Acquisition of carcinoma and paracancerous tissue
The CRC tissues and their paracancerous tissues were collected from 22 patients with colorectal cancer who had not undergone chemoradiotherapy in Yijishan Hospital. The tumor tissue and the surrounding normal tissue within 0.5cm were collected during the resection and stored in liquid nitrogen immediately, with the informed consent of the patient and the approval of the medical committee and the ethics committee of Yijishan Hospital.
Four human CRC cell lines HT29, SW480, SW620, HCT116 and human normal colon epithelial cell lines FHC were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cell complete medium contained 10% FBS (Gibco, Waltham, America) and RPMI-1640(Gibco, Carlsbad, America) or DMEM (Hyclone, Logan, America). Four colorectal cancer cell lines were cultured in complete medium of RPMI-1640 while normal colon epithelial cells FHC were cultured in DMEM. The environment of the cell incubator was kept at a constant temperature of 37 ° C with 5% carbon dioxide. The cells were given fluid exchange or passage for 1-2 days.
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA of tissues and cells was extracted using Trizol (Invitrogen, Carlsbad, America) as per the merchant's step-by-step instructions. Nanodrop 2000 Spectrophotometer (Thermo Fisher, Waltham, America) was used to measure RNA concentration, and then 2 microliters were taken for reverse transcription using a reverse transcription kit (Takara, Dalian, China) to obtain the corresponding cDNA. Real-time fluorescence quantitative PCR was detected using qPCR kit (Takara, Dalian, China). The sequence of primers used in the experiment is as follows：U6, CTCGCTTCGGCAGCACA-forward and AACGCTTCACGAATTTGCGT-revers；GAPDH, GAACGGGAAGCTCACTGG-forward and GCCTGCTTCACCACCTTCT-reverse；miR-495-3p，AAACAAACAUGGUGCACUUCUU-forward and GAAGUGCACCAUGUUUGUUUUU-reverse；HMGB1，TGCTGATTAGTTACCACAGTTCTGA-forward and CTCGGGTACACAGGACACACAA- reverse. The primers above were all purchased from Ribo Biology（Guangzhou，China）, in which U6 was used as the internal reference of miR-495-3p and GAPDH as the internal reference of HMGB1. The expression of miR-495-3p and HMGB1 in tissues or cells was analyzed by 2−ΔΔ CT method.
MiR-495-3p mimics, inhibitors and their respective negative controls (control mimic and control inhibitor), the short hairpin RNA(shRNA) HMGB1 and negative control (sh-NC) was synthesized by Ribo Biology (Guangzhou, China). The HMGB1 overexpression plasmid and the empty plasmid was supplied by GenePharma Co. Ltd (Shanghai, China). The original medium was replaced with the serum-free medium Opti-MEM (Gibco) when the confluence degree of cells in the six-well plate reached 60%-70%. Then Lipofectamine 3000 Kit (Invitrogen) was used for transfection according to the manufacturer's instructions. After 6 hours, the Opti-MEM medium was replaced with medium containing 2% FBS or complete medium. 48 hours after transfection, cells were collected for subsequent experiments.
Cell Survival Test (CCK-8 assay)
We used Cell Counting Kit-8 Kit (Keygen Biotech Co. Ltd,Nanjing, China) to analyze the viability of cells according to the instructions. After the corresponding transfection treatment, HT29 and SW480 cells were seeded into 96-well plates with 1x104 cells per well.10μl of CCK-8 reagent was added to each well at 12, 24, 36, 48, and 60 hours after transfection. The cells were put back into the incubator for further incubation for 2 hours and the absorbance was measured at 450nm.
Cell Proliferation Test (EDU assay)
Six hours after transfection, the cells were inoculated into 24-well plates. When the cells grew to 70%-80% concentration, EDU reagent was added to each well at a ratio of 1:1000 and incubated for two hours. Then the cells were fixed and stained according to the manufacturer's instructions (EDU Cell Proliferation Kit, Ribo, Guangzhou, China). Finally, fluorescence microscope was used to observe the cells and image them.
Cell migration Test (Transwell assay)
24 h after transfection, the cells were digested, and the concentration of cells was adjusted to 1×105 cells/ml with serum-free RPMI-1640. 100μl cell suspension were added to the upper compartment of Transwell chamber, while RPMI-1640 medium containing 20% serum was added to the lower compartment. After incubation for 48 hours, the transmembrane cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, and the non-transmembrane cells were lightly wiped off with a cotton swab. Finally, an inverted microscope was used for observation and imaging.
Cell apoptosis Test (flow cytometry)
To detect apoptosis rates, we purchased apoptosis detection kits (BD Biosciences, CA, America). 48 hours after transfection, the cells were collected, washed with PBS for three times and resuspended with an appropriate amount of Binding Buffer. 1μl Annexin-V-Fluorescein isothiocyanate (FITC) was added for 15 min, and then 1μl Propidium iodide (PI) was added. (Each cell line was set with FITC staining alone, PI staining alone and blank control to adjust the drawing gate.) Finally, apoptosis was detected by flow cytometry.
Correlation between miR-495-3p and HMGB1 (dual-luciferase reporter gene assay)
To evaluate the direct binding of miR-495-3p to HMGB1, we used the dual luciferase reporter assay. After the wild-type and mutant-type vectors of HMGB1 3 '-UTR were designed, miR-495-3p mimic or control mimic and wild-type or mutant-type vector plasmid were co-transfected into 293T cells (it is a very common cell line expressing foreign genes for study, which is relatively easy to transfection). The co-transfection groups were as follows: miR-495-3p mimic + MUT, miR-495-3p mimic + WT, control mimic + MUT, and control mimic + WT. After 24 hours, a dual luciferase reporter assay kit was used for experimental analysis according to the instructions.
In vivo experiment of nude mice (armpit tumor formation)
We purchased 3-week-old SPF (special-solution-free) grade male nude mice from Hangzhou Ziyuan Laboratory Animal Science and Technology Co., Ltd. (Hangzhou, China). A one-week quarantine was conducted in the quarantine room of SPF animal laboratory of Wannan Medical College. About 2×106 HT29 cells were injected subcutaneously into the left axilla of each nude mouse. After that, the tumor volume was observed and measured every three days. After 15 days, the mice were sacrificed with cervical dislocation, and the tumor bodies were isolated and weighed. All operations have passed the experimental animal welfare and ethics review of Wannan Medical College.
Protein extraction and Western blotting
After 48 hours of transfection, the culture medium was sucked and discarded. 200μl of Radioimmunoreception (RIPA) lysis buffer (Thermo Fisher, Ma, America) containing protease inhibitor was added to each well of the six well plate, and then the protein in colorectal cancer cells was dissolved at 4 ℃ for 30 minutes. Then, the supernatant containing protein was centrifuged at 4℃ under 7500 centrifugal force for 5 min. Proteins were isolated by SDS-PAGE constant pressure 80-120V electrophoresis, and then transferred to PVDF membrane at constant current of 300mA. After sealed at room temperature for one hour, the required HMGB1 (24kDa) and internal reference actin (42kDa) protein bands were cut and placed into a 1:1000 ratio of primary antibody (ABclonal, Woburn, America) reaction solution for overnight incubation at 4℃. The next day, after washing the membrane with TBST for three times, the membrane was incubated in 1:5000 secondary antibody solution at room temperature for 2 hours. Exposure analysis was performed using a luminescent solution after washing the membrane again. Image J software is used to process the gray value of strips.
Data analysis method
According to the mean and standard deviation of the three independent repeated experiments, GraphPad Prism-8 software was used for analysis and mapping. The corresponding P value was calculated by t-test. P<0.05 indicated statistically significant difference, and was represented by *,P<0.01 was denoted by **,P<0.001 is denoted by ***.