Tissue sections in immunohistochemistry were enrolled at Affiliated Liutie Central Hospital of Guangxi Medical University and provided informed consent for tissue to be used for research studies. All tissues were snap-frozen and stored in liquid nitrogen immediately after surgical resection. Tissue microarray slides were purchased from Shanghai Outdo Biotech Co., Shanghai, China. The clinicopathologic features of the patients are described in Table 1. Each patient was diagnosed based on World Health Organization criteria, and tumor differentiation grade was classified according to Edmondson and Steiner (21). Liver function was evaluated using the Child-Pugh scoring system. Tumor stages were classified based on the International Union against Cancer TNM classification system. OS was defined as the time from surgery to death or surgery to last observation time. The OS data were obtained from the last follow-up for surviving HCC patients. Time to tumor recurrence was calculated from the surgical resection to the date of any relapse, including both extrahepatic metastasis and intrahepatic recurrence (22).
Table 1
Correlation between the clinicopathologic characteristics and miR-1915-3p expression in HCCs
Clinical parameters
|
miR-1915-3p
|
P value
|
Low (n = 45)
|
High (n = 45)
|
Age (years)
|
|
|
|
0.138
|
|
≤ 50
|
24
|
17
|
|
|
> 50
|
21
|
28
|
|
Gender
|
|
|
|
0.270
|
|
Male
|
39
|
35
|
|
|
Female
|
6
|
10
|
|
HBsAg
|
|
|
|
0.764
|
|
Positive
|
38
|
39
|
|
|
Negative
|
7
|
6
|
|
AFP (ng/ml)
|
|
|
|
0.274
|
|
≤ 400
|
31
|
26
|
|
|
> 400
|
14
|
19
|
|
ALT (U/L)
|
|
|
|
0.398
|
|
≤ 40
|
19
|
23
|
|
|
> 40
|
26
|
22
|
|
GGT (U/L)
|
|
|
|
0.288
|
|
≤ 54
|
17
|
22
|
|
|
> 54
|
28
|
23
|
|
Cirrhosis
|
|
|
|
0.535
|
|
No
|
7
|
5
|
|
|
Yes
|
38
|
40
|
|
Tumor size (cm)
|
|
|
0.398
|
|
≤ 5
|
26
|
22
|
|
|
> 5
|
19
|
23
|
|
Tumor number
|
|
|
|
0.270
|
|
Solitary
|
39
|
35
|
|
|
Multiple
|
6
|
10
|
|
Tumor differentiation
|
|
|
0.006*
|
|
Ⅰ/Ⅱ
|
31
|
18
|
|
|
Ⅲ/Ⅳ
|
14
|
27
|
|
TNM stage
|
|
|
0.020*
|
|
Ⅰ/Ⅱ
|
42
|
34
|
|
|
Ⅲ/Ⅳ
|
3
|
11
|
|
Chi-square test was used in all analysis |
HBsAg hepatitis B surface antigen, AFP alpha-fetoprotein, ALT alanine aminotransferase,GGT gamma glutamyl transferase, TNM tumor–node–metastasis |
*P < 0.05,Bold values signify P-value < 0.05 |
Cell lines and animals
Human HCC cell lines (SMC-7721, QGY-7701, QGY-7703, MHC-97H, HuH7, HepG2) and human normal liver cell lines LO2 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle medium (DMEM)(Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific) in 5% Co2 at 37 ℃.
Male Balb/c-nude mice (four-week-old) were purchased from the Shanghai Institute of Material Medicine and raised under specific pathogen-free conditions. The use of laboratory animals conformed to the NIH guide for Care and Use of Laboratory Animals, 8th ed.
Immunohistochemistry
Situ hybridization (ISH) was evaluated with miRCURY locked nucleic acid (LNA) detection probe for miR-1915-3p 5’-CCCCAGGGCGACGCGGCGGG-3’ to detect the miR-1915-3p expression in HCC tissue arrays, as previously described with modifications(23). Briefly, digoxigenin double-labeled LNA-modified probe (500 nmol/L) was added to the hybridization solution and hybridized according to the manufacturer’s protocol. The sample images were captured with 200X magnification after incubation with 4-nitroblue-tetrazolium and nuclear fast red. ISH signals for miR-1915-3p were scored by combining the proportion of positive cells and staining intensity. The intensity of staining cells was scored as: strong (3), intermediate (2), weak (1), and negative (0). The proportion of positive cells was scored as follows: 4 points: 75 to 100% of positive cells, 3 points: 50 to 74% of positive cells, 2 points: 25 to 49% of positive cells, 1 point: 1 to 24% of positive cells, 0 point: no positive cells. The staining index (SI) was calculated as staining intensity score times the proportion of positive tumor cells. High expression was explained by a SI ≥ 5, while low expression was defined as an SI < 5.
Vectors and cell transfections
miR-1915-3p ectopic expression level lentiviral vector (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin-miR-501-3p) and anti-miR-1915-3p and its negative control were purchased from Genomeditech (Shanghai, China). Bcl2L11 expression vector (CMV-MCS-SV40-Neomycin-Bcl2L11) and shRNA Bcl2L11 vector, as well as its negative control, were also purchased from Genomeditech (Shanghai, China). The transfected clones were selected with puromycin and G418 (Sigma-Aldrich, USA), according to the manufacturer’s protocol, and the transfected efficiency was verified by quantitative PCR (qPCR) and western blot assays. wt or mt 3’-UTR were sequenced from Bcl2L11 and were cloned into the pGL3-promoter vector (Promega, Madison, WI). All transfections were performed as previously described (24).
RNA extraction and quantitative real-time PCR (q RT-PCR)
Total RNA was extracted from cell lines and fresh-frozen HCC tissues with Trizol reagent (Thermo Fisher Scientific). A synthesis of complementary DNA (cDNA) was performed by PrimeScript RT Reagent Kit (Takara, Shiga, Japan) and detected at the ABI7500 Sequence Detection System (Applied Biosystem, Foster City, CA, USA), according the manufacturer’s guidelines. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for mRNA and U6 was used for microRNA. The gene expression relative to the endogenous control was analyzed by ∆Ct and 2-∆∆Ct. The following primers were used for qRT-PCR detection:
Western blot
Western blot was performed with a modified procedure as previously described (25). Briefly, total protein was extracted from HCC tissues and cells by using a RIPA Lysis Buffer (Thermo Scientific, USA) containing 1% phenylmethanesulfonyl fluoride on ice. The cell lysates (50 μg of protein) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane that was blocked with Tri-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin (BSA) for two hours at room temperature. They were then incubated with the following antibodies at 4℃ overnight: anti-caspase3 (Abcam, ab4051, Cambridge, UK), anti-caspase8 (Abcam, ab108333, Cambridge, UK), anti-Bcl2 (Abcam, ab32124, Cambridge, UK), anti-BAD (Abcam, ab32445, Cambridge, UK), anti-Bcl2L11 (Abcam, ab32158, Cambridge, UK), anti-P53 (Abcam, ab131442, Cambridge, UK), and anti-GAPDH (Proteintech, 60004-1-Ig, USA). The membrane was incubated with the secondary antibodies (Proteintech, SA00001-1, USA) at room temperature for one hour. The immunoblots were detected by enhanced chemiluminescence assays and GAPDH was used as the loading control.
Wound-healing, cell proliferation and transwell invasion assay
The wound healing assay was conducted with a modified method as previously described (25). Briefly, hepatocarcinoma cells were grown to 90% confluence and then starved for 12 hours. A scraped line was created with a p200 pipette tip and washed twice with PBS. The cells were subsequently incubated for 48 hours with a starvation medium (DMEM without FBS). Cellular migration toward the scratch was observed and recorded after 48 hours, and the percentage of open area was assessed. The CCK8 assay was used to measure the proliferation of HCC cells. Briefly, cells (104/well) were inoculated into 96-well assay plates. Then, the cells were incubated with 10 μl CCK-8 solution (Dojindo, Tokyo, Japan) for two hours. Absorbance was acquired by using an automatic microplate reader at 450 nm (Thermo Fisher Scientific, Inc, Waltham, MA, USA). For the transwell invasion assays, the cells were plated in Transwell upper chambers (Corning, New York, USA) at a density of 2 × 104 in 200 μl of serum-free medium, while 800 μl of normal-containing culture medium was placed in the lower chambers. After 48 hours of incubation at 37℃, the cells that invaded the lower surface were fixed with 4% paraformaldehyde for 15 minutes and stained with0.1% crystal violet dye (Ameresco, LIC, USA) for six minutes. They were photographed in an inverted microscope (×200 magnification) and counted by using Image J software.
Luciferase reporter assay
For the luciferase assay, cells were seeded in 96-well plates and cultured for 24 hours. In all, 10 ng of Bcl12L11 wild-type (WT) or mutant (MUT) 3’-UTR reporter vectors were transfected into HCC cells using the Lipofectamine 2000 (Invitrogen, USA), according to the manufacturer’s instructions. After an incubation of 48 hours, cells were harvested and a luciferase reporter gene assay was conducted using the Dual Luciferase Reporter Assay Kit (Promega).
Animal experiments
Approximately 1×107 HCCSMMC-7721 cells, which stably transfected with either miR-1915-3p or the NC vector, were suspended in 100 μl Dulbecco’s modified Eagle’s medium and then cutaneously injected into the left flank of each BALB/c nude mouse. After injections for four weeks, the subcutaneous tumors were cut equally into small pieces and transplanted into the livers of nude mice. Transplanted mice were sacrificed six weeks later and tumor volume (V) was calculated with the following formula: (length × width2)/2 (26). The paraffin-fixed livers were serial sectional and tunel assay was performed using a DeadEndTM Fluorometric TUNEL System (Promega), according to the manufacturer’s instructions.
mRNA-seq
Total RNA was extracted from cells by Trizol reagent (Invitrogen, USA) and RNA samples were purified by magnetic oligo (dT). mRNA samples were then reverse transcribed into first-strand cDNA, followed by second strand synthesis. Fragmented DNA samples were blunt ended and adapters were ligated to construct a library. DNA was quantified with Qubit (Invitrogen, CA, USA). Sequencing was carried out using an Illumina HiSeq 3000 SBS instrument according to the manufacturer’s recommendations. We quantified transcript expression levels with a fragment per kilobase of transcripts per million fragments mapped (FPKM), and analyzed the gene transcripts between different samples.
Statistical analysis
All quantified data are presented as mean ± the standard deviation (SD). To compare quantitative data between groups, students’ t-tests were used. For categorical parameters, the chi-square test was used to analyze the data. Kaplan-Meier survival analysis was used to estimate OS. The correlation between groups was determined using Pearson’s correlation tests. All statistical analyses in this study were performed using SPSS-24 (IBM, NY, USA) and Prism 6 (GraphPad). P < 0.05 was considered significant.