Ethics statement
The use of human tissue samples and clinical data was approved by Committee on Ethics of Medicine, Navy Military Medical University (Shanghai, China). All patients provided the informed written consent. The animal welfare guidelines for the care and use of laboratory animals were followed and the experimental protocol was approved by the Animal Care Committee of Navy Military Medical University (Shanghai, China). The mice were maintained in SPF environment of Experimental Animal Center and sacrificed by anesthetization.
Clinical tissue and cell culture
HCC tissues were obtained from patients who are treated with surgical resection in Eastern Hepatobiliary Surgery Hospital, and each patient had specific clinical-pathological information. The clinicopathological characteristics of all patients are summarized in Table 1. All patients had postoperative follow-up by the same team of surgeons. A follow-up was conducted as previously described. The median follow-up was 37.1 months (range, 2-60 months). Human normal liver cell lines WRL-68 and L02, HCC cell lines Hep3B, Huh7, HepG2, MHCC97L, PLC/PRF/5 and HCCLM3 (LM3) were cultured in DMEM (Gibco) with10% FBS, propagated at 37 in 5% CO2. All the cells used in this study had been identified and confirmed by STR profiling.
Table 1
Clinical characteristics of the HCC patients according to the expression of JAG2
Variables
|
JAG2 expression
|
P value
|
Low
|
High
|
( n = 49 )
|
( n = 131 )
|
Sex
|
Female
|
10
|
32
|
0.57
|
Male
|
39
|
99
|
Age (years)
|
≥50
|
18
|
45
|
0.765
|
<50
HBV status
|
31
|
86
|
Yes
|
28
|
82
|
0.504
|
No
|
21
|
49
|
Cirrhosis
|
Yes
|
15
|
60
|
0.066
|
No
|
34
|
71
|
AFP (ng/mL)
|
>20
|
32
|
89
|
0.738
|
≤20
Tumor size (cm)
|
17
|
42
|
>5
|
20
|
44
|
0.367
|
≤5
Tumor number
|
29
|
87
|
Multiple
|
11
|
22
|
0.455
|
Single
|
38
|
109
|
Tumor encapsulation
|
Yes
|
27
|
61
|
0.308
|
No
|
22
|
70
|
Vascular invasion
|
Yes
|
18
|
80
|
0.004
|
No
|
31
|
51
|
TNM stage
|
I-II
|
30
|
55
|
0.021
|
III-IV
|
19
|
76
|
HBV, hepatitis B virus; AFP, α-fetoprotein; TNM, tumor lymph node metastasis. |
p < 0.05 was considered statistically significant. |
Cell transfection
The miR-2392 mimic, inhibitor, and their corresponding negative controls (miR-NC) were purchased from Ribobio (Guangzhou, China). Human siRNA-JAG2 (si-h-JAG2-1, si-h-JAG2-2, si-h-JAG2-3) and si-h-JAG2-NC were designed and synthesized by RiboBio (Guangzhou, China). The target sequences of siRNA-JAG2 are listed in Table S1. The optimal working concentration of miR-2392 mimic (100 nM) and inhibitor (200 nM) were selected. miR-2392 mimic (100 nM) and inhibitor (200 nM) were transfected into Huh7 and LM3 respectively, and their effects on the proliferation, migration, invasion and neoplasia were measured according to the manufacturer instructions (RiboBio, Guangzhou, China)
RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA from HCC serum was obtained by miRNeasy Serum/Plasma Kit (lot. No. 217184, QIAGEN, Germany) according to the instruction of manufacturer. miRNeasy Serum/Plasma Spike-In Control (C.elegans miR-39 miRNA mimic, lot. No. 219610, QIAGEN, Germany) was used as control. Total RNA from HCC tissues and cell lines were obtained by miRNeasy Mini Kit (lot. No. 217004, QIAGEN, Germany) according to the instruction of manufacturer. Then, we reversely transcripted RNA into cDNA according to the instruction of miRcute plus miRNA cDNA First-Strand Synthesis Kit (KR211-02, Tiangen, Beijing, China). Next, cDNA was quantified by application of miRcute enhanced miRNA fluorescence quantitative detection kit (FP411-02, Tiangen, Beijing, China) with gene-targeted or miR-specific primers. We applied the delta-delta Ct method to conduct quantification as well as calculation of the relative expression of each mRNA or miRNA. The relative expression levels of miRNA and mRNA expression were normalized to U6. Primer sequences are listed in Table S2. Each sample was carried out three times.
Transcriptome analysis
Total RNA was extracted from three pairs of fresh human HCC tissues (tumor and para-tumorous tissue) by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80 °C. The RNA with RIN (RNA integrity number) > 8.0 is acceptable for cDNA library construction. RNA-seq and bioinformatic data analysis were performed by Shanghai Novelbio Ltd.
RNA-seq mapping
RNA-Seq reads were filtered and mapped to Human genome (GRCh38, NCBI) utilizing HISAT2. HTSeq was used to calculate the gene count. miRNA-Seq reads were filtered and mapped to Human miRNA database (miRBase v21.0) and Human genome (GRCh38, NCBI) to achieve the miRNA expression. Differentially expressed genes analysis was applied utilizing DESeq2. The RNA-seq data was deposited in GEO (Accession code: GSE146719). The target gene of miR-2392 was predicted using the TargetScan database (http://www.targetscan.org/vert_71/). The expression of JAG2 and Notch3 in HCC samples of the Cancer Genome Atlas (TCGA) database was searched using the UALCAN database (http://ualcan.path.uab.edu/analysis.html).
Luciferase activity assay
The wild-type (WT) sequence containing the predicted target sites of miR-2392 in the 3’UTR of JAG2 mRNA. We mutated the target sites from CCAUCCU to AACGUUG. The wild-type (WT) or mutant (MUT) sequences of JAG2 was inserted into the pGL3 Basic vector (Promega, Madison, WI). Huh7 cells were seeded in 24-well plates at a density of 5000 cells per well. After 24 h, the cells were transiently transfected with a mixture of 0.1 μg of luciferase reporter plasmid containing 3’-UTR segment of JAG2, 0.02 μg of Renilla luciferase reporter plasmid, and 0.4 μg miRNAs by using X-tremegene HP transfection reagent (Roche, Germany) as described by the manufacturer. Luciferase activity was detected 48 h after transfection by using the Dual Luciferase Reporter Assay System as described by the manufacturer (Promega, Madison, WI, USA). All samples were independently repeated three times.
Establishment of stable miR-2392 knockdown and overexpressing cells
miR-2392 was stably knocked down (LV-hsa-miR-2392 inhibitor) and overexpressing (LV-hsa-miR-2392 mimics) in HCC cell lines by means of lentiviral approach which was designed and produced by GeneCopoeia (Rockville, MD). LV-hsa-miR-2392 NC was used as non-target control. The information of sequences is listed in Table S3.
Western blotting
Protein from fresh HCC samples were obtained with RIPA lysis buffer added with protease inhibitors. After quantification with bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China), we separated each protein through 10% SDS-PAGE and then moved them onto PVDF membranes (Millipore, USA). Then, samples were blocked with 5% nonfat milk. After incubation with primary antibodies against GAPDH and JAG2 (1:200, ab109627, Abcam, UK) and secondary antibodies, protein levels were detected with Image J (GE Healthcare Life Sciences). Each sample was analyzed three times.
The total cellular protein and the nuclear protein were extracted according to instructions of nuclear and cytoplasmic extraction reagents kit (Beyotime, Jiangsu, China). The protein concentrations were determined by bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China). 20 μg of protein was loaded per lane and separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). GAPDH was used as internal control. Antibody information: PCNA (1:1000, ab92552, Abcam, UK), MMP2 (1:1000, ab92536, Abcam, UK), MMP9 (1:1000, ab76003, Abcam, UK), Vimentin (1:1000, ab8979, Abcam, UK).
Cell proliferation assay
Cell proliferation assay was conducted with Counting Kit-8 (CCK-8) (Beyotime, Jiangsu, China). 5000 cells (transfected with miR-NC, miR-2392 mimic, miR-2392 inhibitor) were planted into 96-well plates for 24h, 48h and 72h respectively. Then 10% CCK-8 solution was added. The absorbance of each sample was assessed by a microplate reader set at 450 nM. Each sample was performed for three times.
Cell migration and invasion assay
The methods of cell migration and invasion assays were constructed as previously described. Confluent cell monolayers which transfected with miR-2392 mimic, inhibitor and miR-NC in 6-well plates were wounded by manual scraping with a sterile pipette tip. The imagines were analyzed by Image J (NIH). Cell invasion assay was performed on Matrigel-coated transwell chambers (Corning, NY, USA) with the treatment of miR-2392 mimic, inhibitor and miR-NC.
Colony formation assay
Cells (2000 cells/well) which transfected with miR-NC, miR-2392 mimic and miR-2392 inhibitor were seeded in each well of a six-well cell culture plate. After 2 weeks, the plates were fixed in 4% paraformaldehyde and stained with 1% crystal violet. The colony numbers were counted to assess cell proliferation. The assays were performed in three independent experiments.
Immunohistochemistry
Tissue microarrays consisting of 180 cases of HCC tissues and matched para-cancerous tissues were constructed. Tissue sections were deparaffinized, rehydrated, and treated with 3% H2O2 for 15 min to inhibit endogenous peroxidase activity. Following heat-induced epitope retrieval in 10 mM citrate buffer (pH 6.0) in a microwave for 15 min, the slides were incubated at 4 °C overnight with a pre-diluted primary antibody (JAG2, 1:200, ab109627; Notch3, 1:200, ab23426, Abcam, UK). After incubation with a secondary antibody, the signal was developed with 3,3’-diaminobenzidine tetrachloride.
The staining scores were evaluated by two individuals in a blinded fashion. A quick scoring system from 0 to 12 that combined the intensity and percentage of the positive signal was used. Briefly, a signal of 0 indicated no staining, 1 indicated weak staining, 2 indicated intermediate staining and 3 indicated strong staining. Percentage scores were assigned as follows: 0 corresponded to 0%, 1 to 1-25%, 2 to 26-50%, 3 to 51-75%, and 4 to > 75%. The median value of total staining scores was identified as the optimal cut-off value. If the evaluated score was lower than the median, the indicator expression of in those HCC samples was classified as low; otherwise, it was classified as high [17, 18].
CTC enrichment
CTC enrichment of whole blood samples was conducted according to the method previously described. Briefly, following density gradient centrifugation (Ficoll-Paque PLUS,GE Healthcare, WI, USA), CTCs were enriched by extracting CD45-expressing leukocytes with magnetically beads labeled anti-CD45 monoclonal antibody (130-045-801, Miltenyi Biotec, Germany) according to the instructions. The remaining cells were cyto-centrifuged on polylysine-coated slides for identification and enumeration of CTCs, or resuspended in 150 μL Dulbecco modified Eagle medium (DMEM) (high glucose) (Thermo Fisher Scientific, MA, USA) for the in vitro three-dimensional (3D) culture of CTCs.
Three-dimensional (3D) culture in vitro
The enriched CTCs were cultured using a 3D cell culture assay. Briefly, Matrigel (BD Biosciences, San Jose, CA, USA) was thawed according to the manufacturer's instructions and mixed equally with the CTC-containing DMEM. When HCC cell lines are used for 3D culture, 1×103 cells suspended in DMEM were mixed with Matrigel. The prepared mixture was then incubated in a 24-well plate for 30 min at 37°C. Subsequently, 500 μL DMEM supplemented with 10% fetal bovine serum (FBS) were added to the plate, and the media was supplied every two days during culture. Spheroid formation was observed under a microscope every day and counted on day 7. A spheroid was defined as 3D cell structure > 100 μm in diameter that was determined using images taken on a microscope.
Immunofluorescence staining
Cell spheroids were slightly detached, picked, and fixed on glass slides with 4% paraformaldehyde. Blocking was performed with normal goat serum. Slides were incubated for 1 h with the primary antibodies, including the mouse anti-human CPS1 (1:200, ab110303, Abcam, UK), mouse anti-human ASGPR (1:400, sc-52623, Santa Cruz, USA), rabbit anti-human EpCAM (1:200, ab213500, Abcam, UK) and rabbit anti-human CD133 (1:200, #64326, CST, USA). CPS1 and ASGPR antibodies were used for detection of hepatocytes (29, 30), EpCAM and CD133 antibodies were used for detection of stem-like cells. After washing 3 times with PBS, the secondary antibodies, including the Cy3-conjugated goat anti-mouse/rabbit IgG (Beyotime, China), Alexa Fluor 647-conjugated goat anti-mouse/rabbit IgG (Beyotime, China) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Beyotime, China) were added and incubated at room temperature for 40 min. Cells were then counterstained with 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) and the images were captured using an Olympus-IX71 fluorescent microscope (Olympus Inc., Center Valley, PA, USA).
In vivo tumor growth and metastasis model
BALB/c male nude mice 4-6 weeks old were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). Mice were raised under pathogen-free conditions. All in vivo experiments were done according to approved protocols from the Institutional Animal Care and Use Committees, according to national and institutional guidelines. Briefly, to establish in vivo lung metastasis model, 2 × 106 viable cells (LV-hsa-miR-2392 inhibitor or LV-hsa-miR-2392 NC) were injected into the tail veins of mice. One week after inoculation, si-h-JAG2-3 (2’OMe + 5’Chol modified) was injected into the tail vein three times a week (10nmol/time/mouse) for treatment. After monitoring for 6 weeks, mice were sacrificed, and their lungs were dissected, fixed with phosphate-buffered neutral formalin, and prepared for standard histological examination.
Statistical analysis
Data was reported as mean ± SD. Data analysis was conducted by IBM SPSS Statistics Version 19.0 and Graphpad Prism 5. Analysis of Variance (ANOVA) was used for comparison between groups. The gray value was analyzed by Image-Pro Plus 6.0 software. Survival analysis was estimated by the Kaplan-Meier survival method. Variables with a P value of < 0.05 in the univariate analyses were further tested using multivariate Cox proportional hazards model. Results were determined to be statistically significant when 𝑃 < 0.05.