Tissue samples collection
Tumor and adjacent normal tissue samples of 50 CRC patients admitted to Liu Zhou People’s Hospital from July 2016 to January 2018 without any treatment were collected.
Cell culture and transfection
CRC cell lines (SW480 and SW620) and normal human colonic epit CON) were bought from the American Type Culture Collection (ATCC, Manassas, VA, USA). Culture medium consists of RPMI-1640 (Gibco, Waltham, MA, USA), 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in 5% CO2 at 37℃. Small interfering RNA (siRNA) against SNHG16 (si-SNHG16) and SNHG16 overexpression plasmid (pcDNA-SNHG16) or their negative controls (si-NC and pcDNA), ubiquitin specific peptidase 22 (USP22) overexpression plasmid (pcDNA-USP22), miR-132-3p mimic (miR-132-3p), miR-132-3p inhibitor and their negative controls (miR-NC and inhibitor-NC) were purchased from RiboBio (Guangzhou, China). Lentiviral short hairpin RNA (shRNA) targeting SNHG16 (sh-SNHG16) and negative control (sh-NC) were synthesized by Genechem (Shanghai, China). All oligonucleotides or plasmid vectors were transfected into SW480 and SW620 cells using Lipofectamine 3000 (Invitrogen).
Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol reagent (Invitrogen) was utilized for extracting the total RNA. The Reverse Transcription Kit (Takara, Dalian, China) was used to synthesize cDNA. SYBR Green (Takara) was used to quantify the levels of SNHG16, miR-132-3p and USP22 using ABI7500 system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls. The primers were listed as below: SNHG16: F, 5’-CAGAATGCCATGGTTTCCCC-3’, R, 5’-TGGCAAGAGACTTCCTGAGG-3’; USP22: F, 5’-GGCGGAAGATCACCACGTAT-3’, R, 5’-TTGTTGAGACTGTCCGTGGG-3’; GAPDH: F, 5’-CCACATCGCTCAGACACCAT-3’, R, 5’-ACCAGGCGCCCAATACG-3’; miR-132-3p: F, 5’-GCGCGCGTAACAGTCTACAGG-3’, R, 5’-GTCGTATCCAGTGCAGGGTCC-3’; U6: F, 5’-CTCGCTTCGGCAGCACATATACT-3’, R, 5’-CGCTTCACGAATTTGCGTGT-3’. The fold-change was calculated using 2−ΔΔCt method.
Cell vitality assay
The proliferation of CRC cells was determined using 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) Assay Kit (Beyotime, Shanghai, China). The transfected SW480 and SW620 cells were cultured with MTT solution for 4 h. After the cells were washed with phosphate buffer saline (PBS), dimethylsulfoxide (DMSO) was added for shock dissolution for 10 min, and the absorbance at 490 nm was tested.
After digestion with trypsin, SW480 and SW620 cells were re-suspended with PBS and collected into the centrifuge tube. Then, the cells were stained using Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, Shanghai, China) away from light for 20 min. The rate of apoptosis was detected by Flow cytometry (Merck KGaA, Darmstadt, Germany).
Transwell assay was performed using 8 μm polycarbonate membrane of chambers (Corning Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA) to detect cell invasion, but chambers without Matrigel wwere ultilized to detect cell migration. The transfected SW480 and SW620 cells were collected and seeded into the upper chambers containing the serum-free medium. The lower chambers were filled with RPMI-1640 medium containing 10% FBS. After 24 h, the lower chamber cells were fixed with paraformaldehyde and stained with crystal violet. Finally, images were taken under a microscope (Shoif, Shanghai, China) to observe and count the number of metastasis of cells (× 200).
Dual-luciferase reporter assay
The SNHG16 wild-type (SNHG16-WT) and ) reporter vectors containing the miR-132-3p binding sites and mutant binding sites were synthesized by General Biosystems (Anhui, China). Similarly, USP22 3’UTR-WT/MUT reporter vectors were constructed in the same way. The above reporter vectors were co-transfected with miR-132-3p mimic and miR-NC into SW480 and SW620 cells. After 48 h, Dual-luciferase Reporter Assay Kit (Promega, Madison, WI, USA) was performed to detect the luciferase activity.
Western Blot (WB) analysis
SW480 and SW620 cells were lysed with lysis buffer (Beyotime). Protein concentration was measured using the BCA Assay Kit (Yeasen). Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blockage with non-fat milk, the membranes were cultured with the primary antibodies against USP22 (1:2,000, Invitrogen) or β-actin (1:5,000, Invitrogen) overnight at 4℃. Then, the membranes were cultured with horseradish peroxidase-coupled secondary antibody (1:2,000, Invitrogen). Protein signals were observed with an enhanced chemiluminescence (ECL) solution (Beyotime).
Mice xenograft models
Twelve male BALB/c-nude mice (5-week-old) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). SW620 cells transfected with sh-SNHG16 or sh-NC (n = 6 per group) were subcutaneously injected into the mice. From d 7, the tumor length and width were recorded to calculate tumor volume every 5 d. After 27 d, the tumor was removed for weight measurement and qRT-PCR.
Data were presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad 5.0 software (GraphPad Software, La Jolla, CA, USA). The Student’s t-test and one-way analysis of variance (ANOVA) were used to analyze the differences between the paired groups and multiple groups, respectively. Significant difference was defined as P < 0.05.