Human GC tissues and paired adjacent normal tissues were collected from 30 patients with GC who underwent radical gastrectomy at the Department of General Surgery, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, China. After surgical excision, the sample was quickly frozen in liquid nitrogen for experiments. This study was approved by the ethics committee of Shandong Provincial Hospital. The collection of gastric cancer and para-cancer tissue and its use was approved by the Institutional Review Board of the Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Shandong, China.
Six gastric cancer cell lines (AGS, BGC-823, HGC-27, MGC-803, SGC-7901, MKN-45) and GES-1 cell lines were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA). And cells were incubated in moist air at 37 °C and 5% CO2.
Hsa-miR-875-5p mimics and mimics negative control, hsa-miR-875-5p inhibitor and inhibitor negative control were purchased from GenePharma (Shanghai, China). At least 24 h before transfection, the cells were cultured in complete medium without antibiotics. Cells were planted in six-well plates, when the cell fusion degree reached 50–70%, the cells were washed with 1 × PBS (PH7.4), 50 nM miR-875-5p mimics or miR-mimic NC and 100 nM miR-875-5p inhibitor or miR-inhibitor NC were transfected into AGS and MKN-45 cells through Lipofectamine™2000 (Invitrogen). Si-USF2 and si-NC were purchased from Genomeditech (Shanghai, China) and transfected as above.
RNA extraction and qRT-PCR
Total RNA was extracted from GC tissues and cells using Trizol reagent (Takara, Japan) according to the manufacturer’s instructions. The cDNA was synthesized using the Evo M-MLV RT Premix for qPCR reagent (AG, China). QRT-PCR was performed on Roche LightCycler 480 II fluorescent quantitative PCR instrument with qPCR SYBR Green Pro Taq HS Master Mix(AG). β-actin was used as the endogenous control. The primers used in this study are as follows: USF2 forward: 5′-AAAGGAGGGATCCTGTCCAA-3′, USF2 reverse: 5′-CAGGGCGTTCTCATTCTTCA-3′; β-actin forward: 5′-GCATCGTCACTGGGGAC-3′ and β-actin reverse: 5′-ACCTGG CCGTCAGGCAGCTC-3′. In addition, unchained curves were used to evaluate specific amplification. QRT-PCR reaction procedures are as follows: 95 ℃ for 30 s, 40 cycles at 95 °C 5 s and 60 °C 30 s. All procedures were carried out in triplicate and relative expression was calculated by the 2−ΔΔCT method.
Total RNA was extracted as mentioned above. According to the manufacturer's instructions, using the Mir-X miRNA first-Strand Synthesis Kit (Takara, Japan), RNA (2 µg) was converted to cDNA. PCR reaction was performed using TB Green® Premix Ex TaqTM II (Takara, Japan), and U6 was used as the endogenous control. The primers of miR-875-5p and U6 were purchased from RiboBio (RiboBio Co., Ltd, Guangzhou, China), the primer sequences used in this study were as follows; has-miR-875-5p forward:5′-GCGGGCGGTATACCTCAGTTTTAT-3′, reverse 5′-ATCCAGTGCAGGGTCCGAGG-3′; U6 forward: 5′-CTCGCTTCGGCAGCACA-3′; U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′. 2−ΔΔCt method was used to calculate the relative expression level. All procedures were also performed in triplicate.
Protein extraction and Western blot
The protein was extracted 72 h after transfection. The cells were flushed with cold PBS, then RIPA buffer (Beyotime, Shanghai, China) containing PMSF (SolarBio, Beijing, China) and phosphatase inhibitors (SolarBio, Beijing, China) was used for cracking. The protein concentration was calculated using the BCA Protein Assay Kit (Beyotime, Shanghai, China) and the protein was separated by SDS-PAGE using a 10% and 12% polyacrylamide gel (20 μg per sample). Proteins are transferred to the immobilon-NC membrane by electrotransfer. The imblotted membrane was sealed in 5% skimmed milk diluted with TBST, and then incubated with appropriate primary antibodies (anti-USF2, anti-p21, anti-p57, anti-Cyclin D1, anti-ZEB1, anti-E-cadherin, anti-Vimentin, anti-TGF-β1, anti-smad2, anti-phospho-smad2, anti-smad3, anti-phospho-smad3 and anti-GADPH obtained from CST) at 4 °C for 12 h.
Dual-luciferase reporting assay
Dual luciferase assay was used to further verify the targeting relationship of miR-875-5p and USF2. Bioinformatics analysis predicted that the possible binding site of miR-875-5p was the 3′UTR site 595–601 of USF2 mRNA.
AGS and MKN-45 cells were grown in 1640 medium containing 10% FBS and transfected with psicheck2-Husf2-3′ UTR reporter plasmid and human miR-875-5p mimics or inhibitor using Lipofectamine™2000. After 48 h of culture, luciferase activity was detected to determine whether the microRNA binds and regulates USF2.
Cell growth was measured using cell proliferation reagent CCK-8 (MCE). Cells were inoculated into a 96-well plate (Corning Costar, Corning, NY) at a concentration of 2.0 × 103 per well, as per manufacturer’s instructions, and 10 μL CCK8 was added to each well at harvest. One hour after CCK8 was added, cell viability was determined by measuring the absorbance of the transformed dye at 450 nM.
The transfected cells were seeded into a 96-well plate at a concentration of 1.0 × 104/well. The EdU solution was diluted with cell complete medium in a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU medium. Add 100 μL 50 μm EdU medium to each well and incubate for 2 h. Then follow the manufacturer’s instructions for the operations. The results were observed using a high-content imaging system.
Transwell migration/invasion assay
MKN-45 and AGS cells grew to approximately 70% confluence in RPMI 1640 containing 10% fetal bovine serum and then transfected. After 24 h, the cells were digested by trypsin and then washed once with PBS. To measure cell migration, culture inserts with an 8 μm aperture (Transwell; CoStar, High Wycombe, UK) were placed in the holes of the 24-well plate and the upper and lower chambers were separated. In the lower chamber, add RPMI 1640 containsing 20% FBS, 600 μl. Then, serum-free medium containing 5 × 104 cells was added to the upper compartment for migration assay, and 1 × 105 cells were used for matrix gel invasion assay (Matrigel Basement Membrane Matrix; BD, NJ, USA). After incubation at 37 °C and 5% CO2 for 24 h, the cells in the upper compartment were removed with a cotton swab. The cells that invaded the base membrane of the inserts were fixed in 4% paraformaldehyde for 10 min, stained with 1% crystal violet for 20 min, rinsed in PBS, and examined microscopically. Each experiment was performed at least three times.
Wound healing assay
To evaluate the motility of GC cells. A total of 1 × 106 cells/wells were inoculated in a 6-well plate and cultured overnight, then transfected with miR-875-5p mimics or NC, miR-875-5p inhibitor or NC and si-USF2 or NC. 8 h later, the confluent cell monolayers were scraped with a sterile pipette head and the plates were washed twice with PBS and adding fresh serum-free medium immediately. An image of the plate is taken under a microscope. The clearance size was analyzed with Image J software.
Tumorigenesis in nude mice
BALB/c nude mice (male, 4–6 weeks old, 16–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Provincial Hospital Affiliated to Shandong University. MiR-875-5p NC or miR-875-5p mimics were transfected into MKN-45 cells, and 5 × 105 MKN-45 cells in logarithmic growth phase were suspended in 100 μL phosphate buffer, and then seeded subcutaneously into the right axillary of nude mice. The experiments were divided into two groups on average (miR-875-5p NC group and miR-875-5p mimics group, n = 6), tumor size was monitored by measuring length (L) and width (W) with a vernier caliper every 4 days, and volume was calculated using the following formula: (L × W2)/2. The mice were fed for 28 days. Tumors were sacrificed and collected. Tumor volume and weight was measured for analysis. Animal experiments conformed to the standards set by the Declaration of Helsinki, and were approved by the ethics review Committee of Shandong Provincial Hospital affiliated to Shandong University, Shandong, China.
Immunohistochemical staining of xenograft tumors tissue
Tumor sections were incubated overnight with commercial rabbit polyclonal antibodies against USF2 at 1:50 dilution at 4 °C. Then, the slices were diluted with horseradish peroxidase (HRP) antibody (1:100; Invitrogen, Thermo Fisher, US), conjugated at room temperature for 2 h and then covered with DAB (SP kit (rabbit streptavidin–biotin method detection system), ZSGB-BIO, China). After rinsing the colored plates with water for a period of time, they were soaked in hematoxylin and dyed, then dehydrated and sealed. Subsequently, the results of IHC staining were scored by evaluating the extent and intensity of staining in 5 fields of view using a microscope (Tissue FAXS Systems, Austria) at × 200 magnification. The staining intensity was divided into four grades: no staining, score 0; pale yellow, score 1; pale brown, score 2; and dark brown, score 3. The positive expression area was also classified into five categories: < 5%, score 0; 6–25%, score 1; 26–50%, score 2; 51–75%, score 3; and 76–100%, score 4. The multiplication of intensity and area scores was used as the final USF2 expression score. All slides were scored by two independent pathologists from Shandong Provincial Hospital who had no knowledge of the grouping and treatment of the slides. When there were discrepancies between the two pathologists, the mean score was used.
The results are presented as means ± SD. Statistical significance was measured by multiple comparisons using Student’s t-test with a significance level of p < 0.05.