EPRS1 correlates with malignant progression in hepatocellular carcinoma

Cell culture and transfection

Human HCC cell lines SNU-387, HepG2, Huh7, and Hep3B were obtained from the Cell Bank, Type Culture Collection, Chinese Academy of Science. Huh7 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). SNU387 cells were cultured in RPMI1640 medium supplemented with 10% FBS. HepG2 and Hep3B cells were grown in MEM supplemented with 10% FBS. Halofuginone (S8144) was purchased from Selleck. The use of mycoplasma-free cells was ensured throughout all experiments.

Cell transfection

Small interfering RNAs (siRNAs) targeting EPRS1 (siEPRS1#1 sense was GGAUGAUACUCCUGCUGAATT, antisense was UUCAGCAGGAGUAUCAUCCTT; siEPRS1#2 sense was CCUGACAACUCGAACUAUUTT, antisense was AAUAGUUCGAGUUGUCAGGTT) were ordered from GenePharma (Shanghai, China). Plasmid overexpressing EPRS1 (NM_004446) based on the GV219 vector was purchased from GeneChem. Lipofectamine 2000 and Lipofectamine 3000 (Invitrogen) transfection reagents were used to transfect siRNAs and plasmids following the manufacturer’s recommendations.

Real-time PCR

Total RNA was extracted using the E.N.Z.A. Total RNA Kit I (R6834, OMEGA) according to the manufacturer’s instructions. cDNA was synthesized from the extracted total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The following primers were used to quantify gene expression using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad): EPRS1 forward, ATCTTCTTCCTTTCGCGGGG; EPRS1 reverse, CAGCAAAGCTCCTAGCGGA; ACTB forward, CATGTACGTTGCTATCCAGGC; ACTB reverse, CTCCTTAATGTCACGCACGAT. EPRS1 expression was quantified in relation to ACTB expression using the 2−ΔΔCt method.

Cell proliferation assay

Cells were seeded at 5 × 103 cells/well in 96-well plates with transparent bottoms. CCK-8 (Dojindo) was used to measure cell proliferation of different wells at the indicated time points, following the manufacturer’s recommendations. Varioskan LUX multimode microplate reader (Thermo Scientific) was used to measure the absorbance at OD450nm.

Hepatosphere formation assay

A total of 2 × 103 cells/well were grown on 24-well ultra-low adherence plates using single-cell suspensions. Serum-free DMEM/F12 media with 20 ng/mL EGF, 20 ng/mL fibroblast growth factor, and 2% B27 were used for the propagation of the seeded cells. The development of hepatospheres was seen under the microscope after 10 days of culture, and both the number of hepatospheres and their size were determined.

Cell migration assay

Using Transwell cell culture inserts with 8.0-µm pores, a cell migration test was conducted in 24-well plates (Corning). Hep3B, Huh7, and HepG2 cells were seeded at 5 × 104 cells/well in the top chamber with serum-free DMEM. The lower chamber contained DMEM with 10% FBS. These cells were permitted to migrate for 48 h. The cells on the inner surface of the inserts were removed using cotton swabs. The inserts were fixed with 4% formaldehyde at 4 °C for 15 min, and the cells situated on the bottom surface of the inserts were stained for 20 min with 0.1% crystal violet before being counted under a light microscope in three randomly selected areas.

Mass spectrometry and data processing

The protein mixtures from sample tissues were extracted, processed, and digested. Briefly, peptide samples are labeled with TMT reagents, and the peptide mixtures were graded using an Agilent 300 Extend C18 column with high-performance liquid chromatography and then evaluated using an Orbitrap Exploris 480 mass spectrometry system (Thermo Fisher). To examine all control and suppressed EPRS1 raw files, we used Proteome Discoverer (v2.4.1.15) to search for proteins in the homo sapiens 9606 SP 20,201,214.fasta database (20,395 sequences) and added them to the reverse database. While the false positive rate resulting from random matching was computed, a co-contamination database was included to reduce the impact of contaminating proteins on those identified.

Western blot

Equal amounts of cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes before immunoprobed with specific antibodies against EPRS1 (HPA026490; Sigma-Aldrich), LAMC1 (sc-13,144; SANTA CRUZ Biotechnology), and β-actin (#3700; Cell Signaling Technology). Immobilon Western Chemiluminescent HRP Substrate (WBKLS0050; Millipore) was used to detect the signals by capturing images with the Clinx ChemiScope Chemiluminescence imaging system.

Human specimens

A total of 107 primary HCC specimens and 95 peri-tumoral tissues (84 matched pairs) were obtained from patients undergoing surgical excision without prior radio- or chemotherapy at the First Affiliated Hospital, Shihezi University School of Medicine. Informed consent was obtained from patients upon research authorization by the Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine.

Immunohistochemistry

Antigen retrieval was performed on paraffin slices after they had been deparaffinized and dehydrated. To eliminate non-specific background staining, sections were blocked with 5% goat serum for 1 h before being treated with the primary antibody to EPRS1 (HPA026490; Sigma-Aldrich) at 4 °C overnight. Thereafter, the sections were washed with TBST and treated with 3% H2O2 to inhibit endogenous peroxidase activity. Afterward, the samples were treated with a secondary antibody coupled to horseradish peroxidase and detected using a DAB kit (ZLI-9018; ZSGB Bio, Beijing, China) before being analyzed. The degree of immunostaining in each sample was determined for each sample according to the level of staining (intensity score: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage of positive cells (extent score: 0, ≤ 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%). The final immunoreactivity score of EPRS1 levels was the product of the intensity score and extent score for each case.

Statistical analysis

Data are presented as mean ± standard error of the mean. GraphPad Prism v8 was used to conduct statistical analyses. The Mann-Whitney test, Wilcoxon matched-pairs signed-rank test, two-tailed Student’s t-test, or ANOVAs with post-hoc testing were used to compare the groups. Survival estimates were obtained by using Kaplan-Meier analysis and the log-rank test. The significance of correlation between genes was evaluated by Spearman’s correlation. P-value < 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.001.

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