Cell culture
Human hepatoma cell line, HepG2, obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in RPMI-1640 (Invitrogen, CA, USA) supplemented with 10% Fetal bovine calf serum (FBS, Invitrogen, CA, USA). 293TN cells, purchased from ATCC (MD, USA), were maintained in Dulbecco minimum essential medium (DMEM, Invitrogen CA, USA) supplemented with 10% FBS. All these adherent cells were passaged by 0.25% trypsin digestion (Invitrogen CA, USA) and incubated in an atmosphere of 5% CO2 at 37 °C.
Assessment of lncRNA-CR594175, hsa-miR142-3p, CTNNB1 protein and Wnt pathway related protein expression levels in HCC tumors and their metastasis
Adjacent non-cancerous tissues, HCC, the metastatic from 24 patients (diagnosed in the First Affiliated Hospital of Zhengzhou University and detailed patient information was shown in Tab.1) were collected, followed by total RNA extraction and Quantitative Real-time PCR (RT-qPCR) for measurement of lncRNA-CR594175 and hsa-miR142-3p level and total proteins were extracted and used for CTNNB1 and Wnt pathway related proteins (E-cadherin, C-myc, CyclinD1 and MMP-9) detection by western blotting.
Lentivirus packaging
A siRNA sequence complementarily binding to lncRNA-CR594175 was chosen. The target sequences of siRNA (5’-GAATCCTCGGAGACAGCAG-3’) are homologous to lncRNA-CR594175.The oligonucleotide templates of these shRNAs were chemically synthesized and cloned into the linear pSIH1-H1-copGFP shRNA Vector (System Biosciences, CA, USA) which was obtained through digestion by BamH I and EcoR I (Takara, Dalian, China) and purification by agarose gel electrophoresis. An invalid siRNA sequence (5’- AATCGTCGAGGGCCAGACA-3’) was used as a negative control (NC). Sequencing was used to confirm the vectors constructed (pSIH1-shRNA-CR594175 and pSIH1-NC). The CDS sequence of human CTNNB1 (NM_001904.3) was amplified by using the primers 5’-GGAATTCGCCACCATGGCTACTCAAGCTGATTTG-3’ and 5’-CGGGATCC TTACAGGTCAGTATCAAACC-3’, which contain an EcoRI cutting site and Kozak sequence and a BamhI cutting site, respectively, with the cDNA prepared by reverse transcription of RNA isolated from 293TN cells. The PCR product was digested and cloned into pcDH1-CMV lentiviral expressing vector; the recombinant vector was named pcDH1-CTNNB1. The products of the vectors were confirmed by DNA sequencing. Endotoxin free DNA was prepared in all cases.
One day before transfection, 293TN cells were seeded into 10-cm dishes (Corning, NY, USA). 2 μg of each pSIH1-shRNA-CR594175 vector or pSIH1-NC and 10 μg pPACK Packaging Plasmid Mix (System Biosciences) were co-transfected using Lipofectamine 2000(Invitrogen) in accordance with the manufacturer’s protocol. The medium was replaced with DMEM plus 1% FBS. Forty eight hours later, the supernatant was harvested and then cleared by centrifugation at 5000×g at 4 °C for 5 minutes, and passed through a 0.45 µm PVDF membrane (Millipore, MI, USA). The titer of virus was determined by gradient dilution. The packaged lentiviruses were named as Lv-shRNA-CR594175 and Lv-NC. Recombinant lentivirus Lv-CTNNB1 and Lv-miR142-3p were packaged by following the same protocol.
Genetic intervention through a lentiviral approach
Cells were divided into four groups: a control group, Lv-NC group (infected with Lv-NC), Lv-shRNA-CR594175 group (infected with Lv-shRNA-CR594175) and Lv-CTNNB1 group (infected with Lv- CTNNB1). HepG2 in logarithmic phase growth were seeded to 6-well plates at 5 × 105 cells/well. One day later, viral solution was added at a multiplicity of infection (MOI) of 10. The infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 hours after infection. Total RNA and protein were isolated from the cells and subjected to RT-qPCR and western blotting for lncRNA-CR594175 and CTNNB1 protein.
Luciferase experiment
Total RNA was extracted from HepG2, reverse-transcribed into cDNA, and 2 μl of the reaction product subsequently used as a template for PCR. Primers targeting the 3′-UTR of the CTNNB1 gene were designed such that flanking XbaI restriction sites were introduced into the 127 bp (base-pair) PCR product containing the 5′-AACACTA-3′ hsa-miR-142-3p target site. The forward and reverse primer sequences were 5′- GCTCTAGATTAAGAATTGAGTAATGG-3′ and 5′-GCTCTAGA ACTAATTGGACCATTTTC-3′, respectively. PCR reaction conditions were as follows: 35 cycles of a 94℃ denaturing step for 30 s, a 55℃ annealing step for 30 s, and a 72℃
elongation step for 10 s. The PCR product was digested with XbaI (Takara) and cloned into the pGL3-promoter luciferase reporter vector (Promega, MI, USA) to generate the vector pGL3-wt-CTNNB1. The hsa-miR142-3p target site in the pGL3-WT-CTNNB1 vector was mutated from 5′- AACACTA -3′ to 5′- CATAACA -3′ to construct the mutated reporter vector, pGL3-mt-CTNNB1. The products of all cloning and mutagenesis reactions were confirmed by DNA sequencing. Endotoxin free DNA was prepared in all cases. The hsa-miR142-3p mimic(5'-UGUAGUGUUUCCUACUUUAUGGAtt-3'), the hsa-miR142-3p inhibitor (5'- UCCAUAAAGUAGGAAACACUACAtt-3), and negative control miRNA (NC,5'- UGUAGUGUUUCCUACUUUAUGGAtt-3') were all chemically synthesized (Invitrogen).
We used Targetscan (http://www.targetscan.org/) to predict whether a hsa-miR142-3p binding site exists within the 3′-UTR of human CTNNB1 mRNA. The results showed that a seven-base hsa-miR142-3p seed sequence is present in the 3′-UTR of CTNNB1 mRNA. The same tool was used to predict the binding sites of hsa-miR142-3p on lncRNA-CR594175.A suspension of 293TN cells in logarithmic phase growth was prepared and the number of viable cells counted using a hemocytometer in conjunction with trypan blue staining. The cells were seeded into six-well plates at a concentration of 2×105 cells per well and maintained in Dulbecco’s Modified Eagle’s medium supplemented with 10 % FBS at 37℃ for 24 hours in a 5 % CO2 atmosphere. The transfection of plasmid DNA and RNA was performed using Lipofectamine 2000 (Invitrogen). Transfection of cells with pRL-TK (100 ng) served as a reference for luciferase detection. Luciferase activity was measured using the dual luciferase reporter assay system (Promega) 48 hours after transfection. The experiment to observe the effect of lncRNA-CR594175 depletion on the inhibition of luciferase by hsa-miR142-3p mimics was carried out in 293TN and HepG2 cells ; the plasmid transfection and luciferase activity assay were the same as used in the validation of target sites of hsa-miR142-3p.
Cellular proliferation assay
HepG2 cells were divided into seven groups: a control group, Lv-NC group, Lv-shRNA-CR594175 group, Lv-miR142-3p group, Lv-shRNA-CR594175 and Lv-miR142-3p group, Lv-CTNNB1 group, and Lv-shRNA-CR594175 and Lv-CTNNB1 group. Fourty-eight hours after infection, HepG2 cells groups were trypsinized, and seeded into 96-well plates at a density of 1×104 cells per well. The cells were cultured under normal conditions and cell viability was examined using CCK-8 at 24-, 48-, and 72-hour time points. Briefly, 10 uL CCK-8 solution (Dojindo, Japan) was added, and the cells then cultured under normal conditions for an additional 4 hours before measurement of absorbance at 490 nm.
Cell invasion assay
Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (Chemicon, International, MI, USA) according to the manufacturer′s instructions. The kit uses an insert polycarbonate membrane with an 8-μm pore size. The insert was coated with a thin layer of EC Matrix that occluded the membrane pores and blocked migration of non-invasive cells. Culture medium (500 μl) supplemented with 10% FBS was used as chemoattractant. Cells that migrated and invaded the underside of the membrane were fixed in 4% paraformaldehyde. The invading cells were stained by Calcein-AM, and the number was then determined by fluorescence and reported as relative fluorescence units (RFUs).
Effect of lncRNA-CR594175 silencing on the protein levels of CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9
HepG2 cells were divided into three groups: a control group, Lv-NC group and Lv-shRNA-CR594175. Cells in logarithmic phase were seeded to 6-well plates at 5x105 cells/well. One day later, viral solution was added and the infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 hours after infection. Proteins were isolated and subjected to western blotting for CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9 protein, respectively.
RT-qPCR
Total RNA was isolated with Trizol Reagent (Invitrogen) according to the manufacturer’s instruction and reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Takara, Japan) and oligo(dT)18 primer (Takara, Dalian, China). The following specific primers were used in RT-qPCR of human lncRNA-CR594175 and β-actin: lncRNA-CR594175: forward 5’-TTATGACACATGCCACAACA-3’ and reverse 5’-GGTACCTGTTATAAGTAGAATCA-3’; β-actin: forward5’-CCTGTACGCCAACACAGTGC-3’ and reverse
5’-ATACTCCTGCTTGCTGATCC-3’. The lengths of amplified products were 109 bp and 211 bp, respectively. RT-qPCR was performed using SYBR Premix Ex Taq kit and TP800 System (Takara). cDNA from 200 ng total RNA was used as the template. The PCR reaction was carried out under the following conditions: 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 20 s. The relative levels of mRNA and hsa-miR-142-3p were normalized using the 2-△△Ct method by using β-actin and U6 as the references. The PCR primers for hsa-miR142-3p or U6 were as follows: hsa-miR142-3p: forward: 5′- TGTAGTGTTTCCTACTTTATGGA-3′ and reverse: 5′-GTCGTATCCAGTGCGTGTCGTG-3′; U6: forward:5′-GTGCTCGCTTCGGCAGCACAT-3′ and reverse: 5′-TACCTTGCGAAGTGCTTAAAC-3′.
Western blotting
The total protein was extracted from the cells and tissues by using
mammalian protein extraction reagent (Pierce, IL, USA). Equal amounts of protein (25 μg per lane) estimated by a bicinchoninic acid (BCA) protein assay kit (Pierce) were loaded onto (11%) SDS-PAGE gels and transferred onto nitrocellulose membranes. The blots were probed with a monoclonal antibody against human CTNNB1 (1:400), E-cadherin (1:200), C-myc (1:300), CyclinD1 (1:400)，MMP-9 (1:250) and β-actin (1:1000) (Santa Cruz, USA), followed by the secondary HRP-conjugated anti-mouse/rabbit antibody (Abcam, Cambridge, UK). After washing, the bands were detected by chemiluminescence and imaged with X-ray films. β-actin was used as an endogenous reference for normalization.
Animal xenografts
Nude mice were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China) and housed at the animal experiment center of Zhengzhou University, where the implantation experiment was performed. All the protocols were previously approved by the Zhengzhou University Animal Ethics Committee. HepG2 cells (1×106) were suspended in 200 μl medium, and injected subcutaneously into the flank regions of 48 female athymic nude mice. Two weeks after inoculation, visible subcutaneous tumors were detected, and the tumors were measured approximately 2.5 mm in diameter 3 weeks after inoculation. All animals were randomly divided into 3 groups (8 mice per group): the Model group, the NC group, and the lncRNA-CR594715-silencing group. For the intervention groups, each animal received 30 μl recombinant lentivirus (1×108 IFU) twice a week since the second week for 4 weeks, while the model group received the same volume of saline instead. Tumor diameter was measured weekly since the second week, and the data was used to plot the tumor growth curves. The formula for calculating the tumor volume was: V=1/2 a×b2, a and b are the long and short diameters of the tumor, respectively.
Statistical analysis
All data are expressed as mean ± SD, and analyzed by one way ANOVA. Least Significant Difference (LSD) was used for multiple comparisons between any two means. P-values < 0.05 were considered statistically significant. All statistical analysis was performed using SPSS 13.0 software.