Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP

Abstract Objectives The current study aimed to investigate the mechanism by which exosomes secreted by CHB patients with PNALT and liver inflammation grade (≥A2) affected the development of liver cancer. Materials and methods Gene expression was assessed by RT‐PCR, Western blotting and immunohistochemistry. CCK‐8, colony formation, transwell, scratch‐wound and flow cytometry assays were used to detect cell viability, proliferation, apoptosis and metastasis. The interaction of TCF21 and HHIP was assessed by co‐immunoprecipitation assay. Luciferase reporter was used to detect the combination of TCF21/HHIP and miR‐25‐3p. Xenograft studies in nude mice manifested tumour growth ability of miR‐25‐3p. Bioinformatics analyses were conducted using TargetScan, EVmiRNA, TCGA, GEO, DAVID, COEXPEDIA, UALCAN, UCSC and the Human Protein Atlas databases. Results CHB‐PNALT‐Exo (≥A2) promoted the proliferation and metastasis of HepG2.2.15 cells. miR‐25‐3p was upregulated in CHB‐PNALT‐Exo (≥A2). miR‐25‐3p overexpression promoted cell proliferation and metastasis and was related to poor survival in patients with CHB‐PNALT (≥A2). The cell proliferation‐ and metastasis‐promoting functions of CHB‐PNALT‐Exo (≥A2) were abolished by miR‐25‐3p inhibitors. TCF21 directly interacted with HHIP. Inhibition of TCF21 or HHIP promoted cell proliferation and metastasis. Knockdown of TCF21 or HHIP counteracted the effects of CHB‐PNALT‐Exo (≥A2) containing miR‐25‐3p inhibitor on cell proliferation, metastasis and the expression of Ki67, E‐cadherin and caspase‐3/‐9. Conclusions Transfer of miR‐25‐3p by CHB‐PNALT‐Exo promoted the development of liver cancer by inhibiting the co‐expression of TCF21 and HHIP.


| INTRODUC TI ON
Primary liver cancer ranks sixth among all the malignant cancers in terms of its morbidity and is the second most common cause of cancer-related deaths, with about 700 000 deaths annually worldwide. 1,2 In China, primary hepatic cancer is the second most common cancer after liver cancer. 3 Hepatic cells secret extracellular vesicles (30 nm-150 nm in diameter) called exosomes that contain numerous DNAs, microRNAs (miRNAs), mRNAs, proteins and lipids. 4 Exosomes have been shown to modulate the exchange of these substances between cells and play a vital role in maintaining hepatic homeostasis. Exosomes secreted at different developmental stages or induced by various stimuli differ, and they play important roles in physiological, cellular and pathological processes. 5,6 Exosomes have been shown to influence hepatic cell proliferation and inflammation. 7 Statistics show that more than 80% of the primary hepatic cancer patients are HBV HBsAg-positive, 8 and liver cirrhosis caused by chronic HBV infection is a major risk factor for hepatocellular carcinoma (HCC). Alanine aminotransferase (ALT) is the most common biochemical indicator used to evaluate liver inflammation, 9 and current guidelines for the prevention and treatment of hepatitis B 10 suggested that chronic hepatitis B (CHB) patients with persistently normal alanine aminotransferase levels (PNALT), and the infection may develop into liver cirrhosis or cancer. The risk of HCC in patients with chronic HBV infection has been estimated to be 100 times than that of non-infected patients. 11 However, the risk of HCC in CHB patients, with ALT levels more than twice the ULN, is higher than that of patients with PNALT. Therefore, the related molecular mechanisms need to be further explored.
MicroRNA (miRNA) is a type of short (~22 nucleotides in length), non-coding single-stranded RNA. Most miRNAs are enriched in exosomes, 12 which are small vesicles (30 nm-100 nm in diameter) containing a large number of miRNAs, mRNAs and proteins. The pool of exosomal miRNAs is more homogeneous and more stable than the pool of free miRNAs. Exosome-derived miRNAs have been shown to have numerous functions, which has been reported that overexpression of miR-18a in exosomes promoted the proliferation of liver cells and decreased the levels of α-estrogen receptor. 13 Additionally, exosomes derived from HBV-associated liver cancer promoted chemoresistance by upregulating chaperone-mediated autophagy. 14 Exosomal miRNAs derived from different cells have distinct effects in various diseases. For example, mesenchymal stem cell-derived exosomal microRNA-133b suppresses the progression of glioma. 12 Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. 15 Additionally, disease-derived exosomal miRNAs have been shown to have distinct effects. For example, it has been reported that cancer-derived exosomal miR-25-3p promotes the formation of a pre-metastatic niche formation by inducing vascular permeability and angiogenesis, 16 and platelet-derived exosomal miRNA-25-3p inhibits coronary vascular endothelial cell inflammation in ApoE −/− mice. 17 Therefore, we hypothesized that exosomal miRNA-25-3p might play a vital role in the genesis and progression of liver cancer.

| Cell culture
HepG2.2.15 (HBV-positive liver cancer cell line) was purchased from the Institutes for Biological Sciences at the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific, Inc). All the cells were cultured in a humidified incubator with 5% CO 2 at 37°C.
Exosomes vesicles were resuspended in 100-200 µL PBS and stored at −80°C for further use. TSG101 (the biomarkers of exosomes) was identified using Western blotting, and the protein in free serum was used as a positive control. The preparations were examined, and images were captured using transmission electron microscopy (TEM; JEM-2100; JEOL, Ltd.). Each isolation was verified by nanoparticle tracking analysis using a Nanosight NS300 (Nanosight Ltd.) to determine the size and quantity of EVs extracted. Then, exosomes (10 μL) were added into PBS (1000 μL) and exosomes' diameter was tested using Zetasizer Nano Series-Nano-ZS.

| Exosome uptake
Purified exosomes from CHB patients with PNALT (CHB-PNALT-Exo) were labelled with 1 μmol/L Dil (Invitrogen) as previously described. Briefly, CHB-PNALT-Exo was mixed with 1 μmol/L Dil, and the exosome-dye suspension was incubated for 5 minutes with regular mixing. After incubation, excess dye was removed from the labelled by ultracentrifugation at 100 000 g for 1 hours at 4°C in a 70 Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspension in PBS. The final pellets were resuspended in PBS. The Dil-labelled exosomes were co-cultured with HepG2.2.15 cells for 6 hours. Then, the HepG2.2.15 cells were washed with PBS and fixed with 4% paraformaldehyde (PFA), and uptake was observed by fluorescence microscopy.

| Cell apoptosis and viability assays
Cells were stained with annexin V and propidium iodide reagents (Annexin V-FITC/PI Apoptosis Detection Kit) to assess apoptosis.
Data were analysed using a FACSCalibur flow cytometer and BD CellQuest Pro software 5.1 (BD Biosciences).
Cell viability was examined according to the CCK-8 assay following the manufacturer's protocol (Beyotime).

| Invasion and migration assays
Cell suspension (100 µL, 5 × 10 5 /mL contained in FBS-free RPMI-1640) had been added into upper transwell chamber (with the pore size of 8 µm), while medium (600 µL) supplemented with 10% FBS had been added into lower transwell chamber. Image-Pro Plus version 6 (Media Cybernetics, Inc) was used for cell counting.
Migratory capacity of HepG2.2.1.5 cells under various treatments was evaluated through scratch assay. Cells (5 × 10 5 /mL) had been cultivated within the 12-well plates for 24 hours. Afterwards, a wound was created by scratching the plate with the pipette tip (200 µL). The wound size was determined, and photographs were taken with the microscope to compare the cell motility. The CKX41 inverted light microscope (Olympus Corporation) was used for image capture.

| Colony formation assay
Cells under various treatments had been subjected to trypsin digestion to prepare the single-cell suspension, which was then planted to the 6 mm incubation plates at 250 cells/well. Thereafter, cells had been cultivated for 14 days before 25 minutes of fixation with glacial acetic acid and methanol (at 1:7) at 25°C and 0.1% crystal violet staining. Colonies containing over 50 cells had been calculated by Image-Pro Plus 6.0 (Media Cybernetics, Inc).

| Tissue immunohistochemistry
Paraffin-embedded were fixed with 4% paraformaldehyde overnight at room temperature and embedded in a paraffin block.
Paraffin-embedded slides were deparaffinized and rehydrated in a series of ethanol solutions. After two washes with PBS for 5 minutes each, antigen retrieval was performed in Citrate Antigen Retrieval Solution (Beyotime) by boiling for 10 minutes. After cooling down, slides were blocked with 10% foetal bovine serum in PBS for 1 hours. Then, various primary antibodies (Ki67, C CASP3 and E-cadherin) were applied in a concentration of 8 μg/mL overnight at 4°C. After washed with PBS, HRP-conjugated secondary antibodies were added on the slides for incubating 1 hour. DAB substrate solution was used to reveal the colour of antibody staining. The intensity was scored as follows: 0, none; 1, weak; 2, moderate; and 3, intense.
Both the input and IP samples were analysed by Western blotting using various antibodies at the following dilutions: TCF21 antibody

| Microarray data and identification of differentially expressed genes
The GSE101728 data set, which includes seven tumours and adjacent tissues, was downloaded from the GEO database. The differentially expressed genes (DEGs) obtained from the data set were screened using the GEO2R online tool, and RNA-Seq data from 424 liver cancers were downloaded from the Cancer Genome Atlas (TCGA) database. The DEGs obtained from TCGA data set were screened using the "Deseq2" package in R. A log fold change (logFC)> 1 and adj. P-value < .05 were considered statistically significant.

| Construction of PPI network
COEXPEDIA database was used to construct the PPI network.

| Functional annotation enrichment
GO provides three categories of defined terms, including biological process (BP), cellular component (CC) and molecular function (MF) categories. GO term analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID; http:// david.ncifc rf.gov) online tool. P < .05 was set as the cut-off criterion.

| Human protein atlas
The direct comparison of protein expression of TCF21 between human normal and liver cancer tissues was performed by immunohistochemistry image, and direct comparison of protein expression of HHIP between human normal liver tissues and other cancers was performed by immunohistochemistry image and immunofluorescence image based on the Human Protein Atlas (https://www.prote inatl as.org).

| UALCAN and UCSC databases
UALCAN (http://ualcan.path.uab.edu) and UCSC (http://xena.ucsc. edu/) databases were used to analyse the mRNA expression of overlapping genes in primary liver cancer tissues and their association with clinicopathological parameters.

| Luciferase reporter assay
miR-25-3p mimics/inhibitors and the luciferase reporter vector containing wild-type (WT) or mutant (MUT) 3′-UTR of TCF21/HHIP were co-transfected into 293T cells. After cell transfection for 48 hours, the cells were lysed and the luciferase activity was detected using the Dual-Luciferase Assay Kit (Promega Corporation).

| Quantitative RT-PCR (qRT-PCR)
The TRIzol reagent was used to extract total RNA in accordance with manufacturer protocols. RNA level was calculated by the spectrophotometer (Beckman Instruments). The PrimeScript RT Reagent Kit was utilized to reversely transcribe 2 μg RNA. Later, the LightCycler Real-Time PCR System (Roche 480) was utilized for qRT-PCR. All results were presented in the form of fold difference compared with actin level according to the 2 −ΔΔCt method. The primers are shown in Table 3. GAPDH and U6 were utilized to be the internal reference.

| Western blotting
The whole-cell protein extracts from cells or tissues were prepared anti-GAPDH (1:1000) at 4°C overnight. Later, the PVDF membranes were rinsed before 2 hours of incubation with specific secondary antibodies (diluted at 1:1000) under ambient temperature, and then, membranes would be rinsed with TBST thrice. Then, the Enhanced Chemiluminescence (ECL) Western Blotting Detection Kit (Amersham Pharmacia Biotech) was utilized to detect the immunobands. All films were canned by the Bio-Rad Molecular Imager in combination with the Image Lab Software. The ImageJ analyser software was adopted to analyse the relative protein band densities. Each protein band density was standardized based on GAPDH.

| Statistical analyses
For miR-25-3p, a relative difference in miR-25-3p expression > 2.7 was considered to be high expression. The correlations of miR-25-3p expression with various clinicopathological features were examined using the chi-square test. Survival curves were constructed using the Kaplan-Meier method and analysed by the log-rank test.

| CHB-PNALT-Exo (≥A2) promotes cell proliferation, invasion and migration, and inhibits apoptosis in HepG2.2.15 cells, and miR-25-3p is closely related to the poor survival in HBV-positive patients with liver cancer
Exosomes were isolated from the peripheral blood of HBV-positive patients with liver cancer and CHB patients with PNALT and liver inflammation grade ≥ A2 or <A2 ( Figure 1A). The volume kurtosis of the exosomes' diameter was 10 nm-100 nm ( Figure 1B). Expression of the exosome biomarker TSG101 was assessed by Western blotting assay ( Figure 1C). The analysis showed that miR-25-3p expression in CHB-PNALT-Exo (≥A2) was lower than that in HBV-positive liver-Exo and was higher than that in CHB-PNALT-Exo (<A2) group ( Figure 1D). Likewise, miR-25-3p expression in the CHB-PNALT (≥A2) group was lower than that in the HBV-positive liver group and was higher than that in the CHB-PNALT (<A2) group ( Figure 1E).
We also found that miR-25-3p expression was significantly correlated with tumour size, tumour number, tumour differentiation, vascular invasion and TNM stage (P < .05; Table 2), but was not significantly correlated with gender, age or AFP level ( Table 2). The Kaplan-Meier analysis revealed that patients with tumours that overexpressed miR-25-3p had shorter tumour-free survival than HBV-positive patients with liver cancer ( Figure 1F). Univariate Cox regression analysis showed that the miR-25-3p expression level and vascular invasion were closely related to overall survival ( Table 4)

| Bioinformatics identification of the target genes of miR-25-3p
A total of 4695 DEGs, including 1262 downregulated and 3433 upregulated genes, were obtained from TCGA database ( Figure 5A), and 824 DEGs, including 459 downregulated and 365 upregulated genes, were obtained from the GSE101728 data set ( Figure 5B). The 14 overlapping genes present in both the DEG data set and the miR-25-3p target genes are shown in Figure 5C. The expression of 14 overlapping genes was assessed using UALCAN ( Figure 5D). The results of GO enrichment analysis using DAVID showed that these genes were mainly enriched in two terms, "BP" and "CC" ( Figure 5E). A heat map of the 14 overlapping genes in human liver cancer and normal tissues was generated using UALCAN ( Figure 5F). The Kaplan-Meier analysis revealed that CPEB3, CNTN4, EZH2, CDK5R1 and CENPF were significantly related to poor survival ( Figure 5G). The co-expression of TCF21 and HHIP was accessed using the COEXPEDIA ( Figure 5H).

| Evaluation of protein expression levels of TCF21 and HHIP in liver cancer tissues using the Human Protein Atlas
Based on analyses using the Human Protein Atlas, TCF21 protein levels were found to be low or absent in liver cancer tissues and were medium in normal liver tissues ( Figure 6A). HHIP expression levels were also medium in normal liver tissues; however, HHIP expression levels in liver cancer tissues were not reported in the Human Protein Atlas database. Our analysis showed that HHIP was mainly located in the nucleoplasm of A-431, U-2 OS and U-251 MG cells ( Figure 6B), and TCF21 and HHIP were significantly downregulated in human tumour tissues when compared to the levels in the corresponding adjacent tissues ( Figure 6C). TCF21 and HHIP expression levels were also significantly lower in tumour tissues than in the non-tumour tissues of the mice ( Figure 6D).

| TCF21 and HHIP are target genes of miR-25-3p
The expression levels of TCF21 and HHIP were upregulated in the tissue of CHB patients with PNALT (≥A2) ( Figure 7A). Co-IP experiments indicated that TCF21 directly interacted with HHIP in   Figure 8F-N).

| The blocking effects of miR-25-3p inhibitors on the effect of CHB-PNALT-Exo (≥A2) on cell proliferation and metastasis in HepG2.2.15 cells are reversed by knockdown of TCF21 and HHIP
A CCK-8 assay showed that the viability of HepG2.

| D ISCUSS I ON
HBV causes a chronic liver infection (called CHB), which is common worldwide. CHB is one of the major risk factors for end-stage liver and enhancer of zeste homolog 2 (E2H2). However, the SYT1 expression data were inconsistent in TCGA database, UALCAN database and GSE101728 data set, and CDK5R1 and CENPF expression levels were upregulated in TCGA database, UALCAN database and GSE101728 data set. In addition, TCF21 and HHIP were mainly enriched in "tube development," whereas CNTN4 had no connection with "tube development" (Figure 5). Therefore, both TCF21 and HHIP, which were identified as target genes of miR-25-3p and were downregulated in liver cancer, were used to follow up studies ( Figure 6). The results showed that TCF21 directly interacted with HHIP and was positively correlated with HHIP in CHB patients with PNALT and HBV-positive patients with liver cancer (Figure 7). TCF21, which is located on chromosome 6q23-q24, encodes a member of the basic helix-loop-helix (bHLH) TF family. 22 TCF21 plays a critical role during embryogenesis and in the development of numerous cell types in the heart, lung, kidney and liver. 23,24 TCF21 functions as an anti-oncogene and can inhibit tumour cell proliferation and metastasis, and vascular production in breast cancer, 25 ovarian cancer, 26 lung cancer 27 and liver cancer. 24 HHIP is also an anti-tumour gene and a negative feedback factor in the HH pathway that directly inhibits HH. HHIP, which is encoded by a gene located at 4q31.21-31.3, can compete with PTCH for binding to hedgehog (Hh) protein, thereby blocking HH signalling. 28 Studies have shown that while HHIP mRNA is expressed in normal tissues, its expression is decreased in some tumour tissues. 29 It has been reported that overexpression of HHIP inhibited tumour cell proliferation and metastasis of lung cancer 28 and gastric cancer. 30 However, the related mechanisms and the role of HHIP in liver cancer have not been re-

| CON CLUS IONS
The expression of miR-25-3p was upregulated in exosomes secreted by CHB patients with PNALT and liver tissue inflammation grade ≥ A2 and was closely related to poor survival in HBVpositive patients with liver cancer. Exosomes derived from CHB patients with PNALT and liver tissue inflammation grade ≥ A2 promoted the occurrence and development of HBV-positive liver cancer, and these effects were reversed by inhibition of miR-25-3p. TCF21 and HHIP are two target genes of miR-25-3p that have anti-tumour effects in HBV-positive liver cancer. Exosomes secreted by CHB patients with PNALT and liver tissue inflammation grade ≥ A2 promoted the development of liver cancer by transferring miR-25-3p to inhibit the co-expression of TCF21 and HHIP.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (No. 81800532).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.