Samples collected
In this study, we collected 6 pairs of CRC tissues and corresponding paratumoral normal tissues from CRC patients who received surgical resection at the Department of Gastrointestinal Surgery at the Jiangmen Central Hospital, Guangdong Province, China between 2019 and 2020. Patients were diagnosed following the introduction of AJCC 8th Edition: Colorectal Cancer. The samples included three pairs of low metastasis CRC tissues (without lymph node metastasis) diagnosed as pT1N0M0 and their corresponding adjacent tissues, three pairs of high metastasis CRC tissues (with lymph node metastasis) diagnosed as pT4N2M0 and their corresponding adjacent tissues. Informed consent was obtained from all patients. All procedures were approved by the Institutional Research Ethics Committee of the Jiangmen Central Hospital(decision no. JXY202229).
miRNA sequencing
Low metastasis CRC tissues (M_Low group), high metastasis CRC tissues (M_High group), and their corresponding adjacent tissues were collected for miRNA sequencing, and each group contained three samples. After RNA isolation, RNA from the three samples in the same group were mixed, and the RNA from the adjacent normal tissues of the two groups was also mixed as the Normal Control group (Normal group). Then libraries were prepared following the protocol of the TrueSeq Small RNA Sample Prep Kit (Illumina). Subsequently, the libraries were used for sequencing on the Illumina platform. The sequencing data were analyzed using the R package.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Tissues and cells were collected for RNA isolation using TRIzol (Thermo). Then RNA was reversed into cDNA following the protocol of HiScript® Reverse Transcriptase (Vazyme, Nanjing, China). PCR was performed on the ABI 7900 platform using AceQ® qPCR SYBR Green (Vazyme) as per the manufacturer’s instructions. For calculating the relative miRNA expression, U6 was used as the internal standard; for calculating the relative PSMD10 expression, GAPDH was used as the internal standard. The primer sequences used in this study are shown in Table 1.
Droplet digital polymerase chain reaction (ddPCR) assay
The PSMD10 expression was also detected by ddPCR. ddPCR was performed as described in a previous study with a few modifications[14]. In brief, RNA was extracted from tissues and reverse-transcribed into cDNA following the protocol of HiScript® Reverse Transcriptase (Vazyme, Nanjing, China), which was used as template. ddPCR was performed on the MicroDrop-100 droplet digital PCR system (Forevergen, China). The primers are shown in Table 1.
Cell culture
Human low metastasis colon cancer cell line SW480 and Human high metastasis colon cancer cell line SW620 were purchased from the Shanghai Chinese Academy of Science Cell Bank (China). All cells were cultured in DMEM medium (Gibco, Grand Island, NY, USA), containing 10% FBS at 37℃ in a 5% CO2 atmosphere. The cells were passaged at a ratio of 1:4.
Plasmid and Transfection
The human miR-1248, miR-1248-sponges, PSMD10, and sh-PSMD10 were PCR-amplified from cDNA and cloned into lentiviral vectors. All primers used in the reactions for clone PCR are shown in Table 2. Cell transfection was performed using the Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s instructions. Briefly, cells were digested, seeded, and cultivated for 24 h before transfection until the attainment of 60%–70% confluence. Then, lipofectamine 3000 and relative plasmid were diluted in Opti-MEM individually followed by mixing at 37℃ for 5 min. The medium was replaced with a new medium containing the DNA-lipid mixture. After forty-eight hours, cells were collected for further analysis.
Colony formation assay
For colony formation assay, vector-transfected CRC cells were plated in 6-well plates at a density of 500 cells per well and cultured in a humidified atmosphere containing 5% CO2 at 37℃ for 2 weeks. Cell colonies were washed with phosphate-buffered saline (PBS), fixed with methanol for 30 min, and stained with 0.1% crystal violet (1 mg/mL) for another 30 min. The colony morphologies were captured and manually counted under a light microscope.
CCK-8 analysis
Cell viability was assessed using Cell Counting Kit-8 (CCK-s8) assay. In brief, the indicated CRC cells were seeded on 96-well plates containing complete medium at a density of 1000 cells per well. After cell attachment, the medium was changed, and cells were transfected with relevant vectors, and cultured for 5 days. Cell proliferation was detected every 24 h. Then 10 μL CCK-8 solution was added to each well and the cells were incubated at 37 for 4 h. CCK-8 absorbance was measured at 450 nm.
Invasion and migration assays
Transwell assays were performed to assess the invasion and migration of SW480 and SW620 cells. SW480 and SW620 cells transfected with relevant vectors were first starved for 24 hours. On the next day, the cells were suspended in serum-free L-15 medium, and 4000 cells were seeded in the upper chamber. The lower chamber was filled with 600 µL of L-15 medium containing 20% FBS to induce cell invasion and migration. Following incubation at 37℃ in a 5% CO2 atmosphere for 48 hours, the membranes with the cells invaded or migrated were fixed with methanol and stained with crystal violet. Then the cells in the membranes were counted under a microscope.
Luciferase activity assay
miRDB was used to predict the potential binding site in target mRNA PSMD10 of miR-1248. The wild-type sequence of PSMD10 (PSMD10-WT) and mutant type sequence of PSMD10 (PSMD10-MUT) were cloned into the pmirGLO vector (Promega) respectively, which was performed by General Biol (Anhui, China). Subsequently, PSMD10-WT or PSMD10-MUT was co-transfected with anti-miR-1248 or anti-miR-NC into SW480 cells. They were also co-transfected with miR-1248 or miR-NC into SW620 cells as well. After transfection for 48 hours, the luciferase activity was measured following the procedure of TransDetect® Double-Luciferase Reporter Assay Kit (Transgen, Beijing, China).
Western blot analysis
Cells were lysed by RIPA buffer (Beyond) containing protease inhibitor. Then, protein concentration was evaluated using the BCA method. Protein (25 µg) was separated by 10% SDS-PAGE gel and was transferred to PVDF membrane. Afterward, the membrane was blocked with 5% skim milk and incubated overnight with PSMD10 (ab182576, Abcam), α-tubulin (CST, 3873s, 1:2000) at 37℃. Secondary antibody (SA0004-1, Porteintech) was added to the membrane and incubated for 2h at 25℃. ECL plus (PE0010, Solarbio) and Tanon-5200CE (Biotanon, Shanghai, China) were used to observe the protein bands. α-tubulin served as the loading control.
Statistical analysis
All data were analyzed using Graphpad 8.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 10.0 (IBM, Armonk, NY, USA). Continuous variables are presented as mean ± standard deviation and between-group differences were assessed using the Wilcox. test. P values < 0.05 were considered indicative of statistical significance.