SUPPLEMENTARY MATERIAL AND METHOD
miRNA array.miRCURY LNA™ microRNA chips (version 8.0, Exiqon, Vedbaek, Denmark) with 840 miRNA probes were used to profile the differences for miRNA expression among three HCC subtypes as previously described[1]. All miRNA array data have been deposited in ArrayExpress database. The ArrayExpress accession number is E-MTAB-3347.From the data of miRNA arrays, we only selected miRNAs that were up- or down-regulated more than three-fold in HCC compared with the corresponding ANLTs. The miRNA array results were confirmed in another group of fresh HCC and ANLT tissues (n = 15) and miR-130b was one of the most highly up-regulated miRNAs. The miRNAs significantly up- or down-regulated were listed in our previous article[2].
Lentivirus and Vector Construction.The miR-130b overexpression and inhibition lentivirus were construct by GeneChem Corporation (GeneChem, Shanghai, China). The PTEN expression vector was constructed by inserting its CDS sequence (AH007803.1) into the pEX-2 vector (GeneChem). PTEN-shRNA were also purchased from GenePharma Company and the functional sequences were GCGCTATGTGTATTATTATCTCGAGATAATAATACACATAGCGC. The efficiency of PTEN overexpression vector and PTEN-shRNA vector were detected by western blot (data not shown).
Cell Proliferation and Colony Formation Assays. For cell proliferation assay, 2×104 cells were seeded into 35mm dishes(Corning Costar Corp, Corning, NY) and cultured at 37°C. Everyday, cells was obtain from 3 dishes and counted by using hemacytometer. For colony formation assays, 500 cells were seeded into 35mm dishes (Corning Costar Corp) and cultured for 2 weeks at 37°C. The numbers of colonies per dish were counted after staining with crystal violet. Only positive colonies (diameter > 40 um) in the dishes were counted and compared.[3] These experiments were performed in triplicate.
In VitroWound Healing. Cells were seeded onto 35mm dishes (Corning Costar Corp) coated with fibronectin. After cells reached 100% confluence, to inhibit cell proliferation, which could confound the analysis of cell migration, cells were preincubated with mitomycin (Sigma, St. Louis, MO, 10μg/ml) for 1h at 37°C.[4] Wound healing assays were performed with a sterile pipette tip to make a scratch through the confluent monolayer. Medium was changed and cells were cultured for 24 hours. The percent wound closure was calculated for five randomly chosen fields.
Invasion Assays. For the invasion assay, 1 × 105 cells in serum-free medium containing 0.1% bovine serum albumin were placed into the upper chamber of the insert with Matrigel (BD Biosciences, Franklin Lakes, NJ). After 24 hours of incubation at 37°C, we removed the cells remaining in the upper chamber or on the upper membrane. The number of cells adhering to the lower membrane of the inserts was counted after staining with a solution containing 0.1% crystal violet (Beyotime Institute of Biotechnology, Beijing, China) and 20% methanol. For each experiment group, the invasion assay was performed in triplicate, and three randomly fields in each replicate were chosen for the cell number quantification.
Luciferase Reporter Assay.The wild type or mutant of 3’ UTR sequences of PTEN were inserted into the pGL3 vector (GeneChem). Luciferase activity was assessed according to the Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI) using a Veritas™ 96-well Microplate Luminometer (Promega) with substrate dispenser (Promega). 293T cells infected with miR-130b lentivirus or control virus were seeded in 96-well plates with 70% confluence. 12 hours later, the cells were cotransfected with 50 ng pGL3-UTR and 10 ng pRLTK by using Lipofectamine LTX. 24 hours after transfection, firefly and Renilla luciferase activities were measured. The Renilla luciferase activities were used to normalize transfection efficiency.
HCC Mouse Model. The hepatocellular carcinoma model in mice was constructed as described.[5] Briefly, 5 × 106 HCCLM3 cells of different experimental groups were injected subcutaneously into the left upper flank regions of nude mouse (3-4 weeks of age, male, BALB/c). After 1 month, the subcutaneous tumor tissues were removed and implanted into the liver of nude mouse (5 in each group). After 6 weeks, mice were sacrificed, the tumor size was calculated as follows: tumor volume (mm3) = (L × W2)/2,[6] where L = long axis and W = short axis. Livers and lungs were harvested, fixed with phosphate-buffered neutral formalin, sectioned serially and stained with hematoxylin and eosin (H&E) for standard histological examination. Mice were manipulated and calcured in the Animal Institute of CSU according to the protocols approved by the Medical Experimental Animal Care Commission.
Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR).Total RNA was reverse-transcribed into complementary DNA with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) by using a specific reverse-transcription primer (Applied Biosystems, CA). Quantitative PCR was performed to quantify the expression of miR-130b and U6 small nuclear RNA with TaqMan microRNA assays (Applied Biosystems, CA). Relative fold changes of expression in tumor tissues against ANLT and among different cell lines were calculated using the comparative Ct (2-△△Ct) method with U6 small nuclear RNA (Ambion, TX) as endogenous control.
Cell Lines and Cell Culture. In this study, liver cell lines L02 and five HCC cell lines (HepG2, SMMC7721, MHCC97-L, MHCC97-H, HCCLM3) were used. The source and characters of these cell lines were described in our previous work.[7] These cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics at 37°C with 5% CO2.
Western Blot. Total protein was extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membrane (Millipore, Bedford, MA). Primary antibodies used in this study were shown as follow: Rabbit monoclonal anti-PTEN antibody (ab32199), Rabbit polyclonal anti-Snail antibody (ab180714), Mouse monoclonal anti-β-actin (ab8226) were purchased from abcam (Abcam, Cambridge, MA). Mouse monoclonal anti-Akt antibody (#2910), Rabbit monoclonal anti- Phospho-Akt (Ser473) antibody (#4060) were purchased from Cell Signaling Technology (Cell Signaling, Danvers, MA). mouse monoclonal anti-Vimentin antibody (sc-6260), rabbit polyclonal anti-E-cadherin antibody (sc-7870), mouse monoclonal anti-N-cadherin antibody (sc-8424), mouse monoclonal anti-HIF-1α were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA).
Immunohistochemistry.Formalin-fixed paraffin sections of HCC were stained for protein using the streptavidin-peroxidase system (ZSGB-BIO, Beijing, China). The Immunohistochemistry was performed as describe.[8]
Immunofluorescence. Stably transfected cells were seeded in the 8-well culture plate to prepare and incubated with TRITC Phalloidin for 30 min and DAPI for 30s. The slides were visualized using a microscope or inverted fluorescence microscope.
Statistical Analysis. Statistical analysis was performed using the SPSS (version 13.0, Chicago, IL). The differences between groups were analyzed by Student’s t test when only two groups were compared or by one-way analysis of variance (ANOVA) when more than two groups were compared. Spearman correlation test was used for analyzing the correlations between miR-130b expression and the clinical and pathological variables. Survival curves were constructed using the Kaplan-Meier method and evaluated using the log-rank test. The Cox proportional hazard regression model was used to identify factors that were independently associated with overall survival and disease-free survival. P < 0.05 (two-tailed) was considered statistically significant.
Reference
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