Human Clinical specimen:
Between 2016 and 2020, a total 40 pairs of the hepatoblastoma tissue including the adjacent matching normal hepatic tissue were obtained from pediatric patients who underwent hepatic surgery in Shanghai Tenth Peoples Hospital, China in between. Table 1 shows the clinicopathological characteristics of individual patients in detail. This study was sanctioned by the Institutional Research Ethical Review Committee. None of the patients undertaken in this study had never received the radiotherapy and chemotherapy. A verbal and written consents were obtained from all the pediatric patient’s parents prior to collection of human clinical sample for research.
Hepatoblastoma Cell lines cultures:
ATCC Hepatoblastoma cell lines (HUH6 & HepG2) and normal hepatic cell line (QSG7701) were obtained from the Shanghai Chinese Academy of Cell Collection. HUH6, HepG2 and QSG-7701 cells were cultured DMEM, Minimum Essential Medium (MEM) and RPMI 1640 respectively, incorporated with 10% FBS and 100U/ml Penicillin G/Streptomycin. These cell lines were incubated at 370c in 5% CO2 incubator. Culture medium (DMEM, MEM & RPMI) and FBS were purchased from Gibico (Grand Island, NY, USA).
Hepatoblastoma (HUH6 and HepG2) cell lines transfections:
For transient hepatoblastoma cell lines transfection three candidates of si-SNHG9 were chemically synthesized by Gene Pharma (Shanghai). Meanwhile, for the stable transfection pLvx-SNHG9-shRNA was synthesized from Keli Biotechnology (Shanghai, China). In additional miR23a-5p mimic, miR23a-5p inhibitor and matched negative controls (miR-NC) were synthesized by Shanghai Gene Pharma Inc (Shanghai China). HB cell transfection was carried out according to manufactured instructions. Briefly, the 2.5 x 105 hepatoblastoma cell (HUH6 & HepG2) cells are seeded on 6-well plate and incubated for 18 – 24 h at 370c to allow 30 – 40% confluency growth. On the 2nd day old cultured medium from 6 well plate was pipetted out and washed with 1x ice cold PBS. Next, 1000 µl of Serum free Opti-MEM is added to each well. 200µl of siRNAs/miRNA mimics/inhibitors-Lipofectamine 2000 mixture was prepared by adding 2µg (6µl 2 OD) of siRNA/miRNA mimics/inhibitors and 4µl of Lipofectamine-2000 in200µl of serum free OPTI-MEM medium and to respective 6-well plate and was incubated 4 – 6 h 370c. After 4 – 6 h OPTI-MEM medium was substituted with DMEM/MEM medium and incubated at 370c for 48 – 72 h. The knockdown efficacy of siRNA/miRNA mimics/inhibitors was validated by performing the qRT-PCR from the total mRNA extracted from transfected HB cell lines. The sequences of SNHG9 siRNA, hsa-miR-23a-5p/mimics/inhibitors are enlisted in Supplementary file1.
Total mRNA isolation and Quantitative real-time PCR (qRT-PCR):
Total mRNA was isolated from the hepatoblastoma tissues and cell lines using the Trizol regent (Invitrogen), as per the manufacturer`s guidelines. The Prime Script RT reagent kit (Takara, Dalian, P.R China) is then used to reverse-transcribe the mRNA into cDNA. Meanwhile, for the microRNAs, mRNA was reversed transcribed in cDNA using miRNA-specific loop RT primer synthesized by Ribobio (Guangzhou, China). cDNA of specific target gene (SNHG9, Wnt3a c-MYC, β-catenin and miR23a-5p) was amplified using SYBR Premix Ex Taq II (Takara Biotechnology, China) on qRT-PCR ABI Prism 7500 machine (Applied Biosystems, Thermo scientific). GAPDH and U6 are used as the internal control. Differential expression of target genes was calculated by 2-∆∆CT method. The primers sequence of the target genes is enlisted in supplementary file 1.
Proteins extraction and Western SDS-PAGE electrophoresis:
Protein from the stably/transiently knocked from hepatoblastoma cell lines (HUH6 & HepG2 cells) was extracted using the RIPA lysis buffer (Biyuntin, China) with the protease inhibitor (PI) cocktail (Cell Signaling Technology, USA) and phenylmethanesulfonylfluoride (PMSF) (Biyuntin, China). The BCA kit (Biyuntin, China) was used to determine the protein concentration and denatured at 1000c for 10 mins. 40 – 80 µg denaturated total protein sample was separated on 10 -12% SDS-PAGE. Separated protein were blotted into the nitrocellulose membrane. Nitrocellulose membrane were then transferred to 5%BSA blocking solution for 60 min and subsequently the nitrocellulose membrane was incubated with specific primary antibodies Wnt3a (ab2194120, β-catenin (ab32572), β-Actin(ab170325) (1:5000), C-Myc (ab32072), Survivin (ab76424), Blc-2 (ab182858), Bax (ab32503), Cleaved Caspase -3 (E83-77), Cleaved Caspase-9 (ab2324) at 40c for overnight.
Following the overnight incubation, the nitrocellulose membrane was rinsed four times with PBST and then incubated with secondary, HRP- conjugated goat anti-rabbit antibody (ab6721) at room temperature for 90 -120 mins. Next, nitrocellulose membrane is treated with ECL western blotting substrates and the chemiluminescence’s signal generated from nitrocellulose membrane was detected using AmershamTM A600 chemiluminescence film scanner (GE Healthcare Life Sciences). Beta actin was used as internal control for the validation of protein loading samples. All the antibodies related to gene of interest were purchased from Abcam.
Cell proliferation test:
Cell proliferation activity of hepatoblastoma cell (HUH6 & HepG2) after the subsequent transfection with SNHG9 siRNA/miR23a-5pmimics/inhibitors/SNHG9 OE plasmid was assed by CCK-8 assay (CCK-8, Biyuntian, China) assay. Hepatoblastoma cell lines (HUH6 & HepG2 cell) transfected with si/shRNA/miRNA mimics/inhibitors were seeded t a density of 1x103 cells/well in 96 wells plate and incubated humidified 5% Co2 incubator at 370c for 5 days. 10µl of CCK8 solution (Biyuntian, China) was added to each well and the absorbance of the colorimetric reaction of successive 5 days was determined using BioTek multi-mode microplate reader (BioTek, USA). Cell proliferation activity was normalized with zero hours’ time absorbance. Cell viability was calculated and plotted using Graph Prism. Each experiment was repeated thrice.
Clonogenic Assay:
Transfected hepatoblastoma (HUH6 & HepG2) cells were plated at the density of 1 x 103 per wells into a 6-well plate and incubated for 14 days at 370c. Briefly, following the 14 days incubation, the culture medium from the 6-well plates pipetted out and washed with 1x PBS. In 6 well plate cell colonies were fixed with 4% paraformaldehyde, then wash with PBS before staining with 0.05% crystal violet. Eventually, the crystal violet from 6 well plate is removed and washed with tap water and allow the plate to dry. Using the Image J software, the number of colonies in each well were counted and presented in bar chart using the Graph prism.
Flow Cytometric analysis for Apoptosis Assay:
To determine the Cellular apoptosis activity of transfected hepatoblastoma (HUH6 & HePG2) cell we utilized the Annexin V fluorescein isothiocyanate (FITC)/ propidium iodide double staining Apoptosis Detection Kit. After the trypsinization, transfected HB cells were collected in tubes and centrifuged at 1000 RPM for 5 mins. The cells pellet collected on bottom of tube was washed twice with ice cold 1x PBs before being suspended in Annexin binding buffer. The cell suspension was then distributed distinct tube at the density of 1x105 cells/tube followed by the double staining solution. Initially, cells were stained with Annexin-V FITC for 15 minutes and then, with propidium iodide (PI) for 5 minutes. Eventually, flow cytometry (BD Biosciences company, USA) was utilized to detect the apoptotic cell. The Flow Jo Software was used to calculate the percentage of the cellular apoptosis. Each experiment was performed in triplicate.
Isolation of Cytoplasmic and Nuclear RNA:
The Ambion PARIS Kits (Invitrogen, NY, USA) has been used for the isolation of cytoplasmic and nuclear fractional RNA from hepatoblastoma mammalian cell lines (HUH6 & HepG2). The relative concentration/fractional distribution of SNHG9, U6, 18s and GAPDH was calculated based on qRT-PCR findings. U6, 18s and GAPDH were used as nuclear and cytoplasmic control transcript.
RNA Immunoprecipitation Assay (RIP) Assay:
The EZMagnna RNA-bindings protein immunoprecipitation kit (Millipore, MA, USA) was utilized to validate the interaction between the SNHG9 and miR23a-5p and was performed in accordance with manufactured guidelines. In brief, pCDNA-SNHG9 or miR23a-5p mimics transfected HUH6 and HepG2 cells were plated in 6 well plate and incubated 48 h. After 48 h of transfection, HB cell lysate was obtained after the subsequent treatment of HUH6 and HepG2 cell with RIP lysis buffer. Magnetics beads coated with Ago2 antibody (Millipore’s, USA) or anti rabbit IgG (Milipore`s, USA) antibody mixed with cell lysate buffers and incubated for 6 h at 40c. After the 6 h immunoprecipitated RNA was extracted with the subsequent elution of protein beads. Eventually, qRT-PCR was performed to analyze the extracted precipitated RNA.
Biotin Pulldown Assay:
Biotin label antisense and sense SNHG9 RNA and DNA probes has designed, synthesized and purchased from Sangon Biotech (Shanghai, China). Hepatoblastoma (HUH6) cell lysate were mixed with the biotinylated SNHG9 RNA/ DNA probes and was incubated approximately at 250c for 1h. The streptavidin-agarose beads (Invitrogen) were mixed to mixture to elute the biotin-coupled RNA complexes. Eventually, qRT-PCR was performed to assess the abundance of SNHG9 and has-miR23a-5p in pull-down materials.
Xenograft Tumors:
The vivo animal tumorigenicity experiment was performed to validate the oncogenic potential of SNHG9. 4 weeks old BALB/c nude mice of was used for this experiment. BALB/c nude mice were purchased and randomly classified into two major groups Lvsh-NC and lv-shSNHG9. Each group consisting of 6 BLAB/C nude mice. For the tumorgenicity assay, lvsh-NC and lv-shSNHG9 transfected HUH6 were collected, centrifuged and resuspended in ice cool 1X PBS. And then, 5x106 of lv-shNC and lv-shSNHG9 transfected HUH6 were injected subcutaneously into the posterior flank of BALB/c nude mice. After six days of injection of HUH6 cell suspension in mice the tumors growth on mice was observed and evaluated in every three days. The volume of the tumors was measured using an equation V=0.5 × D × d2 where V= volume, D is the longitudinal diameter and d is latitudinal diameter of tumors). The mice were killed by cervical dislocations after 21 days and the weight of tumors and volume of tumor is measured. All the animal experiment was carried out in compliance to the NIH guidelines for the care and use of laboratory animals.
2.7.1 Bioinformatics analysis:
Similarly, we utilized the RegRNA 2.0 (http://regrna2.mbc.nctu.edu.tw/detection.html) database to identify the possible miRNA binding to SNHG9. Similarly, we utilized the RNA hybrid database to possible binding site of SNHG9 to miR-23a-5p. Similarly, we utilized the Targets (http://www.targetscan.org/vert_72) can database to identify the miR-23a-5p targeting genes. Similarly, we utilized the Starbase (http://starbase.sysu.edu.cn/) and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) to identify the binding sites of miR-23a-5p in Wnt3a target gene.
Dual Luciferase Assay:
The putative target binding site of miR23a-5p on SNHG9 and Wnt3a wild and mutant variants were cloned into pmirGLO(PsiCheck2) firefly luciferase vectors (Promega, Madison WI, USA). HUH6 and HepG2 cells were seeded at the density of 1×105 cells in 12 well pate and incubated at 370c for 24 h. SNHG9 {SNHG9-Wt & SNHG9-Mut} and Wnt3a [Wnt3a-WT & Wnt3a-Mut] constructed vector was co-transfected into HepG2 and HUH6 cells with miR23a-5p mimics/inhibitors using the Lipofectamine 2000 (Invitrogen, USA). Forty-eight hours after the post transfection, the HepG2 and HUH6 cell was lysed using passive lysis buffer. The cell lysate was then collected and centrifuged. And eventually used to measured luciferase activity. Luciferase activity was determined using the Dual-Luciferase Reporter Assay system (Promega, China).
Statistical analysis:
All the finding of this study was presented in mean ±SD. Statistical analysis was conducted using SPSS version 16.0 (IBM, NY, USA) and GraphPad prism version 8.0 (GraphPad Software, La Jolla, CA). Student`s t-test and one-way ANOVA was used to calculate and evaluate the two and more groups. Spearman`s rank correlation coefficient test was used to determine the correlation between two groups. Study finding with p value ≤ 0.05 was defined as statistically significance.