u003e(c) \u003c/strong\u003eHE staining of metastatic lung nodules in two groups of mice. \u003cstrong\u003e(d) \u003c/strong\u003eKaplan–Meier curves of recurrence/relapse-free survival (RFS) for patients with low versus high expression of α1-integrin and FAM167A in the GEO and TCGA databases. \u003cstrong\u003e(e) \u003c/strong\u003eStatistical chart of the number of metastatic lung nodules. \u003cstrong\u003e(f) \u003c/strong\u003eImmunohistochemical analysis of the differences in metastatic lung nodules between the two groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/a8dde683bfc387afcd044375.png"},{"id":29789502,"identity":"4ed2b92b-2019-487e-b2e0-7d53dd7cf9a2","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":928536,"visible":true,"origin":"","legend":"\u003cp\u003eSpleen deficiency changes the immune status of mice and affects the overall survival of liver cancer patients. \u003cstrong\u003e(a) \u003c/strong\u003eFlow cytometry gating strategy for detecting tumor-infiltrating leukocytes. \u003cstrong\u003e(b) \u003c/strong\u003eFlow cytometric quantification showing the decreased infiltration of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003e(c) \u003c/strong\u003eStatistical chart of the percentages of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the splenic lymphocytes (%). \u003cstrong\u003e(d) \u003c/strong\u003eDifferences in overall survival rate between liver cancer patients with spleen deficiency symptoms (SD-LC) and those without spleen deficiency symptoms (LC). \u003cstrong\u003e(e) \u003c/strong\u003eDifference in relapse-free survival rates between patients in the SD-HCC group and those in the HCC group. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. \u003cstrong\u003e(f)\u003c/strong\u003e Schematic illustration of the pathway through which exosomal miR-29a-3p promotes lung metastasis from liver cancer under spleen deficiency conditions.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/40b2ca8f8ceac119f92b6739.png"},{"id":30149676,"identity":"755b6506-c0d2-4474-910f-dc59803b1da8","added_by":"auto","created_at":"2022-12-10 10:00:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3995799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/3777069e-f3db-43d8-b10a-6046a0b137c5.pdf"},{"id":29789496,"identity":"34e69064-6b3a-4819-94a5-69cf01f40186","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12159,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. Primers for the qRT-PCR assay.\u003c/p\u003e","description":"","filename":"TableS1primerdesign.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/32283cd42d8ac8b1313ed0c8.xlsx"},{"id":29789989,"identity":"284d89bd-f174-4222-b659-c2ec86887df2","added_by":"auto","created_at":"2022-12-01 19:25:20","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11922,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2. siRNA primer sequences for plasmid construction.\u003c/p\u003e","description":"","filename":"TableS2.siRNAprimersequencesforplasmidconstruction.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/4984b180b8816b311764ef21.xlsx"},{"id":29789501,"identity":"33c69ae2-6eca-4e25-acf3-b6e89a3e5f36","added_by":"auto","created_at":"2022-12-01 19:17:20","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2154060,"visible":true,"origin":"","legend":"","description":"","filename":"WBOriginalmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2266609/v1/5a055da841e3097a4d63164e.pdf"}],"financialInterests":"","formattedTitle":"Exosomal miR-29a-3p in the immune microenvironment of spleen deficiency promotes hepatocellular carcinoma lung metastasis by activating FAM167A-α1-integrin-NF-κB signaling axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is the second leading cause of cancer mortality. Metastasis of tumors is one of the most common causes of mortality in HCC. Its etiology and exact molecular mechanisms remain unclear, and therapeutic strategies against it remain unsatisfactory. In patients with HCC, lung metastasis is the most common form of distant disease invasion and progression as well as a leading cause of death[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].Lung metastasis of HCC is affected by many factors, the most important of which is the tumor microenvironment. The liver tumor microenvironment is generally divided into cellular and non-cellular components, including fibroblasts, hepatic stellate cells, immune cells, endothelial cells, mesenchymal stem cells, growth factors, cytokines, extracellular matrix, hormones, and viruses[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u0026ldquo;seed\u0026ndash;soil\u0026rdquo; theory holds that the growth[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], invasion, and metastasis of tumors are closely related to the tumor microenvironment\u0026mdash;\u0026ldquo;soil\u0026rdquo;[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the overall body environment eventually reaches a level that is conducive to the survival of tumor cells but not of normal cells. When the body environment changes during development, the \u0026ldquo;big soil\u0026rdquo; of the internal environment of the organism plays a decisive role in many physiological processes. According to the theory of traditional Chinese medicine (TCM), spleen deficiency (SD) is a core syndrome underlying the pathogenic mechanisms of HCC, with the SD internal environment corresponding to the tumor microenvironment, which as mentioned above provides the \u0026ldquo;soil\u0026rdquo; for the occurrence and development of the malignant disease.In clinical practice, we have found that many patients with tumors also have SD symptoms, such as pale complexion, excessive sweating, loss of appetite, abdominal distension, cold body, fatigue, cold hands and feet, and diarrhea[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, therapeutic strategies targeting components of the tumor microenvironment have become a popular choice for combating tumor metastasis[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExosomes, which are extracellular vesicles with a diameter of approximately 30\u0026ndash;200 nm, are secreted by almost all cells. These vesicles carry a variety of substances, including DNA, RNA, lipids, proteins, and metabolites. Studies have shown that exosomes are involved in various physiological and pathological processes in the human body and play a role in material and information transfer between cells. Exosomal microRNAs (miRNAs) are closely associated with various human diseases[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].Specific proteins (e.g., TSG101, CD81, HSP70, CD63, and CD9) that are highly enriched in exosomes are often used to identify the vesicles[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Numerous studies have shown that exosomes can mediate communication between cells and tumor-related proteins in the tumor microenvironment and promote tumor metastasis[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003emiRNAs are small non-coding RNAs that inhibit the translation of messenger RNAs (mRNAs). It had been shown that exosomes contain high levels of miRNAs, which have been demonstrated to contribute to tumor immune regulation, metastasis, and chemotherapy resistance [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the relationship between plasma-derived exosomal miRNAs extracted from HCC mice with SD and lung metastasis remains unknown.\u003c/p\u003e \u003cp\u003eIn this study, we divided mice into two groups: those with HCC with SD symptoms (designated the SD-HCC group) and those with HCC alone (designated the HCC group). A C57 mouse model of SD was also successfully established and its exosomes were extracted from the plasma. The exosomes extracted from the SD-HCC mice and HCC mice were injected into immunodeficient mice via tail vein injection. In the lung metastasis model, we found that exosomal miRNAs in the SD-HCC mice promoted lung metastasis by secreting more pro-inflammatory cytokines. Our RNA sequencing results suggested that the gene that was differentially expressed between SD-HCC and HCC mice was \u003cem\u003eFAM167A\u003c/em\u003e (encoding family with sequence similarity 167 member A). Therefore, we speculated that mice with liver cancer and SD could secrete exosomal miR-29a-3p, directly targeting \u003cem\u003eFAM167A\u003c/em\u003e and changing the immune microenvironment of the body, thereby activating the α1-integrin\u0026ndash;nuclear factor-kappa B (NF-κB) signaling pathway and ultimately promoting cancer metastasis. These results highlight potential therapeutic targets for the prevention and treatment of cancer metastasis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003eEstablishment of the mouse model of spleen deficiency and classification criteria for the spleen deficiency index\u003c/h2\u003e\n\u003cp\u003eAll 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\u0026deg;C as a stock solution. Each mouse was injected subcutaneously with approximately 100 \u0026micro;L of the solution once a day (i.e., 0.1 mg∙kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e∙d\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) for 14 consecutive days to establish the SD model. The control group was injected subcutaneously with 100 \u0026micro;L 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[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] 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\u0026thinsp;=\u0026thinsp;no SD symptoms; 8\u0026ndash;14\u0026thinsp;=\u0026thinsp;mild SD; 15\u0026ndash;21\u0026thinsp;=\u0026thinsp;typical SD; and 22\u0026ndash;28\u0026thinsp;=\u0026thinsp;severe SD (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClassification criteria for determining the spleen deficiency index\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIndex/Score\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eGrading standards\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBody odor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOdor-free\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMild odor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMedium odor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSevere odor\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMental state\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStable\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIrritable\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFatigued\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSomnolent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFever \u0026amp; chills\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCowered\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChills\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eArched back \u0026amp; trembling\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRespiration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePanting\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTachypnea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFaint\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFur\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlossy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMatted\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFluffy \u0026amp; erect\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBrown \u0026amp; erect\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStool\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWet\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWet \u0026amp; rotten\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMucous texture\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAppetite\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eReduced to 50%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eReduced to 25%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNone at all\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: A total score of 7 represents no spleen deficiency, 8\u0026ndash;14 represents mild spleen deficiency, 15\u0026ndash;21 represents typical spleen deficiency, and 22\u0026ndash;28 represents severe spleen deficiency.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eCell lines and culture\u003c/h2\u003e\n\u003cp\u003eHuman-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\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, Thermo Fisher Scientific, St. Peters, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eExtraction of exosomes\u003c/h2\u003e\n\u003cp\u003eThe 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 \u0026micro;L of reagent C that had been completely thawed on ice was added to 200 \u0026micro;L of mouse plasma, with up and down pipetting and vortex mixing performed until a homogeneous mixture was obtained. Then, 50 \u0026micro;L of extraction reagent A and 50 \u0026micro;L of reagent B were added to the suspension, and exosomes were extracted according to the steps outlined in the manufacturer\u0026rsquo;s instruction manual. Finally, the obtained pellet was resuspended in 50\u0026ndash;120 \u0026micro;L of sterilized 1\u0026times; phosphate-buffered saline (PBS), following which the exosome-containing suspension was aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further use and analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e\n\u003cp\u003eThe 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\u0026deg;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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eLuciferase reporter gene assay\u003c/h2\u003e\n\u003cp\u003eWe predicted the targeting relationship between miR-29a-3p and the \u003cem\u003eFAM167A\u003c/em\u003e gene using the bioinformatics databases TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_80/\u003c/span\u003e\u003c/span\u003e) and miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirdb.org/\u003c/span\u003e\u003c/span\u003e). The 3'-untranslated region (3'-UTR) of the \u003cem\u003eFAM167A\u003c/em\u003e promoter region constituted the miR-29a-3p-binding site for construction of the wild-type (WT) plasmid of \u003cem\u003eFAM167A\u003c/em\u003e 3'-UTR (FAM167A-WT).\u003c/p\u003e\n\u003cp\u003eBased 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 \u003cem\u003eFAM167A\u003c/em\u003e 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\u0026trade; 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).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eELISA detection\u003c/h2\u003e\n\u003cp\u003eWe 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\u0026ndash;8\u0026deg;C for approximately 20 min (2,000\u0026ndash;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\u0026rsquo;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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted from the cells using the MolPure\u0026reg; Cell/Tissue Total RNA Kit (Cat: 19221ES50, Yeasen, Shanghai, China) and reverse transcribed using SuperScript\u0026trade; 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eWound-healing and Transwell assay\u003c/h2\u003e\n\u003cp\u003eThe 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\u0026ndash;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\u0026ndash;400,000 were seeded into 2 mL of serum-free DMEM in 6-well plates and cultured at 37\u0026deg;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 \u0026micro;L 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.\u003c/p\u003e\n\u003cp\u003eFor the cell migration assay, 800 \u0026micro;L of DMEM containing 10% fetal bovine serum was added to the lower chambers of a 24-well Transwell plate, and 200 \u0026micro;L 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\u0026ndash;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 \u0026micro;L of methanol was added to the lower chamber and 200 \u0026micro;L 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 \u0026micro;L of crystal violet was added to the lower chamber and 200 \u0026micro;L 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eNude mouse tumor xenograft models\u003c/h2\u003e\n\u003cp\u003eTo generate tumor xenograft models, 0.1 mL of a precultured Hepa1-6 cell suspension (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/mL) was injected subcutaneously into the armpit of BALB/C nude mice. When the subcutaneous tumors were approximately 1 cm in diameter (after ~\u0026thinsp;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 mm\u003csup\u003e3\u003c/sup\u003e 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 mm\u003csup\u003e3\u003c/sup\u003e 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\u0026deg; 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\n\u003cp\u003eThe 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, \u0026ldquo;gel preparation\u0026ndash;electrophoresis\u0026ndash;transfer membrane\u0026ndash;blocking and incubation antibody\u0026ndash;incubate secondary antibody\u0026ndash;ECL luminescence,\u0026rdquo; 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),\u0026alpha;1-integrin Ab (1:1000), Alix Ab (1:1000), HSP70 Ab (1:1000), CD81 Ab (1:1000), NF-\u0026kappa;B Ab (1:1000), phosphorylated-NF-\u0026kappa;B Ab (1:1000), and GAPDH mAb (1:1000). Secondary antibodies were anti-rabbit (1:2000) and anti-mouse (1:2000).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eSurvival analysis\u003c/h2\u003e\n\u003cp\u003eThe Kaplan\u0026ndash;Meier plotter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kmplot.com/\u003c/span\u003e\u003c/span\u003e), which is an interactive online tool for investigating survival correlations, can assess the effects of 54,000 genes on survival in 21 cancer types [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. To determine the clinical significance of the \u0026alpha;1-integrin and \u003cem\u003eFAM167A\u003c/em\u003e 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\u0026ndash;Meier plots and the log-rank \u003cem\u003ep\u003c/em\u003e-value (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA interference and plasmids\u003c/h2\u003e\n\u003cp\u003eSmall interfering RNAs (siRNAs) (viz., siNC and siRNAs targeting \u003cem\u003eFAM167A\u003c/em\u003e or \u0026alpha;1-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).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunohistochemistry and immunofluorescence staining\u003c/h2\u003e\n\u003cp\u003eThe 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 \u0026micro;g/mL 4\u0026prime;,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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eEstablishment of the lung metastasis model via tail vein injection\u003c/h2\u003e\n\u003cp\u003eTwenty 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 10\u003csup\u003e6\u003c/sup\u003e/100 \u0026micro;L per mouse, and the exosome dose was 10 \u0026micro;g∙mouse\u003csup\u003e\u0026ndash;1\u003c/sup\u003e∙week\u003csup\u003e\u0026ndash;1\u003c/sup\u003e. 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 \u003cem\u003ein vivo\u003c/em\u003e imaging system. Thereafter, the nude mice were euthanized, and lung tissue was harvested for hematoxylin and eosin (HE) staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eHematoxylin and eosin staining\u003c/h2\u003e\n\u003cp\u003eTissue 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eFlow cytometry\u003c/h2\u003e\n\u003cp\u003eThe 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\u0026rsquo;s instructions. The difference in immune cell proportions between the two groups of mice was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eGraphPad 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\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The \u0026chi;\u003csup\u003e2\u003c/sup\u003e and Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test were used to assess the statistical significance of differences between the various mouse groups. Analysis of variance with Tukey\u0026rsquo;s multiple comparisons post-hoc test and Pearson\u0026rsquo;s correlation analysis were performed for statistical comparisons. Kaplan\u0026ndash;Meier analysis and log-rank tests were applied for the survival analyses. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eSpleen deficiency changed the body weight, appetite, Mental state and other indicators in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo groups of normal male specific-pathogen-free C57BL/6 mice were used: one group was injected with reserpine (0.1 mL/10 g, once a day for 14 d), and the other group was injected with an equal volume of normal saline. One week later, the pre-prepared HCC tumor from a nude mouse was cut into 1 mm\u003csup\u003e3\u003c/sup\u003e pieces and orthotopically transplanted into the livers of the above two groups of mice, thereby generating mice with HCC only (HCC group) and those with both SD and HCC (SD-HCC group).\u003c/p\u003e\n\u003cp\u003eWe analyzed the body weight and daily feed amount of the C57BL/6 mice before, during, and after SD modeling and found that there was no statistical difference between the two groups before modeling (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, d). However, statistically significant differences were found between the two groups during and after the SD modeling process (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, c, e, f). At the same time, the SD scores between the two groups were significantly different before and after modeling (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpleen deficiency promoted the secretion of more exosomes in mouse plasma, thereby promoting cell migration, invasion, and wound healing\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003ein vitro\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThere is an abundance of evidence that tumor metastasis is closely related to the tumor microenvironment[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].In this study, exosomes were extracted from mouse plasma using an exosome isolation kit, and their cup-shaped structure, size, and number were determined using electron microscopy and Nanosight particle tracking analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). We found that the C57 mice that had been injected with reserpine for 14 d secreted more exosomes after successful SD modeling than the normal C57 mice did (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). The exosome markers were all detected on the western blot, where their protein contents were significantly higher than those of the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Next, we added exosomes extracted from the plasma of mice with SD to the liver cancer cell lines MHCC97H and Hepa1-6 for co-culture and tested their migratory activity using the Transwell assay. Surprisingly, the liver cancer cells in the exosome-treated group had stronger migration and invasion abilities than those in the blank control group. Moreover, the cancer cells treated with exosomes from the mice with SD had stronger migration and invasion abilities than the cells treated with exosomes from a normal mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, f, g, h). Additionally, we added the plasma-derived exosomes from the SD-HCC and HCC groups of mice to Hepa1-6 cells to conduct scratch experiments and found that the extracellular vesicles from mice with SD symptoms could better promote cell wound healing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ei, j).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-29a-3p released by exosomes from mice with spleen deficiency affected FAM167A\u0026ndash;\u0026alpha;1-integrin activation of the NF-\u0026kappa;B pathway and the promotion of inflammatory cytokine release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sent the two groups of exosomes for sequencing and obtained 15 miRNAs with statistically significant differences between the groups, among which the up-regulation of miR-29a-3p was the most obvious (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Then, we performed qRT-PCR analysis of the two groups of exosomes to obtain the multiple differences in miR-29a-3p between them on the RNA level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). We performed RNA sequencing analysis of the liver cancer tissues from the HCC and SD-HCC groups of C57 mice. The conditions for screening differentially expressed gene sequences were a fold change value of greater than 1 and a \u003cem\u003ep\u003c/em\u003e-value of less than 0.05. As a result, the genes coding for FAM167A, \u0026alpha;1-integrin, and serglycin were found to differ significantly between the two mouse groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). (Serglycin has been studied in depth in another article and is not discussed herein.) A heatmap of the sequences was generated using TBtools software[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].Using miRDB and TargetScan, the target gene of miR-29a-3p was predicted to be \u003cem\u003eFAM167A\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). Alignment of the miR-29a-3p sequence with the full-length \u003cem\u003eFAM167A\u003c/em\u003e sequence confirmed that the \u003cem\u003eFAM167A\u003c/em\u003e coding sequence was a potential target of this miRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). Subsequently, we cloned the binding sites of WT and MUT miR-29a-3p into the luciferase vector for transfection into HCCLM3 cells. The luciferase assay results showed that in HCCLM3 cells, the vector containing the WT binding site caused a significant decrease in luciferase activity, whereas cells transfected with the MUT binding site did not show this trend (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef). Additionally, we transfected cells with the mimics and siRNAs of miR-29a-3p and \u0026alpha;1-integrin for 48 h and then extracted the cellular proteins for analysis. The western blot results showed that the miR-29a-3p mimic or knockdown of \u003cem\u003eFAM167A\u003c/em\u003e promoted the expression of \u0026alpha;1-integrin, and miR-29a-3p high expression or \u003cem\u003eFAM167A\u003c/em\u003e inhibition also promoted NF-\u0026kappa;B phosphorylation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg). More importantly, knockdown of \u0026alpha;1-integrin attenuated the effect of miR-29a-3p on NF-\u0026kappa;B phosphorylation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh). To further investigate the effect of miR-29a-3p, HCC cells were stably transfected with an miR-29-3p inhibitor. As a result, the impact of miR-29a-3p on the cells was abolished by its specific inhibitor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ei, j). Additionally, we found that the miR-29a-3p mimic also contributed to the motility of HCC cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ek, l). It is well known that interleukin (IL)-1\u0026beta;, IL-6, and IL-8 are targets of NF-\u0026kappa;B. We found that the mRNA levels of IL-1\u0026beta;, IL-6, and IL-8 in cells co-cultured with exosomes from the mice with SD were higher than the levels in cells co-cultured with exosomes from mice without SD and in the blank control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003em). The ELISA results confirmed that the levels of the IL-type factors in the supernatant of the cells were increased to varying degrees (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003en). Collectively, these results suggest that exosomal miR-29a-3p from SD-HCC mice mediates \u0026alpha;1-integrin and \u003cem\u003eFAM167A\u003c/em\u003e gene expression and activates the NF-\u0026kappa;B pathway. Specifically, it promotes NF-\u0026kappa;B phosphorylation and increases the levels of the IL-type factors, thereby promoting cancer cell migration and invasion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpleen deficiency changed the tumor immune microenvironment and promoted tumor growth\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore how the internal environment of the body changes under SD conditions, we generated Hepa1-6 tumors in the armpits of BALB/C mice and then transplanted the tumor tissue into the liver of spleen-deficient C57 mice. We found that the tumor size in mice with SD was significantly larger than that in the mice without the syndrome (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). We then used qRT-PCR to detect the \u0026alpha;1-integrin expression level in the liver cancer tissues of the two groups of mice and found that the level was significantly higher in the SD-HCC group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). The same trend was obtained in the western blot analysis of this protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Immunohistochemical analysis of tissue sections of the tumors also verified that the \u0026alpha;1-integrin expression level was higher in the SD-HCC group(The positive rate of tumor cells was higher in the SD-HCC group) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). We used immunofluorescence staining to detect the difference in FAM167A levels between the two groups and found that it was lower in the SD-HCC group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg), which was opposite to the \u0026alpha;1-integrin expression level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh). HE staining of the two groups of tissues showed that the SD-HCC group had a higher cell density, a disordered arrangement, more multinucleated cells, destruction of the hepatic cord and sinusoids, and a higher degree of liver stasis and cirrhosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eSecreted exosomes in SD-HCC mice accelerated the lung metastasis process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to previous studies, exosomes and integrins are closely associated with cancer metastasis[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, mouse exosomes and integrins showed apparent changes under the SD internal environment. Therefore, to further clarify whether these changes were related to tumor metastasis, we established a lung metastasis model in immunodeficient mice. To this end, the HCC group was injected with MHCC97H-luc liver cancer cells and plasma-derived exosomes from mice with liver cancer only, whereas the SD-HCC group was injected with MHCC97H-luc liver cancer cells and plasma-derived exosomes from mice with liver cancer and SD. The exosomes were respectively mixed with the hepatoma cells and injected into immunodeficiency mice via the tail vein. After 6 weeks, fluorescein sodium salt was administered intraperitoneally into each mouse for in vivo imaging of the animals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The metastatic lung nodules in the SD-HCC group had enhanced fluorescein intensity and were larger in area. We euthanized the mice and stained the lung tissue with HE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Surprisingly, the SD-HCC group not only had an increased amount of lung metastases (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee) but also a higher degree of tissue differentiation and a higher cell density. The increased disorderly arrangement occurred mostly with the multinucleated cells. Immunohistochemical analysis of the differences in FAM167A, \u0026alpha;1-integrin, and p-NF-\u0026kappa;B gene expression between the two groups of tissues revealed consistency of their levels with the previous \u003cem\u003ein vitro\u003c/em\u003e results, in that the \u0026alpha;1-integrin and p-NF-\u0026kappa;B levels were increased and the FAM167A level was decreased in SD-HCC lung metastasis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). We selected cases from the Gene Expression Omnibus and The Cancer Genome Atlas databases and analyzed the OS and RFS rates in relation to the \u003cem\u003eFAM167A\u003c/em\u003e and \u0026alpha;1-integrin genes. The results, which were calculated from the time the disease was first diagnosed, also revealed that low FAM167A and high \u0026alpha;1-integrin expression levels were associated with shorter OS and RFS in liver cancer. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpleen deficiency changed the immune state of mice and affected the overall survival and relapse-free survival of patients with liver cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further explore the specific mechanism of action, we did a further experiment and follow-up of patients with liver cancer. We did flow cytometry to analyze the changes in the relevant immune cells. Flow cytometric analysis of the level of spleen cell infiltration in the two groups of mice showed decreased infiltration of T cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e) in the SD-HCC group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, c).We had followed up on 70 liver cancer patients at the Department of Traditional Chinese Medicine of the First Affiliated Hospital of Sun Yat-sen University in the past 10 years.We found that the patients without symptoms of SD (LC group) had significantly longer OS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed) and RFS rates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee) than the patients with symptoms of SD (SD-LC group).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe tumor microenvironment, a dynamic system coordinated by intercellular communication, is closely related to tumor progression and metastasis [\u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].Clinically, we have found that patients with SD symptoms are more likely to develop cancer, and cancer patients with SD symptoms have higher rates of metastasis and mortality and a shorter tumor-free survival time after surgery than those without such symptoms. The malignant disease progresses rapidly in patients with SD.\u003c/p\u003e \u003cp\u003eBecause there is a paucity of data on the effects of an SD internal environment on HCC metastasis, we constructed animal models of SD-HCC and of orthotopically transplanted liver cancer (HCC), extracted plasma exosomes from the two groups of mice, and injected them into immunodeficient mice through the tail vein to observe the effects of lung metastasis. We found that the SD-HCC mouse-derived exosomes had a significantly enhanced ability in promoting lung metastasis compared with the HCC mouse-derived exosomes. We performed cell experiments with the extracted exosomes and also found that the SD-HCC group could better promote the migration and invasion abilities of liver cancer cells. RNA sequencing analysis of the liver cancer tissues revealed apparent differences between the two mouse groups. Finally, miRDB and TargetScan predicted \u003cem\u003eFAM167A\u003c/em\u003e as the target gene of miR-29a-3p. FAM167A is found in some leukemias and lymphomas and is typically associated with a poor prognosis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSubsequently, we added the mimic and siRNA of miR-29a-3p and α1-integrin to cancer cell cultures and extracted cellular proteins 48 h after transfection. Western blot analysis showed that knockdown of the miR-29a-3p mimic or \u003cem\u003eFAM167A\u003c/em\u003e promoted the expression of α1-integrin, and miR-29a-3p expression or FAM167A inhibition also promoted NF-κB phosphorylation. More importantly, knockdown of α1-integrin attenuated the effect of miR-29a-3p on NF-κB phosphorylation. Integrins are cell surface receptors composed of α and β subunits. Studies have shown that integrins accelerate cell migration by promoting the nuclear transport of NF-κB and activating the NF-κB signaling pathway, which is closely related to tumor metastasis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To further investigate the effects of miR-29a-3p, HCC cells were stably transfected with an miR-29-3p inhibitor, which abolished the effects of the miRNA on cells, as expected. Additionally, we found that the mRNA levels of IL-1β, IL-6, and IL-8 in cells exposed to SD-HCC mouse-derived exosomes for 72 h were higher than the levels in cells exposed to HCC mouse-derived exosomes and cells in the blank control group. These findings reveal that exosomal miR-29a-3p in the SD-HCC environment regulates the gene expression of α1-integrin and \u003cem\u003eFAM167A\u003c/em\u003e, activates the NF-κB pathway, promotes phosphorylation, and increases inflammatory IL-type factor levels, thereby ultimately promoting cancer cell migration and invasion and eventually leading to lung metastasis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFirst invoked by Paget, the seed and soil hypothesis suggests that the successful growth of metastatic cells depends on the interactions and properties of cancer cells (seeds) and their potential target organs (soil). The \u0026ldquo;seed\u0026ndash;soil\u0026rdquo; theory holds that cancer cells (seeds) must find their appropriate soil to growth, invasion, and metastasis. The \u0026ldquo;soil\u0026rdquo;, the body\u0026rsquo;s internal environment, which is also called as the body with SD syndrome in TCM theory. It is the core pathogenic mechanism for the occurrence and development of malignant disease.\u003c/p\u003e \u003cp\u003eIn previous studies, there have been many discussions about exosomes promoting tumor growth and metastasis [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Metastatic organogenesis has been one of the greatest mysteries since Stephen Paget\u0026rsquo;s 1889 \u0026ldquo;seed\u0026ndash;soil\u0026rdquo; hypothesis was introduced. Exosomal proteomics has revealed distinct integrin expression patterns, and numerous studies have demonstrated that exosomal integrins can be used to predict organ-specific cancer metastasis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are new exciting strategies in the fight against cancer, with changing the tumor microenvironment and implementing immunotherapy methods being breakthroughs in cancer treatment development [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Many studies have shown that the human body is in a state of immunodeficiency when SD symptoms are present [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, it is of great significance to improve the symptoms of SD and change the human body environment for improving immunity. We also found that exosomal miR-29a-3p in the SD internal environment promotes the metastasis of HCC via activation of the FAM167A\u0026ndash;α1-integrin\u0026ndash;NF-κB signaling pathway. It has been reported [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], that the \u003cem\u003eFAM167A\u003c/em\u003e gene can activate the NF-κB pathway and induce BCR\u0026ndash;ABL-independent tyrosine kinase inhibitor resistance. The FAM167A protein activates the non-canonical NF-κB pathway by binding to desmoglein-1 (DSG1), a cell adhesion protein. Under the state of SD, exosomes can activate the NF-κB signaling pathway in liver cancer cells and significantly promote the expression of phosphorylated NF-κB. Studies have shown that exosomes and integrins are closely related to tumor metastasis [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e],Our study showed that the levels of exosomes and integrins will increase significantly under the state of SD, leading to activation of the NF-κB pathway and the promotion of NF-κB phosphorylation, which also changes the tumor immune microenvironment and reduces the proportion of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e immune cells.\u003c/p\u003e \u003cp\u003eThese findings have important guiding significance for our clinical practice. For patients with TCM symptoms of SD, we can apply Chinese herbs for strengthening the spleen and replenishing the \u0026ldquo;qi\u0026rdquo; for symptomatic treatment, starting on the basis of the \u0026ldquo;seed\u0026ndash;soil\u0026rdquo; theory to change the microenvironment that tumors rely on for survival. Our follow-up project will be to study the therapeutic mechanisms of related TCMs. Such as Spleen strengthening medicine ingredient atractylodes enol, quercetin, etc. One particularly exciting candidate is quercetin, an active ingredient in the Chinese medicinal plants \u003cem\u003eLobelia\u003c/em\u003e and \u003cem\u003eScutellaria barbata\u003c/em\u003e. It is hoped that more effective ingredients for both SD and cancer treatment can be found in natural medicines to overcome the global problem of malignant diseases in humans.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study showed that under conditions of SD, the body releases more miRNA-containing exosomes, which mediate the gene expression of α1-integrin and \u003cem\u003eFAM167A\u003c/em\u003e, activate the NF-κB pathway, and promote the release of more IL-type inflammatory factors. At the same time, SD changes the immune microenvironment of the body and ultimately promotes tumor metastasis and growth. TCM strategies for strengthening the spleen and its active components can start from the \u0026ldquo;seed\u0026ndash;soil\u0026rdquo; theory; that is, to change the immune microenvironment or \u0026ldquo;soil\u0026rdquo; wherein tumors survive. The mechanism of such therapeutic strategy needs to be further studied and explored before it can be applied clinically.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATCC: American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eDMEM: Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e\n\u003cp\u003eELISA, enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eHCC: Hepatocellular carcinoma\u003c/p\u003e\n\u003cp\u003eHE: hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eLC: liver cancer\u003c/p\u003e\n\u003cp\u003emiRNA: microRNA\u003c/p\u003e\n\u003cp\u003emRNA: messenger RNA\u003c/p\u003e\n\u003cp\u003eMUT: mutant\u003c/p\u003e\n\u003cp\u003eOS: overall survival\u003c/p\u003e\n\u003cp\u003ePBS: phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003eqRT-PCR: real-time reverse transcription-polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRFS: recurrence/relapse-free survival\u003c/p\u003e\n\u003cp\u003eRIPA: Radio Immunoprecipitation Assay\u003c/p\u003e\n\u003cp\u003eSD: spleen deficiency\u003c/p\u003e\n\u003cp\u003eSD-HCC: Hepatocellular carcinoma in spleen deficiency syndrome\u003c/p\u003e\n\u003cp\u003esiRNAs: small interfering RNAs\u003c/p\u003e\n\u003cp\u003eTCM: traditional Chinese medicine\u003c/p\u003e\n\u003cp\u003eUTR: untranslated region\u003c/p\u003e\n\u003cp\u003eWT: wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals received humane care throughout the experiments. The study protocols were approved by the Committee on the Use of Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University (Number: Ethical review [2020] No.402).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 81873248, 82174173, 81903967, 82104962, 82104647, 81972785, and 81773162) and the Postdoctoral Science Foundation of China (No. 2021M700964). The study was also supported by the Project of Inheriting Famous TCM Masters of Guangdong Provincial Administration of Traditional Chinese Medicine (No. [2020]1), China (No. 2017A030313866 and 2022A1515012298 to B.H.) and the Open Funds of State Key Laboratory of Oncology in South China (No. HN2021-09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJL, QC, and PL designed the experiments, analyzed the data, and prepared the manuscript. SZ, BH, HH, LY, and CQ conducted the experiments. ZM, BL, ZM, YW, MZ, ML, and HY devised the methodology, collected data, and provided the specimens. All authors confirmed the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eN.N. Pavlova, J. Zhu, C.B. 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Peng, Prognostic value of integrin variants and expression in post-operative patients with hbv-related hepatocellular carcinoma, Oncotarget 8(44) (2017) 76816-76831.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exosomes, hepatocellular carcinoma, miR-29a-3p, Spleen deficiency internal environment, Lung metastasis","lastPublishedDoi":"10.21203/rs.3.rs-2266609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2266609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHepatocellular carcinoma (HCC), a common type of cancer, has a strong metastatic ability and poor prognosis. The tumor microenvironment is the \u0026ldquo;soil\u0026rdquo; for the occurrence and development of tumors, with exosomes playing an important role in these processes. In traditional Chinese medicine(TCM), the tumor microenvironment corresponds to the internal environment of the syndrome known as spleen deficiency (SD). Numerous studies have shown that exosomes contain high levels of miRNAs, which have been shown to contribute to tumor immune regulation and metastasis. The aim of this study was to explore the mechanisms underlying the changes in the tumor microenvironment under the condition of spleen deficiency in order to find better treatments for cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe effects of exosomal miR-29a-3p on lung metastasis from hepatocellular carcinoma (HCC) were evaluated using the scratch test, migration test, mouse SD model, HCC model, and tail-vein injection model of lung metastasis. The western blot assay, ELISA, flow cytometry, luciferase reporter gene analysis, qRT-PCR and immunofluorescence staining were among the methods used to study the molecular mechanism of lung metastasis promotion under the SD internal environment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with the mice with HCC only, the mice with HCC and SD symptoms secreted more miR-29a- 3p-enriched exosomes, and their tumor tissue expressed significantly higher levels of α1-integrin and lower levels of FAM167A. These changed the immune microenvironment of mice (Decreased infiltration of T cells (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e), activated α1-integrin-NF-κB signaling pathway, and secreted more interleukin inflammatory factors(IL-1β, IL-6, and IL-8), which promoted the invasion and infiltration of HCC and its lung metastasis both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. In a series of patients with liver cancer, SD was found to have affected their overall survival and relapse-free survival.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study showed that under conditions of SD, the body releases more miRNA-containing exosomes, changes the immune microenvironment of the body, and ultimately promotes tumor metastasis and growth. These results highlight potential therapeutic targets and methods for the prevention of cancer metastasis, which may help to screen possible anticachexia TCMs and elucidate its mechanism in the future.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-29a-3p in the immune microenvironment of spleen deficiency promotes hepatocellular carcinoma lung metastasis by activating FAM167A-α1-integrin-NF-κB signaling axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-01 19:17:15","doi":"10.21203/rs.3.rs-2266609/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbcb2fac-9848-4e0a-bea2-4e8a60ed62b9","owner":[],"postedDate":"December 1st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-10T09:59:55+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-01 19:17:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2266609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2266609","identity":"rs-2266609","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}