Data acquisition and preprocessing
Public high-throughput RNA microarray and RNA sequencing from HCC and adjacent non-HCC tissues were collected from public databases, including the Genotype-Tissue Expression (GTEx, https://gtexportal.org), the Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/) and Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih). The retrieval formula is: ((hepatocellular OR liver) AND (Neoplas* OR Tumo* OR Cancer OR Malignan*)) OR ((HCC) OR (Hepatocellular Carcinoma)). The inclusion criteria are as follows: (1) All the subjects are Homo sapiens; (2) The collected samples contain HCC tissues and non-HCC tissues; (3) The number of tumor and non-tumor samples ≥3; (4) PLIN1 expression levels were observed in the dataset. All extracted data were normalized and merged by log2 transformation. Furthermore, the remove BatchEffect() function from the “Limma” package was utilized for removing the batch effects among different platforms.
Clinical application potential of PLIN1
The standardized mean differences (SMD), receiver’s operating characteristics (ROC), Summarized receiver’s operating characteristics (SROC), and the pooled sensitivity and specificity were used to assess the clinical potential of PLIN1. Stata Version 12.0 was used to conduct the aforementioned analysis. The Kaplan-Meier survival curve was generated by the Gene Expression Profiling Interactive Analysis (GEPIA2, http://gepia2.cancer-pku.cn/#analysis) to provide insight into PLIN1's prognostic potential in HCC.
Sample and clinical pathological data collection
80 paraffin HCC specimens and 80 para-cancerous tissues were collected from the Department of Pathology, the First Affiliated Hospital of Guangxi Medical University for use in immunohistochemistry staining. Furthermore, 84 cases of HCC and 84 matching cases of para-cancerous tissues were taken from the sample bank of the first affiliated Hospital of Guangxi Medical University. The associated clinicopathological characteristics, such as tissue type, sex, age, microsatellite focus, alpha-fetoprotein content (alpha-fetoprotein, AFP), tumor thrombus, Edmenson grade, vascular invasion, tumor number, and extrahepatic metastasis, were also gathered from the medical records. All the subjects have provided appropriate informed consent. The First Affiliated Hospital Ethics Committee of Guangxi Medical University gave its approval to the project.
Cell culture
A hepatocyte (HL-7702, L-02) and human HCC cell lines (SK, Huh7) were acquired from the Chinese Academy of Sciences' Institute of Biological Sciences in Shanghai, China. The cell lines L-02, SK, and Huh7 were cultivated in DMEM media (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin combination (Solarbio, China) in a humidified incubator containing 5% CO2 at 37°C, and the cells were subcultured when they reached around 80% confluence. As soon as the cells entered the exponential growth phase, experiments were carried out.
Immunohistochemical staining
Following surgical resection, tissue samples were preserved for 24 hours in 10% neutral formalin, then dehydrated and embedded in paraffin. The paraffin-embedded tissues were then cut into 4µm sections in preparation for IHC staining. PLIN1 polyclonal antibody was purchased from Abcam (Cambridge, UK). The IHC staining procedure was performed as in previous studies[20, 21]. Under a microscope, 10 randomly chosen high-magnification fields were evaluated for the staining intensity and the percentage of positive cells. Based on the staining intensity, 0 indicates no staining, 1 indicates weak staining, 2 indicates moderate staining, and 3 indicates strong staining; According to the average percentage of positive cells, the scores were as follows: 0 (<5%), 1 (5%~25%), 2 (26%~50%), 3 (51%~75%), 4 (76%~100%). Total immunohistochemical staining score = staining intensity * percentage of positive cells, ≥6 was positive, and <6 was negative samples. Immunohistochemical results were determined independently by three senior pathologists.
RT-qPCR
The RNAeasy Plus Animal RNA Extraction Kit (Beyotime Biotechnology, China) centrifugal column was used to extract total RNA in accordance with its instructions. The RNA was reverse-transcribed following the instruction of Prime-Script RT Master Mix (TaKaRa, Japan). For RT-qPCR, the ABI7500 system (Thermo Fisher, USA) was utilised with TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Japan). ACTB functioned was used as the internal control. The relative expression of PLIN1 mRNA in each sample was calculated as 2-ΔΔCT. Primer Premier created the sequences of the specific primers utilized in this investigation, of which sequences are listed below:
H- ACTB -F: 5 ' - CCTGGCACCCAGCACAAT-3 '
H- ACTB -R: 5 ' - GGGCCGGACTCGTCATAC-3 '
H-PLIN1-F: 5 ' -GCA GCA TTG AGA AGG TGG TGG AG-3 '
H-PLIN1-R: 5 ' -ATC GAG AGA GGG TGT TGG TCA GAG-3 '
Lentivirus transfection
Lentiviral plasmids were purchased from Genechem (Shanghai, China). HCC cells were inoculated in 6-well plates and pre-cultured for 24h. Following the experimental guidelines, lentivirus was used to transfect SK and Huh7 cells when the cell fusion reached approximately 30%. The cells transfected with PLIN1 overexpression lentivirus were designated as the experimental group (SK-PLIN1, Huh7-PLIN1), while the cells transfected with negative lentivirus were designated as the control group (SK-NC, Huh7-NC). Puromycin was used to screen the cells that were transfection-positive.
Transwell cell migration and invasion detection
HCC cells in the logarithmic growth phase were gathered, including the cells stably transfected with PLIN1 overexpression lentivirus (SK-PLIN1, Huh7-PLIN1) and the corresponding negative control cells (SK-NC, Huh7-NC). To conduct the migration test, fill the 24-well plate with 500μl of 10% FBS-containing media, then insert the chamber into the plate. Next, each well was infected with 100µl of serum-free cell suspension containing 3×104 cells. For invasion assay, the transwell chamber was preincubated with Matrigel for 2 hours. Next, 100µl of serum-free cell suspension with a cell concentration of 4×104 was inoculated in each well, and 500µL of media containing 10% FBS was poured into each well of 24-well plates in the lower chamber. Afterwards, the chamber and 24-well plates were incubated in a humidified incubator at 37°C with 5% CO2 for 24 hours. The following day, the chamber was taken out, and any non-migrating or non-invading cells on the upper surface were carefully removed with a cotton swab. The cells that were migrating or invading were fixed for 30 minutes with methanol and stained for 30 minutes at room temperature using 0.3% crystal violet; the staining time could be extended based on the staining of the cells. Five visual fields were randomly selected under the microscope to observe and count the number of invasions and migrations.
Wound-healing assay
Each group of logarithmic growth HCC cells was seeded in 6-well plates (Corning, USA) with a density of 5×105/well for SK and 6×105/well for Huh7. Once the cells reach 90% fusion, use a sterile 200μl pipette tip to make a scratch on the bottom of the plate and replace it with a new medium devoid of serum to carry with the culture process. The ability to migrate was evaluated by examining the migration of cells to the scratched location, which was captured on a microscope at various times following the scratch. The software Image-J was utilized to determine the healing rate of each group.
Flow cytometric analysis of cell cycle
The cell cycle distribution of each group was examined using a cell cycle staining kit (Multi Sciences, China). For each group, 2×105–1×106 cells were collected, and 1 ml of PBS was added for resuspension. Then, the cells were gradually added to 3 ml of already chilled 75% ethanol and fixed at -20℃ overnight. On the day of detection, the fixed cells were centrifuged, the ethanol was disposed of, and the tube wall was gently bounced to release the precipitate. After adding 5 ml of PBS, the cells were left for 15 minutes to hydrate again. After adding 1 ml of the DNA staining solution, vortex for 5–10 seconds. Keep the inoculum dark and at room temperature for 30 minutes. Using a flow cytometer, choose the test with the lowest loading rate.
CCK 8 cell proliferation assay
The CCK-8 kit (Beyotime Biotechnology, China) was used to assess the vitality of the cells. HCC cells in the logarithmic phase were collected from each group and inoculated into 96-well plates with 100µl of serum-free cell suspension containing 4×104 cells. A second set of cell-free wells with only culture media was used as a blank control, with 4 replicates per group. The 96-well culture plate was cultivated in a constant temperature incubator with 5% CO2 at 37°C until the cells attached to the wall. Fill each well with 10μl of CCK-8 solution, then incubate for 1 hour at 37°C in an incubator. The absorbance of every hole was then measured using a Multiskan FC Microplate spectrophotometer at 450 nm. We created four time points: 24 hours, 48 hours, 72 hours, and 96 hours.
Functional enrichment analysis of DCGs
We used R software to screen for PLIN1 co-expressed genes using Pearson's correlation approach based on the previously combined data from various platforms. The screening criteria were correlation coefficient r > 0.7 and P value < 0.05. Simultaneously, the "Limma" package of R was utilized to determine the HCC differentially expressed genes (DEGs). Genes with |log2 (fold change) | >1 and Adjusted-P < 0.05 were recognized as DEGs of HCC. By intersecting the two genes, a set of overlapping DCGs can be obtained. The underlying functional pathways regulated by DCGs were found using the GO functional annotation and KEGG pathway analysis, which was carried out using the "clusterProfiler" package with a significance threshold set at P value < 0.05.
Statistical analysis
Under appropriate circumstances, the experiment was conducted independently 3 times. Data is displayed as mean ± standard deviation (M±SD). Student t-test was used to compare the means between two groups, and a one-way analysis of variance was used to compare the means between multiple groups. The χ2 test was used to examine the relationship between PLIN1 expression and clinicopathological characteristics. Kaplan-Meier method was employed for survival analysis, and the log-rank test was selected for verification. The ROC curve was generated to evaluate the utility of PLIN1 as a biomarker for HCC, and the area under the curve (AUC) was utilized to evaluate its accuracy. SPSS22.0 was used for most statistical analysis, and GraphPad Prism Version 8.0 was employed for plotting. All statistical P-values were based on two-tailed statistical tests, and P-value < 0.05 denotes statistical significance.