Cell culture
The human HCC cell lines HepG2, Hep3B and human embryonic kidney cell 293T were purchased from the Cell Bank:China Academy of Sciences (Shanghai, China). HepG2, Hep3B and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% (v/v) FBS. All cells were cultivated at 37 °C with 5% CO2.
Generation of drug-resistant cells
Hep3B and HepG2 were cultures with sorafenib at the concentration of 10 μM for 48h. Viable cells remaining continue to treat with sorafenib concentration increasingly. When the resistant drug index examined up to 3 that cell lines were considered to be sorafenib resistant.
Transfection and stable cell line generation
The miR-138-1-3p mimics, mimics-NC, miR-138-1-3p inhibitor, inhibitor-NC (miR-138-1-3p, 5′-GCUACUUCACAACACCAGGGCC-3′) and PAK5 siRNA (si-PAK5, 5′-CAAAGTCTTCGTACCTGAATC-3′) were obtained from GenePharma (Shanghai, China). They were transfected in Hep3B and HepG2 cells using siLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA). The PAK5/Vector and Myc-PAK5 expression plasmids were purchased from Guangzhou FulenGen Co. Hep3B and HepG2 cells transiently transfected with PAK5 plasmids using X-tremeGENE HP DNA Transfection Reagent (Roche, Indianapolis, IN, USA). All transfection procedure follows the manufacturer’s instructions. The LV-miR-138-1-3p-NC and LV-miR-138-1-3p (miR-138-1-3p, 5’-GCUACUUCACAACACCAGGGCC-3’) were established by GenePharma (Shanghai, China). HepG2 transfected with lentivirus for 48 h, and then selected via puromycin (Vicmed, China) for 30 days.
Cell viability assay
Cell viability was assayed by the Cell Counting Kit-8 (CCK-8) kit (Beyotime, China). Post-treatment Hep3B and HepG2 cells (2 × 103) were cultured in 96-well microplate (Corning Incorporated, NewYork, USA) in quintuplicate at 37 °C with 5% CO2. Then, CCK-8 reagent (10 μl) and 90μl serum-free medium added to each well at 24, 48, 72, and 96 hours, respectively. Measure the absorbance at 450 nm after incubated for 1 hours at 37 °C by a multi-function enzyme-linked analyzer (Biotek Instruments, Winooski, VT, USA).
RNA isolation, reverse transcription and quantitative real-time PCR (qRT-PCR)
Total RNA isolation used Trizol Reagent (Takara, Dalian, China) following the manufacturers’ instructions. RNA reverse transcription performed with the PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara, Dalian, China) and program set according to the operating instructions. MiRNA levels determination utilized the BrightGreen Express 2X qPCR MasterMix (Abm, Canada) with a 7500 Real-time PCR System (Life Technologies, NY, USA). Relative quantitation of miR-138-1-3p (forward: 5′-CGCGGCTACTTCACAACACC-3′, reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′) was normalized to U6 (forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse: 5′-CGCTTCACGAATT TGCGTGTCAT-3′) levels, PAK5 (forward: 5'-GGC GTCCTCTTGTGTCTTC-3', reverse: 5'-GTACTGAGTCCTTCTGATTTGC-3'). ABCB1 (forward: 5′-GGCCTAATGCCGAACACATT-3′, reverse: 5′-CAGCGTCTGGCCCTTCTTC-3′), ABCG2 (forward: 5′-CAGGTGGAGGCAAATCTTCGT-3′, reverse:5′-ACACACCACGGATAAACTGA-3′), LRP (forward: 5′-GTCTTCGGGCCTGAGCTGGTGTCG-3′, reverse:5′-CTTGGCCGTCTCTTGGGGGTCCTT-3′) and MRP2 (forward: 5′-CCAAAGACAACAGCTGAAA-3′, reverse:5′-TACTTGGTGGCACATAAAC-3′) were normalized to GAPDH (forward: 5′-TGGTATCGTGGAAGGACTCAT-3′, reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′) levels (Sangon, Shanghai, China). The relative RNA expression was calculated via the comparative threshold cycle (Ct) method (relative geneexpression = 2-(ΔCtsample − ΔCtcontrol)).
Plate colony formation assay
Equal numbers of post-treatment Hep3B and HepG2 single-cell suspension cells seeded into 6-well plates and cultured for 2 weeks with 5μM sorafenib fixed in the medium. 4% paraformaldehyde fixed the clones for 15 min, and then 0.1% crystal violet stained clones for 20 min.
Flow cytometric analysis
Post-treatment Hep3B and HepG2 cells were resuspended in PBS. Annexin V-FITC Apoptosis Detection Kit (Beyotime, China) and flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) were visualized to evaluate the percentage of apoptotic cells. Data were analyzed using a FACSCalibur Flow Cytometer (Becton Dickinson).
Antibodies and Western Blot
Cells lysis and total protein extraction were using the RIPA lysis buffer (KeyGen BioTECH, Jiangsu, China) and protein concentrations gauged by Enhanced BCA Protein Assay Kit (KeyGen BioTECH). Nuclear and cytoplasmic protein was distilled via Nuclear and Cytoplasmic Protein Extraction Kit (KeyGen BioTECH). SDS-PAGE electrophoresis and nitrocellulose blotting membranes (thermo fisher scientific) were used to proteins transfer. The specific primary antibodies incubation overnight at 4 °C, including anti-PAK5 (1:1000, Abcam, Shanghai, China), anti-ABCB1 (1: 500, Abcam, Shanghai, China), anti-β-catenin (1:1000, Santa Cruz, USA), anti-p-β-catenin (S675) (1:1000, Cell Signaling Technology, USA) and β-actin (1:5000, proteintech, China). Anti-rabbit HRP or Anti-Mouse HRP (1:10,000, Vicmed) incubated at room temperature for 2 hours afterwards. The Western Blot images were detected via Chemistar™ High-sig ECL Western Blot Substrate (Tanon, shanghai, China).
Rhodamine 123 efflux assay
Rhodamine 123 throughly dissolved in DMSO at a concentration of 5 mol/L. Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. 5 μM Rho123 added to the medium and incubated for 30 min at 37 °C with 5%CO2. Transpose new complete medium. Images were observed by immunofluorescence confocal laser scanning microscopy (Zeiss LSM 880).
Immunofluorescence
Seed post-treatment cells on Glass Bottom Culture Dishes (NEST, Wuxi, China) one day in advance. Cells were fixed with 4% paraformaldehyde 20 min, and then blocked with TBS (0.3% Triton X-100 and 0.25% BSA) at room temperature for 2 hours. Afterwards, they were incubated overnight at 4 °C with a primary antibody: anti-PAK5 (1:100, Abcam, Shanghai, China) and β-catenin (1:100, Santa Cruz, USA) Afterwards, they were washed three-fold with PBD. Stained with fluorescent secondary antibody: CoraLite488–conjugated Affinipure Goat Anti-Mouse IgG(H+L) and CoraLite594–conjugated Goat Anti-Rabbit IgG(H+L) (1:200, proteintech, China) at room temperature for 60 min. Nuclei were deal with 4′, 6-Diamidino-2-phenylindole (DAPI) (KeyGen BioTECH) for 10 min. Pictures were taken by immunofluorescence confocal laser scanning microscopy (Zeiss LSM 880).
Co-immunoprecipitation (co-IP)
Cells were lysed in IP lysed buffer (KeyGen BioTECH) with cocktail of protease/phosphatase inhibitors (1:100, Sigma Aldrich, MO, USA), and then added anti-β-catenin (1:100, Santa Cruz, USA) and Mouse IgG (Beyotime) incubation overnight at 4 °C. 40 μl Protein A/G beads (Santa Cruz) were used to bind to antibodies. Wash beads and heat with 2×loading buffer. Immunoprecipitated proteins were examined by Western Blot.
Chromatin immunoprecipitation (ChIP)
CHIP assay kit (Cell Signaling Technology) was employed for CHIP assay. 1 × 107 cells were prepared for the first-step. Then DNA complexes were immunoprecipitated with β-catenin antibody. The resulting precipitated DNA samples were quantified by real-time PCR.
Luciferase reporter assay
The luciferase PAK5/Vector pGL3-Basic and pGL3-ABCB were co-transfected into Hep3B and HepG2 cells that splited in the 48-well plates by using X-tremeGENE HP DNA Transfection Reagent (Roche, Indianapolis, IN, USA). Renilla luciferase activate as normalization. Relative luciferase activity was detected 48 h after transfection following manufacturer’s instructions (Promega, USA) using an Orion Microplate Luminometer (Berthold Detection System).
Xenograft transplantation model
The 4-6 weeks BALB/c female nude mice were customized by HFK Bioscience (Beijing, China) and randomized into four groups (n=6 for each):⑴ hypodermic inject 2×106 LV-ctrl-HepG2-R cells; ⑵hypodermic inject 2×106 LV-ctrl-HepG2-R cells and oral sorafenib 30 mg/kg, twice a day; ⑶ hypodermic inject 2×106 LV-mimics-HepG2-R cells and oral sorafenib 30 mg/kg, twice a day;⑷ hypodermic inject 2×106 LV-siPAK5-mimics-HepG2-R and oral sorafenib 30 mg/kg, twice a day. 2 months thereafter, mice were sacrificed with the neoplasms for immunohistochemical staining. All animal experiments were in conformance with the ARRIVE (Animal Research: Reporting of In VivoExperiments) guidelines and in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals.
Statistical analysis
Statistical analysis was performed by SPSS 21.0 software (SPSS, USA), and images were acquired with GraphPad Prism 5 software (La Jolla, USA). The significance of the differences between the groups was evaluated by paired two-tailed Student’s t-test or one-way analysis of variance (ANOVA). All results that represented were from at least three independent experiments. Quantitative RT-PCR, luciferase reporter and cell proliferation assays were performed with triplicate duplications. Data represent the mean ± standard deviation (SD). Differences were considered statistically significant when P<0.05 (* P<0.05, ** P<0.01, *** P<0.001).