The TCGA (www.tcga-data.nci.nih.gov/tcga), which containing transcript expression data of LIHC and Tissue Expression of healthy tissue, was used. The Expression of multiple mRNAs was analyzed using above data set. Clinical data including survival information of LIHC patients from TCGA was obtained and its clinical correlations with TNRC6A and miR-652-3p were carried out. Genes were ranked by expression level of TNRC6A, followed by differential expression analysis and Gene Otology (GO) analysis.
Human tissue samples
With informed consent from all patients, the liver tumor tissues and its matched adjacent tissues of HCC patients, were collected from The First Affiliated Hospital of Chongqing Medical University. The ethical guidelines of the 1975 Declaration of Helsinki were followed and protocols were approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University in this study.
miR-652-3p and TNRC6A expression level and survival analysis
The data from TCGA database was used to investigate the expression of miR-652-3p and TNRC6A in HCC and corresponding para-carcinoma tissues. GEPIA database (http://gepia.cancer-pku.cn/) and Starbase database were used to investigate the expression of TNRC6A and miR-652-3p, respectively. The correlations between miRNA (including miR-652-3p) or TNRC6A expression and overall survival (OS), disease-free survival (DFS) were compared by Starbase database (http://starbase.sysu.edu.cn/) and Kaplan–Meier plotter (https://kmplot.com/analysis/), respectively. Kaplan–Meier survival plot was used to perform comparison in two groups.
Human hepatocellular carcinoma cell lines (HCC-LM3, Huh7) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology). All these cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS) (Moregate Biotech, Australia) at 37℃ in a humidified chamber with 5% CO2.
Plasmids, siRNA, miRNA mimics and inhibitors transfection
The miR-652-3p mimics and inhibitors used in this study were listed: The miR-652-3p mimics and miR-652-3p inhibitors (Ruibo biotechnology, Guangdong, China) were purchased from Ruibo bio-company. The mature sequence of has-miR-652-3p: AAUGGCGCCACUAGGGUUGUG, TNRC6A siRNA, and its negative control siRNA (Sunya, China) were purchased from Sunya Biotechnology Company. E2F1 overexpression plasmids (Repobio, China) were purchased from Repobio Company. According to the manufacturer's instructions, all mimics, inhibitors, and siRNA, plasmids were transfected into HCC cells using jetPRIME® transfection reagent (Polyplus Transfection, France).
Cell proliferation and colony formation assays
Transfected HCC cells (2×10^3 cells/well) were seeded into a 96-well plate and incubated overnight for the cell proliferation assay. According to the manufacturer's instructions, cell counting kit-8(CCK8) was used to test cell viability every 24 hours for 72 hours. As for colony formation assay, transfected cells (1000 cells/well) were seeded into 6-well plates and maintained in completed DMEM media with 10% FBS. The culture medium was changed every 3-4 days. After three weeks, the colonies were fixed in 4% Paraformaldehyde Fix Solution, stained with 0.1% crystal violet (Sigma), and the clone number was counted and recorded.
Cell cycle analysis
HCC cells (2x105) were digested and seeded into 6-well plate. After overnight incubation, cells were transfected with miR-652-3p mimics or negative control mimics and incubated for 48h. Cells were harvested and fixed in 75% ethanol at -20˚C for 24-48h. After fixation, PBS was used to wash cells one time. Then, 200μl DNA PREP Stain (Beckman Coulter, Inc., Brea, CA, USA) was used to resuspend cells, and the suspension was incubated in the darkroom for 30min at room temperature. BD LSR II instrument (BD Biosciences, San Jose, CA, USA) was used to perform cell cycle analysis. Flowjo software version 10 was used for further analysis.
The transfected HCC cells were seeded into a 6-well plate and incubated until about 100% confluence. Then, a pipette tip was used to scratch cell surface, and then phosphate-buffered saline (PBS) was used to remove cell debris. To evaluate the healing effect of cells with different treatments, cell images were observed and captured under a microscope at a particular time (the time for Huh7 and HCC-LM3 were 72h and 120h, respectively).
Migration and Invasion Assay
For migration assay, transfected HCC cells (5.0 x 104 cells per well) were digested and seeded into the upper chamber of the Transwell plate (24-well, eight μm pore size, Millipore, USA), cultured in 200μL DMEM without FBS. 800μL DMEM with 10% FBS were used to fulfill the lower chamber. Moreover, for invasion assay, before cell seeding, upper chambers were pre-coated with a 40μL coating medium, which was consisted of 32μL DMEM medium and 8μL Matrigel (BD Biosciences, USA) for 3 hours. Equal amounts of cells were seeded into the upper chamber, and the following were performed as described above. After a particular incubation time, cells were fixed by 4% paraformaldehyde and stained by 0.2% crystal violet, and the number of cells was calculated.
Western Blotting and antibodies
Cells were lysed in the RIPA Lysis buffer (Beyotime Biotechnology, China) containing Protease Inhibitor Cocktail (ThermoFisher, USA). Protein concentration was measured by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, USA). Western blots were performed as previously described . Antibodies for western blot were listed as below: N-Cadherin (13116T, CST, USA), E-Cadherin (3195T, CST, USA), Snail (3879T, CST, USA), GAPDH (10494-1-AP, protein tech, China), and TNRC6A (A6115, ABclonal Technology, China).
RNA extraction and quantitative real-time polymerase chain reaction
TRIzol reagent was used to extract total RNA. HiScript II Q RT SuperMix (Vazyme Biotech, Nanjing, China) and Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Kyoto, Japan) were used for mRNA and miRNA cDNA synthesis, respectively. Furthermore, miRNA reverse primers (universal primers) are also provided in the kit. For mRNA, SYBR Green (Vazyme Biotech, Nanjing, China) was used to perform a real-time quantitative polymerase chain reaction (q-PCR) to measure mRNA expression. The relative expression level of genes was normalized with GAPDH internal controls. For miRNA, TB Green® Premix Ex Taq™ (Tli RNase H Plus) (Takara, Kyoto, Japan) was used to perform qPCR to measure the expression of miRNA. The relative expression level of miRNA was normalized with U6 controls. All q-PCR was performed in triplicate on the Bio-Rad QX100 Droplet Digital PCR system (USA). All primers were obtained from Tsingke Biological Technology (Beijing, China).
Total RNA was extracted by using TRIzol reagent following the instruction of the manufacturer. And further mRNA sequencing was performed by GenePharma Company (GenePharma, China). Briefly, mRNA was purified from total RNA using magnetic beads with Oligo (dT) and fragmented into ~200 bp short fragments, and then cDNA libraries were constructed. RNA-seq was performed on the Illumina HiSeq 2000 platform according to the manufacturer's instructions, and reads were generated.
Luciferase reporter assay
A nucleotide sequence of TNRC6AmRNA 3ʹUTR containing the binding site for has-miR-652-3p seed sequence was synthesized and inserted to construct psi-TNRC6A-3UTR (WT). The mutation of the above nucleotide sequence lacking the seed sequence was inserted to construct the psi-TNRC6A-3UTR (MUT). Psi-TNRC6A-WT/MUT plasmids and miR-625-3p mimics/NC mimics were co-transfected into HCC-LM3 cells. After 24 hours, cells with different treatments were harvested and lysed by passive lysis buffer. A dual-luciferase reporter system (Promega, USA) using LB 960 Centro (Berthold) was used to measure luciferase activity. The luminescence intensity of Firefly luciferase was normalized to that of Renilla luciferase.
Chromatin Immunoprecipitation (ChIP) assay
The ChIP kit (9003, CST, USA) was purchased from CST company. According to the protocol from manufacturer, the ChIP grade antibody against E2F1 (66515-1-Ig, Proteintech, China) was used for ChIP assay. The ChIP assay was performed as previously described in our previous study .
Male nude mice (4-6 weeks old) were purchased from the Zhejiang Academy of Medical Sciences animal center and used in all experiments. All animal experiments were performed according to the authorized procedures and approved by the ethical committee of the Zhejiang Academy of Medical Sciences animal center. And all animal experiments were carried out conforming to the requirement of the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals, 2011). To detect the metastatic ability of HCC cells, 100ml PBS contained 1.0 x 106 HCC-LM3 cells treated with antigomiR-652-3p were injected into mice via tail vein to construct lung metastatic models. After that, TNRC6A siRNA or negative control siRNA (5nmol per mice) and polyplus transfection reagent (Polyplus Transfection, France) were injected through the tail vein to knockdown TNRC6A in vivo according to the manufacturer's instructions. After eight weeks, the lungs of mice were collected after euthanization, and lung metastatic nodules were calculated. To detect the EMT change in vivo, 100ml PBS contained 5.0 x 106 HCC-LM3 cells were injected subcutaneously into nude mice to construct HCC model. After that, TNRC6A siRNA or negative control siRNA (5nmol per mice) and polyplus transfection reagent (Polyplus Transfection, France) were injected into tumorsaccording to the manufacturer's instructions. After four weeks, tumors were harvested after euthanization, and IHC was performed.
The SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analyses. All data were presented as the mean ± standard deviation. A two-tailed Student's t-test was used to assess comparisons between two groups. The Kaplan-Meier method was used to assess the overall survival rate of patients. Statistically significant difference was considered when P<0.05.