Upregulation of MicroRNA-19b predicts good prognosis in patients with hepatocellular carcinoma presenting with vascular invasion or multifocal disease

We retrospectively investigated 81 patients diagnosed with HCC and HBV who had either BCLC stage B or stage C disease without extrahepatic metastases who received curative surgery between June 2007 and October 2013 at National Cheng Kung University Hospital. For each case, the diagnosis, histologic grade, and presence of liver cirrhosis were confirmed by pathologists. HBV infection was diagnosed by the presence of serum HBV surface antigen. None of these patients had received chemotherapy or radiotherapy before surgery. Snap-fresh HCC tissues and paired adjacent non-tumorous liver tissues were obtained from each patient during surgery. Tissues were stored in liquid nitrogen after surgical resection until use. HCC tissues were collected from surgical resected samples presenting with tumorous features macroscopically. Adjacent non-tumor tissues were collected > 2 cm away from the edge of the tumors. Clinical parameters including the serum α-fetoprotein (AFP) level at diagnosis, age, TNM stage, and gender were obtained from the database of National Cheng Kung University Hospital Cancer Center. An abdominal computed tomography scan or magnetic resonance imaging was performed every 3 to 4 months after surgery to detect recurrence. The present study was approved by the Institutional Review Board of National Cheng Kung University Hospital (ER-99-251). Written informed consent was obtained from all patients. All specimens were handled anonymously according to legal and ethical regulations, and in accordance with the Helsinki Declaration of 1975, as revised in 1983. The clinicopathological features of the patients are summarized in Table 1 and Additional file 1: Table S1.

Total RNA was isolated from frozen samples using miRNA isolation kits (Qiagen®, Germantown, MD, USA) according to the manufacturer’s protocol. Briefly, around 30 mg of snap-fresh tissue of HCC or adjacent non-tumorous liver were disrupted and homogenized. The lysate was then centrifuged and the supernatant was transferred to the gDNA Eliminator spin column. After centrifugation, the flow-through was transferred to the RNeasy spin column. RNA was extracted using the buffers RPE and RW1. The gDNA Eliminator spin columns, RNeasy spin column and buffers were all supplied in the Qiagen miRNA isolation kits. The concentration and quality of total RNA were measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) at 260 and 280 nm (A260/280) and confirmed by gel electrophoresis.

We selected 5 patients with HBV-associated HCC and performed a miRNA microarray. Two of these patients had liver cirrhosis. RNA labeling and hybridization were completed using a kit from Welgene Biotech Co., Ltd (Welgene Biotech Co., Ltd., Taipei, Taiwan, R.O.C) according to the manufacturer’s instructions. Briefly, RNA was extracted using miRNA isolation kits (Qiagen®) according to the manufacturer’s protocol. RNA purified was quantified at OD 260 nm by an ND-1000 spectrophotometer (NanoDrop Technologies) and analyzed by the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) with the RNA 6000 Nano LabChip kit. During the in vitro transcription process, 1 μg of total RNA was amplified by a low RNA input fluor linear amp kit (Agilent) and labeled with Cy3 (CyDye, PerkinElmer, Waltham, MA, USA). Using incubation with fragmentation buffer at 60 °C for 30 min, 1.65 μg of Cy3-labled cRNA was fragmented to an average size of about 50–100 nucleotides. Correspondingly fragmented labeled cRNA was then pooled and hybridized to SurePrint G3 ChIP/CH3 1X1M array (Agilent) at 60 °C for 17 h. After washing and drying by nitrogen gun blowing, the microarrays were scanned with an Agilent microarray scanner at 535 nm for Cy3. Scanned images were analyzed by Agilent Feature Extraction, version 10.5. Image analysis and normalization software were used to quantify the signal and background intensity for each feature. The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession no. GSE69580.

RNA labeling and hybridization were completed using a kit from Phalanx Biotech Co., Ltd. (Phalanx Biotech Group, Inc., Hsinchu City, Taiwan, R.O.C) according to the manufacturer’s instructions. Briefly, RNA was extracted after miR-19b knockdown in Hep3B. Purified RNA was labeled with fluorescein and hybridized on Human OneArray® (Phalanx Biotech) with 29187 mature human mRNA probes. Finally, hybridization signals were detected, and the images were scanned and quantified. The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE69519.

Complementary DNA was synthetized from the total RNA using gene-specific primers of the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems®, Foster City, CA). For real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), primers for miR-19b and endogenous control U6 were purchased from Applied Biosystems. All reactions were carried out in triplicate according to the manufacturer’s protocol. Briefly, we used 10 ng of RNA sample, 50 nmol/l of stem-loop reverse transcriptase (RT) primer, 10X RT buffer, 0.25 mmol/l each of deoxynucleotide triphosphates (dNTPs), 3.33 U/μl MultiScribe RT, and 0.25 U/μl RNase inhibitor (all from Applied Biosystems’ TaqMan MicroRNA Reverse Transcription Kit®). Reaction mixtures (15 μl) were incubated for 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C and then held at 4 °C (2720 Thermal Cycler; Applied Biosystems®). Real-time PCR was performed using the StepOne™ Plus Real-Time PCR System (Applied Biosystems®). The 20 μl PCR reaction mixture included 1.33 μl of RT product, 1X TaqMan Universal PCR Master Mix, and 1 μl of primer and probe mix from the TaqMan MicroRNA Assay Kit (Applied Biosystems®). Reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Relative quantification of the miR-19b expression was evaluated using the comparative cycle threshold method. The raw data were presented as the relative quantity of miR-19b, normalized with respect to U6.

Complementary DNA was synthetized from the total RNA using gene-specific primers of the TaqMan® Reverse Transcription Kit (Applied Biosystems®, Foster City, CA, USA). For real time qRT-PCR, primers for N-myc downstream regulated 1 (NDRG1), epithelial cell adhesion molecule (EPCAM), hypoxia-inducible factor 1-alpha (HIF1A), high-mobility group protein B2 (HMGB2) and mitogen activated protein kinase 14 (MAPK14) and endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. All reactions were carried out in triplicate according to the manufacturer’s protocol. Briefly, we used 1 ng of RNA sample, 1 μl random primer (random hexamer at a concentration of 0.5 μM as primer, 10X RT buffer, 2.5 mM each of dNTPs, 1 μl of MultiScribe RT™ at a concentration of 50 U/μl, 1.4 μl of 25 mM MgCl₂ and 1 μl of RNase inhibitor at a concentration of 20 U/μl (all from Applied Biosystems’ TaqMan® Reverse Transcription Kit). Reaction mixtures (20 μl) were incubated for 10 min at 25 °C, 30 min at 37 °C, and 5 min at 95 °C and then held at 4 °C (2720 Thermal Cycler; Applied Biosystems®). Real-time PCR was performed using the StepOne™ Plus Real-Time PCR System (Applied Biosystems®). The 10 μl PCR reaction mixture included 1 μl of RT product, 5 μl of 2X TaqMan Universal PCR Master Mix, and 0.5 μl of primer and probe mix from the TaqMan Gene expression Assay Kit (Applied Biosystems®). Reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Relative quantification of the miR-19b expression was evaluated using the comparative cycle threshold method. The raw data were presented as the relative quantity of NDRG1, EPCAM, HIF1A, HMGB2 and MAPK14, normalized with respect to GAPDH.

Human HCC cell line Hep 3B was obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA), was validated in 2014, and was cultured in MEM medium (Invitrogen, Carlsbad, CA,USA) plus 10 % newborn calf serum. Ethics approval was not required.

A quantity of approximately 2 × 10⁵ Hep 3B cells were seeded and cultured in 6-well plates. For each well, 90 pmol of miR-19b inhibitor or control were added to 300 μL Opti-MEM medium and 10 μL of Lipofectamine® 2000 (all Applied Biosystems®). The mixture was added to the cells and incubated for 6 h before replacing the medium. Cells were collected for RNA extraction 24 h after transfection.

The Mann–Whitney test was performed to determine the significance of miRNA levels between the HCC tumor and non-tumor adjacent tissues. Student’s t-test was performed to determine the significance of the AFP level between different groups of patients. Group comparisons of categorical variables were evaluated using the χ² test. Overall survival (OS) was defined from the date of diagnosis to the date of death. Correlation of variables was analyzed using Pearson correlation coefficient. Disease free survival (DFS) was defined from the date of surgery to the date of recurrence. Survival curves were plotted using the Kaplan–Meier method and differences in survival rates were analyzed using the log-rank test. The prognostic relevance of each variable to OS and DFS were analyzed using the Cox regression model. Multivariate analysis of the prognostic factors was performed using the Cox regression model. A P-value less than 0.05 was considered statistically significant. All statistical calculations were performed using SPSS 18.0 for Windows (SPSS Inc., Chicago, IL, USA).