Cell culture
HCC cell lines 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% FBS (Fetal bovine calf serum, Invitrogen). 293TN cells, purchased from ATCC (MD, USA), were maintained in DMEM (Dulbecco minimum essential medium, Invitrogen) supplemented with 10% FBS. All these adherent cells were passaged by 0.25% trypsin digestion (Invitrogen) and incubated in an atmosphere of 5% CO2 at 37 °C.
Assessment of CR594175, hsa-miR142-3p, CTNNB1 protein and Wnt pathway related proteins in HCC tumors and its metastatic tissues
HCC, the metastatic tissues and para-carcinoma tissues 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 real-time PCR for measurement of CR594175 and hsa-miR142-3p and total protein extraction and western blotting for CTNNB1 and Wnt pathway related proteins (E-cadherin, C-myc, CyclinD1 and MMP-9).
Lentivirus packaging
A siRNA sequence complementarily binding to CR594175 was chosen. The target sequences of siRNA (5’-GAATCCTCGGAGACAGCAG-3’) are homologous to CR594175, respectively. 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 NC (negative control). 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 was packaged by the same way.
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 were seeded to 6-well plates at 5 × 105 cells/well. One day later, viral solution was added at an MOI of 10. The infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 hours after infection. And total RNA and protein were isolated from the cells and subjected to real-time PCR and western blotting for CR594175 and CTNNB1 protein, respectively.
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 were designed that targeted the 3′-UTR of the CTNNB1 gene such that flanking XbaI restriction sites were introduced into the 127 bp(base-pair) PCR product containing the 5′-AACACTA-3′ hsa-miR142-3p 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 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 % fetal calf serum at 37℃ for 24 hours in a 5 % CO2 atmosphere. The transfection of plasmid DNA and RNA was performed using Lipofectamine 2000 (Invitrogen, USA). 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, USA) 48 hours after transfection. The experiment to observe the effect of CR594175 depletion on the inhibition of luciferase by hsa-miR142-3p mimics was carried out in HepG2 cells, the plasmid transfection and luciferase activity assay the same as used in validation of target site.
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
HepG2 cells infected with recombinant lentiviruses 72 hours, trypsinized and used for invasion assay. Cell invasion experiments were performed using the QCMTM 24-well Fluorimetric Cell Invasion Assay kit (ECM554, Chemicon International, 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 MatrixTM 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). The grouping and cell treatment were the same as those for cell viability assay. Seventy-two hours after lentiviral infection, cells were trypsinized and the viable cells were counted by using trypan blue staining, and seeded into the upper chamber of transwell at a density of 5 × 105cells/well, and incubated under normal conditions for 48 hours, and then stained and counted. The grouping was the same as in the proliferation assay.
Effect of CR594175 silencing on expression of β-catenin and downstream functional proteins of Wnt pathway
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 5×105 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. Protein were isolated and subjected to western blotting for CTNNB1, E-cadherin, C-myc, CyclinD1 and MMP-9 protein, respectively.
Real-Time PCR
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). The following specific primers were used in quantitative PCR of human CR594175 and β-actin: CR594175: 5’-ACATATAATTGAATATTATT-3’ and 5’-TTAGCCATTTTAAAATGTATGG-3’; and β-actin: 5’-CCTGTACGCCAACACAGTGC-3’ and 5’-ATACTCCTGCTTGCTGATCC-3’. The lengths of amplified products were 321 bp and 211 bp, respectively. Real-time PCR was performed using SYBR Premix Ex Taq™ kit and TP800 System (Takara BIO). cDNA from 200 ng total RNA was used as the template. The PCR reactions 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 mRNA levels of CTNNB1 were normalized using the delta-delta Ct method, to the expression of an endogenous housekeeping gene, β-actin. The expression of hsa-miR142-3p was analyzed with the 2-△△Ct method. For each sample, triplicate determinations were performed, and mean values were adopted for further calculations. All values were normalized to an endogenous U6 control. The PCR primers for mature hsa-miR142-3p or U6 were designed as follows: hsa-miR142-3p sense: 5′- TGTAGTGTTTCCTACTTTATGGA-3′ and reverse: 5′-GTCGTATCCAGTGCGTGTCGTG-3′; U6 sense: 5′-GTGCTCGCTTCGGCAGCACAT-3′ and reverse: 5′-TACCTTGCGAAGTGCTTAAAC-3′.
Detection of protein contents in cell or tissues
The total protein was extracted from the cells using M-PER mammalian protein extraction reagent (Pierce, IL, USA) or from tissues using T-PER tissue protein extraction reagent (Pierce). 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 the secondary HRP-conjugated anti-mouse/rabbit antibody (Abcam, CA, USA). 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 CR594175-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=ab2/2, a and b are the long and short diameters of the tumor, respectively.
Statistical analysis
All data is 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.