HCC patients and tissue collections
This study was approved by Ethics Committee of Inner Mongolia People’s Hospital. A total of 62 HCC patients (38 males and 24 females) were enrolled at the aforementioned hospital from March 2014 to May 2015. Age of these patients ranged from 44 to 69 years (57.1 ± 5.6 years). Other clinical disorders or therapy may also affect gene expression, this study excluded patients with initiated therapy or complicated with other clinical disorders. All the 62 patients were diagnosed for the first time and no recurrent cases were included. Based on their medical record, HBV-positive was observed in 23 cases, HCV-positive was observed in 29 cases, and the rest 10 cases were negative for both. Prior to therapy, HCC and paired non-tumor tissues were collected through fine needle aspiration from all patients. Tissues were confirmed by histopathological exam and were stored in a liquid nitrogen tank before use. Patients signed informed consent.
Treatments and follow-up
The 62 groups were classified into AJCC stage I (n=8), II (n=12), III (n=20) and IV (n=22). Therapies were determined according to their AJCC stages and other medical conditions. From the day of admission, patients were visited monthly to record their survival conditions. Patients died of caused unrelated to HCC were not included.
HCC cells and transient transfections
HCC cell line SNU-182 from ATCC (USA) was included as the cell model of HCC. RPMI 1640 medium supplemented with 10% FBS was used as cell culture medium. At 37 °C, SNU-182 cells were cultivated in a 5% CO2 and 95% humidity incubator.
Backbone expression vector of PCED1B-AS1 or BCL6 was established using pcDNA3.1 vector (Invitrogen). Mimic of miR-10a and negative control (NC) miRNA were synthesized by Sigma-Aldrich. Through lipofectamine 2000 (Invitrogen)-mediated transient transfections, SNU-182 cells were transfected with 1μg expression vector or 35 nM miRNA. NC experiments were performed by transfecting the same amount of empty vector or NC miRNA into the same number of cells. Prior to subsequent experiments, cells were cultivated in fresh medium for 48h.
RNA preparations
Total RNA isolation from SNU-182 cells and tissue samples was performed using Ribozol reagent (Invitrogen). DNase I was used to incubate RNA samples for 2h at 37 °C for complete genomic DNA removal.
RT-qPCRs
Using SS-IV-RT system (Invitrogen), RNA samples were used as template to synthesize cDNA through reverse transcriptions. QuantiFast SYBR Green PCR Kit (QIAGEN) was them used to perform qPCRs with GAPDH as internal control to determine the expression of PCED1B-AS1 and BCL6 mRNA. Mature miR-10a expression was determined using All-in-One™ miRNA qRT-PCR Reagent Kit (Genecopoeia) with all steps performed following manufacturer’s instructions. U6 was used as the internal control of miR-10a. Each qPCR was performed in three technical replicates and 2-ΔΔCq was used to normalize Ct values of target genes to internal controls.
Western blot
Total protein isolation from transfected cells and protein quantification were performed using RIPA solution (Invitrogen) and BCA assay, respectively. At 95°C, protein samples were incubated for 15min for protein denaturation. A 6% SDS-PAGE gel was used to separate proteins, and PVDF membranes were used to transfer proteins. To block membranes, PBS containing 5% non-fat milk was used to incubate membranes for 2h at 25°C. Primary antibodies of BCL6 (ab203619, Abcam) and GAPDH (ab9485, Abcam) were used to incubate membranes at 4°C for 12h. After that, secodary antibody of lgG-HRP (ab6721, Abcam) was used to incubate the membranes at 25°C for 2h. ECL (Invitrogen) was dropped onto membranes to develop signals. Data normalization was performed using Quantity One software.
CCK-8 kit
SNU-182 cells with transfections were harvetsed and were subjected to CCK-8 cell proliferation analysis. Cells were transferred to a 96-well cell culture plate with 4000 cells in 0.1 ml fresh medium. At 37°C, cells were cultivated and CCK-8 solution was added into each well to reach 10% at 2h before the measurement of OD values. At 450 nm, OD values were measured every 24h for a total of 4 days.
Statistical analysis
Gene expression levels in paired tissues from HCC patients were expressed as average values of three technical replicates and data were compared by paired t test. Data of multiple cell transfection groups were expressed as mean±SD values and were compared by ANOVA Tukey’s test. Correlations were analyzed by linear regression. To analyze the prognostic value of PCED1B-AS1 for HCC, patients were grouped into high and low PCED1B-AS1 level groups (n=31). Survival curves were plotted for both groups and log-rank test was used for survival curve comparison. P<0.05 was deemed statistically significant.