VCAN expression level analysis
TCGAportal (www.tcgaportal.org) was used to study the expression of VCAN in different tumor tissues and corresponding paracancerous tissues. Human protein map (https://www.proteinatlas.org/) database contains pathological and genetic information from many reports from a variety of tissues and cells. We used it to detect the expression of VCAN in different tissues and the localization of VCAN mRNA in cells. Next, we used UALCAN (http://ualcan.path.uab.edu/) to compare the expression of VCAN in patients with HCC of different races, ages, and histological subtypes. Finally, the significance of the observed difference was evaluated by Wilcoxon rank sum test.
Relapse and survival analysis
KaplanMeier Plotter (http://kmplot.com/analysis/index.php?p=background) is a free online database, built by using gene expression data and survival data from a variety of cancers patients including HCC(17-19). We used this online database to explore the relationship between the expression of VCAN and OS and RFS of patients with HCC. Kaplan-Meier survival plots were used to compare OS and RFS in HCC patients with high VCAN expression and those with low VCAN expression, and 95% confidence interval hazard ratios and log-rank P values were calculated.
The Cistrome DB Toolkit database (http://dbtoolkit.cistrome.org) is a resource of human and mouse cis-regulatory information, including about 47,000 human and mouse samples with about 24,000 newly collected data sets compared with two years ago. User can use this database to search for TFs related to the regulation of target genes in order to identify binding factors, histone modifications, and chromatin accessibility in a genomic interval of interest up to 2 Mb in length. Once users provide the overlap with the particular genomic interval sets, the similar ATAC-seq, DNase-seq, and ChIP-seq samples can be determined(20,21). We used the Cistrome Database Toolkit to search for TFs that were most likely to increase VCAN expression.
DNA methylation modification analysis
MEXPRESS (https://mexpress.be/), a user-friendly database tool for the the visualization and interpretation of TCGA data, can be used to study TCGA expression, DNA methylation status and clinical data and the relationships between them(22). In this research, we use this database tool to study the methylation status of VCAN mRNA and the relationships between VCAN mRNA expression and different clinical characteristics in HCC patients.
Gene correlations analysis
GEPIA2 (http://gepia2.cancer-pku.cn/#index), an openaccess dataset, can be used to study RNA sequencing expression data from 9,736 tumors and 8,587 normal samples drived from the TCGA and GTEx projects. The dataset provides tumor/normal differential expression analysis, profiling according to cancer types or pathological stages, patient survival analysis, similar gene detection, correlation analysis, and dimensionality reduction analysis. In this study, we used GEPIA2 to synthetically analyzed the correlations between all important genes.
Identification of miRNAs and circRNAs that target VCAN
TargetScanHuman (http://www.target scan.org/vert_71/) has an ability of searching for the presence of conserved 8mer, 7mer, and 6mer sites that match the seed region of each miRNA to predict biological targets of miRNAs (Lewis et al., 2005). starBase v3.0 (http://starbase.sysu.edu.cn/index.php), an open-source platform for the identification of the interactions between miRNA to lncRNA, RBP to lncRNA, miRNA to mRNA, RNA to RNA, ncRNA to RNA,and RBP to mRNA from CLIP-seq, degradome-seq, and RNA-RNA interactome data. These two databases was used to confirm the potentialable miRNAs that bind to VCAN mRNA. In addition, starBase v3.0 was used to perform circRNA prediction, miRNA survival analysis, and analysis of correlations between miRNAs and VCAN mRNA.
Protein-protein interaction and functional enrichment analysis
Metascape (http://metascape.org/gp/index.html#/main/ step1), a web portal, combines 40 independent knowledgebases’s functional enrichment, interactome analysis, gene annotation, and membership search. It promotes comparative analysis of multiple independent and orthogonal experiments across datasets(23). STRING (https://string-db.org/cgi/input.pl) is a database that you can use to search for protein-protein interactions you are interested in, including direct (physical) and indirect (functional) connections; The conclusions obtained are comprehensively calculated and predicted, knowledge transfer between organisms and interactions summarized in other (main) databases(24). We use STRING to create an interaction network between VCAN and other important proteins.
DISIDB (http://cis.hku.hk/TISIDB/index.php) is a web portal. Multiple heterogeneous data types were integrated to analyze the interaction between tumor and immune system in this web portal(25). We use it to analyze the spearman correlations between VCAN expression and immunostimulator, immunoinhibitors, and lymphocyte across human cancers
Cells culture and transfection
We used RPMI 1640 medium, contains 10% fetal bovine serum, to cultivate YY-8103 and LM3 cells lines in 5% CO2 incubator with penicillin(100 IU/mL) and streptomycin(100 mg/mL). The small interfering RNA target to VCAN (si-VCAN) and untargeted control siRNA were producted by HongXin conmpany in Nanjing China. We used the solution manufacturing by Applied biological materials company(Canada) and the Opti-MEM (Gibco, USA) to transfer. We mined the data use the cells after transfection for 48 hours. The target sequence of si-VCAN we obtainedis as follows: si-RNA1: 5‘-GGAAAUGGAAGAUGGCUATT-3’; si-RNA2: 5‘-GGAAAGAUUGAAAGAGATT-3’; si-RNA3: 5‘-GGAUAGGCCUCAAUGACAATT-3’.
Cell proliferation experiments
In CCK8 experiment, we firstly transfected the YY-8103 and LM3 cells line and incubated at 37℃. Then put the CCK8 solution (Biosharp, China) into each hole and incubate for two hours. The absorbance was detected on 450nm at 0, 24, 48, 72 hours. We did all the experiments three times.
Transwell migration and invasion assays
In accordance with the manufacturer instructions, we vaccinated the YY-8103 and LM3 cells line at the upper chamber, and the culture was performed on 200 microliter serum-free 1640 medium. The matrigel mix (BD Biosciences, San Jose, CA, USA) covers transwell chamber (Corning, NY, USA) so that the invasion test can be realized and the matrigel mix is not needed for migration experiment. The HCC cell chemical inducers made by RPMI 1640 medium and 10% FBS was lured to the bottom of the chamber. Incubation for 24 hours, we fixed the colour of upper chamber. Then crystal violet (Kaigen, Nanjing, China) was uesd to dyeing for 15 minutes. We photographed and counted the cells in five fields in order to implement visualization.
Wound healing assay
After culture on a six-well plate，we transfected YY-8103 and LM3. The artificial linear wound in monolayer fused cell was removed by the standard 20μl pipetting device. The free floating cells and debris in well bottom was removed slowly. Inject it into the medium and put the well in an incubator to incubate at 37 degrees celsius. The width of scratch clearance was recorded by inverted microscope and taking pictures at 0, 24, 48 hours. The difference between the width of original wound and the width of process of quantitative cell migration was done three times.
The SPSS 25.0 (IBM, SPSS, and Chicago, IL, USA) and GraphPad Prism 8 were used to analyse the data. We think the data had statistical significance when the p value less than 0.05. Independent t-test was used to compare continuous information between the two groups. Besides, we used chi-square test to dive into categorized data. Corresponding significance level was showed in those figures.