Patients and tissues
The HCC tissue and the paired adjacent sample were acquired from hepatocellular carcinoma patients who received radical surgery in our hospital from June 2019 to September 2020. The specimens and corresponding adjacent tissues were from the same HCC patient. A pathologist with more than 5 years of work experience in this hospital examined each tissue sample to confirm the tissue type. The tumor tissues were immediately stored at -80°C for later use. This study obtained the assent of the ethics committee, and patients agreed with the experiments.
Cell grouping and treatment
Human liver immortalized cells (THLE2), and four HCC lines (huh-7, HepG2, HCCLM3, SK-Hep-1) were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). The huh-7 and HepG2 cells were cultured in a constant temperature incubator, and the medium was DMEM medium with 10% FBS. When the cell density was close to 70%, the miR-205-5p-inhibitor was transfected with Lipofectamine™ 2000 reagent (Invitrogen, USA). The corresponding experimental operation was completed according to the product manual. According to the different transfection, the cells were divided into NC-inhibitor group and miR-205-5p-inhibitor groups. For sh-DLC1 transfection, the lentivirus carrying sh-DLC1 and sh-NC was synthesized by Shanghai Genepharma Co. Ltd (China). The cells were seeded in a confocal dish. When the cells grow to 60%, they were transfected with a lentivirus carrying RFP, and the multiple of infection (MOI) was about 80. The medium was changed 24 hours after transfection, and the culture was continued with DMEM medium (low-glucose), including 10% FBS. After 72 hours, the transfection efficiency was observed under the confocal laser microscope. The rescue assay included 3 groups (NC-inhibitor + sh-NC, miR-205-5p-inhibitor + sh-NC, miR-205-5p-inhibitor + sh-DLC1) based on the transfection.
Q-PCR assay
Trizol reagents were applied to extract tissue and RNA in cells in each group. Ultraviolet spectrophotometers were applied to determine the concentration and purity of RNA. The reverse transcription process used the Prime ScriptTM RT kit (Takara, Japan) and operated based on the instructions. Primer 5.0 software was selected to design primers. The primers were supplied by Shanghai Sangon Biotech Co., Ltd, which was shown as follows, miR-205-5p, Forward, 5'-TCCACCGGAGTCTGTCTCAT-3’, Reverse, 5'-GCTGTCAACGATACGCTACG-3’; DLC1, Forward, 5’-GTTGCCTCAGAGCATCCAG-3’, Reverse, 5’-GGGTGTTGAGATGGAAGAGG-3’; U6, Forward, 5’-ATTGGAACGATACAGAGAAGATT-3’;Reversed, 5ʹ-GGAACGCTTCACGAATTTG-3ʹ; β-actin, Forward, 5’-AAGTACTCCGTGTGGATCGG-3’, Reverse, 5’-ATGCTATCACCTCCCCTGTG-3’. The Sybr Premix Extaq TM kit (Takara, Japan) and Rotor-Gene 3000 systems undertook real-time fluorescence quantitative PCR reactions and measurements. The PCR amplification system includes pre-denaturation at 95°C for 12 min, followed by 95°C for 30 s, 35 cycles, 58°C for 30 s, and final extension at 72°C for 10 min. β-actin was regarded as a reference.
CCK8 assay
The transfected cells were incubated in a plate with 96-wells at 1×103 cells per well, and they were examined regularly at 1 d, 2 d, and 3 d. A total of 10 µL of CCK8 solution (Abcam, Cambridge, UK) was supplied in every well and cultured at 37°C for 2 h. An enzyme-linked immunoassay instrument measured the absorbance value at a wavelength of 450 nm of each well was measured on an enzyme-linked immunoassay instrument. There were 3 parallel replicate wells in all groups.
EdU assay
According to the instructions, the EdU analysis kit (Guangzhou Ruibo Technology Co., Ltd., China) was undertaken to research proliferation. After transfection, the cells were seeded in DMEM with 5 µmol/L EdU and 10% FBS and incubated at 37°C for 2 h. After that, the samples were fixed with 4% formaldehyde at 25°C for 0.5 h and processed with 0.5% Triton X-100 for 20 min for cell membrane permeabilization. After the cells reacted with the 1×Apollo reaction mixture for 0.5 h, they were stained with Hoechst 33342 for 0.5 h. Finally, the samples were recorded under a fluorescence microscope.
Flow cytometry for measuring apoptosis
After 2 d of transfection, the transfected samples were collected and rinsed once by PBS. The cells were centrifuged at 4°C for 5 min and then washed with diluted binding buffer. The diluted binding buffer was used to dilute the cell concentration to 105~106 cell/ml. 5 µl of diluted Annexin-V-FITC solution (Wuhan Hualianke Biology Co. Ltd, China) and 5 µl of PI staining solution were added to the sample and mixed well, kept in the refrigerator at 4°C avoiding light for 10 minutes. A flow cytometer tested the rate of apoptosis.
Wound healing assay
When sample density reached 80–90% in 24 healthy plates, transfection was performed. After 1 d of transfection, a 200 µl pipette tip was taken to make a linear scratch. In order to prevent the cells from entering the proliferation cycle in advance, the cells were incubated in serum-free Opti-DMEM. Scratch healing was photographed at 0, 24, and 48 h. A total of 10 random fields (×200) were selected to calculate the cell migration rate.
Transwell assay for migration and invasion
The base glue was dissolved and diluted, and 50 µl of it was tiled in the upper layer. After 2 d of culture, the cells were digested and counted. About 5×104 cells were resuspended in a 200 µl serum-free DMEM medium and added to the upper chamber. Next, 500 µl of DMEM with 10% PBS was added to the lower chamber and placed in a cell incubator. The upper chamber was obtained, washed 3 times, fixed by 4% paraformaldehyde solution for 0.5 h, and wiped with a cotton swab to clear the excess cells. Then, the cells were stained with crystal violet for 20 min and rinsed with PBS. Finally, an optical microscope (×200) was used to count and photograph the cells. Base glue was not used for invasion ability, and the other steps were the same as above.
Bioinformatics prediction
Starbase is a robust database commonly used for lncRNA/circRNA/microRNA research. Starbase helped to solve the following problems: finding non-coding RNA based on microRNA (such as lncRNA and circRNA), screening mRNA targets based on microRNA, selecting ceRNA regulatory molecules, and mining RNA binding proteins. In this study, the starbase database was used to predict the target connection between miR-205-5p and DLC1
Dual luciferase reporter assay
The wild-type and mutant luciferase reporter plasmids in the 3’UTR region of DLC1 were synthesized by Shanghai Tongke Biotechnology Co., Ltd. First, the 3’UTR region sequence of DLC1 was inserted into the pmirGLO vector (Promega, E1330), which formed the DLC1 wild-type luciferase reporter plasmid after restriction digestion. Then, the sequence mutation that binds to miR-205-5p of DLC1 was inserted into the pmirGLO vector (Promega, E1330) and digested to form a mutant luciferase reporter plasmid for the 3’UTR region of DLC1. Finally, the exact amount of the luciferase mentioned above reporter plasmid and 20 pmol of miR-205-5p mimic were transfected into the cells with lipo2000. After 1 d of culture, the luciferase detection kit was used for detecting.
Western blot assay
Cells were cultured in a 6-well plate with DMEM for 1 d. Transfection of cells and incubated 6 h, and then the medium containing the transfection reagent was discarded. After transfection 48 h, the total protein was isolated and dissolved in the lysate buffer, separated by electrophoresis and transferred to a PVDF membrane (Roche). First, the protein was closed with 2% bovine serum albumin (BSA) at room temperature. Next, the protein was closed at room temperature with 2% BSA at 25°C and then incubated with antibodies (anti-bax, anti-bcl2, anti-cleaved-caspase 3, anti-DLC1, 1: 1000, Santa Cruz) at 4°C. After washing the membrane 3 times, the sample was incubated with the corresponding secondary antibody (1: 2000, Santa Cruz) for 1 h. Finally, the membrane was incubated with the ECL reagent and developed using an ECL kit (US Millipore).
Statistical analysis
SPSS 20.0 statistical software was applied for data analysis. Experiment data were presented as mean ± standard deviation (SD). The Unpaired Student t-test test processed comparison between two groups. P < 0.05 represented the difference was with significance.