Cell culture and miRNA-Seq
The MDCK cell line was obtained from ATCC (CCL-34 P55). MDCK-M-60 (HT-01) and GsAcc 9058 (HT-02) cells were obtained from the master cell bank (MCB) and established from expanded cultures of MDCK cells. Furthermore, MDCK-CA005 (NT-01) and MDCK-CB057 (NT-02) cells were obtained by single-cell cloning technology according to the method described by Guilan Ma[22]. The experiment proved that the tumorigenicity of MDCK-CA005 and MDCK-CB057 cells in nude mice was reduced (data are not shown in this paper). The cells were cultured in DMEM supplemented with 10% foetal bovine serum (FBS, Cellmax, Beijing, China) at 37°C in 5% CO2. The cells were then collected, and RNA was extracted by a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA). After RNA quality was determined using an Agilent Bioanalyzer 2100, RNA molecules in the range of 18–30 nt were enriched by polyacrylamide gel electrophoresis (PAGE). Then, 3’ adapters were added, and 36–44 nt RNAs were enriched. 5’ adapters were then ligated to the RNAs as well. The ligation products were reverse transcribed by PCR amplification, and the 140–160 bp PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeq™ 2500 by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China).
Differential expression analysis of miRNA-Seq data
Fastq files were filtered for low-quality data (< Q20), all clean tags were compared with miRNAs in the GenBank database (Release 209.0) by Blastall 2.2.25 (BLASTN), and then rRNAs, scRNAs, snoRNAs, snRNAs and tRNAs were identified and removed. The expression level of total miRNAs, including existing miRNAs, known miRNAs and novel miRNAs, in each sample was calculated and standardized with transcripts per million (TPM). miRNAs with a fold change ≥ 2 and P value < 0.05 between groups were considered differentially expressed miRNAs (DEMs).
Target gene prediction and miRNA-target gene Regulatory Network
Based on the sequences of the existing miRNAs, known miRNAs and novel miRNAs, candidate target genes were predicted as follows: RNAhybrid (Version 2.1.2) + svm_light (Version 6.01), Miranda (Version 3.3a) and TargetScan (Version 7.0) were used. Cytoscape (Version 3.6.1) software was used to construct the network of interactions between the DEMs and target genes.
Cell transfection
To investigate the effect of miR-2779-x on tumorigenicity in vivo, a miR-2779-x overexpression plasmid and lentivirus packaging auxiliary plasmid (Helper 1.0 and Helper 2.0) were cotransfected into 293T cells. The cell supernatants were collected 48 h after transfection. The lentiviruses were transduced according to the manufacturer's instructions. After concentration and purification, miR-2779-x overexpression lentivirus solution was obtained from GeneChem Co., Ltd. (Shanghai, China). When MDCK cells reached 70–80% confluence, the cells were transfected with the lentivirus at an MOI = 100, and then the positive cells were screened by being cultured with puromycin (4 µg/ml) for two weeks. The lentivirus-modified cell line and its negative control were named lv-miR-2779-x and lv-control, respectively.
In addition, the effects of miR-2779-x on the proliferation, invasion and migration of MDCK cells were investigated by the transient transfection of miR-2779-x mimics or miR-2779-x inhibitors. When MDCK cells in 6-well plates reached 30–40% confluence, miR-2779-x mimics, miR-2779-x inhibitors and their corresponding negative controls (NC) were separately transfected into the MDCK cells according to the instructions of the riboFECT™ transfection reagent. All the mimics and inhibitors were purchased from RiboBio (RiboBio Co. Ltd., Guangzhou, China). The miR-2779-x expression levels in each group were evaluated by RT‒qPCR after transfection for 48 h.
Quantitative real-time PCR (qPCR)
Total RNA was extracted from MDCK cells at 48 h of transfection by the TRIzol-isopropyl alcohol method. For mRNA, cDNA was generated using the Evo M-MLV Reverse Transcription Kit (Accurate Biology, China), and mRNA qPCR was performed according to the instructions of the mRNA qPCR Mix (GeneCopoeia, China). For miRNA, the Bulge Loop™ miRNA Primer (20 µM) was diluted into Bulge Loop™ miRNA Primer (5 µM) using RNase-free water. The reverse transcription reaction was performed at 42°C for 60 min and 70°C for 10 min. The 5s rRNA and miR-2779-x stem loop primers were purchased from Ribo Biotech Co., Ltd. (Guangzhou China), and qPCR was performed according to the instructions of the Bulge Loop™ miRNA qRT–PCR Starter Kit (Ribobio, China). The expression levels of Bak1 and miR-2779-x were calculated with the 2-ΔΔCt method. 5S rRNA and GAPDH were used as the internal references for miRNA and mRNA, respectively. The miR-2779-x and 5S rRNA primers were purchased from Ribo Biotech Co., Ltd. (Guangzhou China). The other primers were as follows: Bak1 forward primer: 5ʹ-CCCTATGGGAAGGCAACGA-3ʹ, reverse primer: 5ʹ-AGGGTGTCAGAAACACAGACCG-3ʹ. GAPDH forward primers: 5ʹ-AGTGACACCCACTCTTCCACCT-3ʹ, reverse primers: 5ʹ-GTGGTCCAGGAGGCTCTTACTC-3ʹ.
Western blotting
MDCK cells were transfected with miR-2779-x mimics and inhibitors and their corresponding negative controls and lysed for 30 min in ice-cold RIPA lysis buffer supplemented with the protease inhibitor PMSF (Solarbio, Beijing, China). Then, the protein concentration was measured with the BCA Protein Assay Kit (Beyotime, Shanghai, China). SDS–PAGE was used to separate the proteins. Then, the proteins were transferred to PVDF membranes. The PVDF membranes were blocked in 5% skim milk powder at room temperature for 2 h and then incubated with diluted primary antibodies (1:1000) for 12 h at 4°C. After washing with TBST three times, the PVDF membranes were incubated in diluted HRP-conjugated goat anti-rabbit IgG (1:2000) at room temperature for 1 h. The reaction was detected with a hypersensitive ECL chemiluminescence kit (Boster, Wuhan, China). The blots were scanned using an Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Inc., MA, USA), and the density of the bands was analysed using ImageJ. Antibodies were obtained from the following sources: Bak1 (catalogue #ab92999), β-actin (catalogue #ab8227), and HRP-conjugated goat anti-rabbit IgG (catalogue #ab205718) were purchased from Abcam (Cambridge, MA).
Colony formation assay
A colony formation assay was used to evaluate the tumorigenicity of the cells in vitro. DMEM (500 cells/ml) was seeded in 6-well culture plates and maintained in a 37°C incubator in 5% CO2 for 6 days. Then, the clones were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. Images of colony formation were scanned, and the numbers of colonies were counted. A cluster containing ≥ 50 cells was considered one clone cluster. The colony formation rate (%) was calculated according to the following formula:
Colony formation rate (%) = (number of clones/number of inoculated cells ×100%).
Apoptosis assay
Lv-miR-2779-x and lv-con were washed with PBS 3 times and digested by trypsin for 3 min; then, 4 ml of complete medium supplemented with 10% FBS was added to terminate the digestion. The supernatant was discarded by centrifugation at 1000 rpm for 5 min, and the pellet was suspended in 3 mL precooled PBS. Annexin V/PI staining was carried out using an Annexin V-FITC/PI Apoptosis Detection Kit (Meilunbio, China). The cells were suspended in 1× binding buffer working solution, 100 µL of the cell suspension was absorbed, 5 µL of propidium iodide (PI) and 5 µL of Annexin V were added, and the cells were gently vortexed. The cells were incubated at room temperature in the dark for 15 min. Then, 400 µL of 1× binding buffer was added to each tube. Finally, the cells were analysed by flow cytometry.
Animals and in vivo tumour growth experiments
Forty female BALB/C nude mice aged 5 weeks were purchased from Beijing Charles River Animal Technology Co., Ltd. The nude mice were randomly divided into four groups and kept in a specific-pathogen-free (SPF) animal room. Each group contained ten rats. The food and water were sterilized. During the feeding period, the health of the mice was observed every day, and no adverse conditions were observed. A total of 107 lv-miR-2779-x or lv-control cells (in 200 µL PBS) were subcutaneously injected between the scapulae of each nude mouse. Positive (HeLa) and negative (MRC-5) controls were processed in the same way. The changes in body weight and tumour volume at the injection site were recorded every five days. After 30 days, the mice were sacrificed by cervical dislocation under isoflurane-induced anesthesia, and the tumour organoids were collected for comparative analysis. The tumour length and width were measured, and tumour volume was calculated according to the following formula: [(length×width)2]/2. The animal experiment research was carried out strictly in accordance with the “Guidelines for the Welfare of Experimental Tumour Animals”. All animal experiments were subject to approval by the Committee on Ethics of Animal Experiments of Northwest Minzu University.
Histomorphometric analysis
The harvested tumours were fixed by incubation with 4% paraformaldehyde for one week. Before sectioning, the tissues were dehydrated with ethyl alcohol at concentrations of 70%, 80%, 90%, 95% (2 h per run) and 100% (thrice, 20 min, 30 min and 70 min per run), made clear by incubation xylene twice (15 min and 30 min per run), and waxed three times (20 min by soft paraffin (two runs) and 40 min by hard paraffin). Finally, the tissues were embedded in paraffin as needed. The paraffin tissue blocks were sectioned 4 µm by an hRM2135 Slicer (Leica, Germany). After dewaxing, the hydrated slices were stained with haematoxylin and eosin (HE) for histomorphometric analysis.
Cell proliferation assay
Cells in the logarithmic phase of growth were diluted to 104 cells/mL in DMEM supplemented with 10% FBS. Two hundred microlitres of cell suspension was added to each well of a 96-well plate. The cells were cultured for 24 h, 48 h, 72 h, and 96 h in a 37°C incubator with 5% CO2. Cell proliferation was analysed using the Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China) according to the manufacturer's manual at each time point.
Wound healing assay
The migration of cells was determined by a wound healing assay. MDCK cells (3×105) were seeded in six-well plates and grown to confluence. The MDCK cells were washed 3 times with PBS, and then a 200 µL pipette tip was used to scratch the monolayer to generate wound. Subsequently, the cells were washed 2 times with PBS again, the supernatants were discarded, and serum-free medium was added. The wound width was recorded and photographed by inverted microscopy every 12 h. ImageJ was used to calculate the wound area. The relative cell migration rate was calculated according to the following formula:
Cell migration (%)=(wound area at 0 h - wound area at 24 h)/wound area at 0 h×100%
Matrigel invasion assay
Matrigel (Biofroxx, Germany) and serum-free DMEM were evenly mixed at a volume ratio of 1:10, and then the mixture was added to the upper chamber of a Transwell insert with 8.0 µm pores (Corning, USA) and incubated at 37°C for 8 h. Cell suspension (200 µl) was added to the upper chamber, and 600 µl DMEM supplemented with 20% FBS was added to the lower chamber. The 24-well plates with Transwell plates were cultured at 37°C in 5% CO2 for 48 h. The cells in the upper chamber were removed, and the cells on the lower surface were fixed with 4% paraformaldehyde at room temperature for 15 min. Subsequently, the cells were stained with 0.1% crystal violet at room temperature for 30 min and washed twice with PBS. The number of stained cells in a single field were counted under the microscope.
Dual-luciferase reporter assay
Primers for PCR amplification were designed according to the sequence of the 3'UTR of Bak1 in NCBI, and the DNA of MDCK cells was used as the template for PCR amplification and 3'UTR sequence fragment synthesis. The pmiR-RB-Report™ vector and PCR products were digested with restriction endonucleases. After overnight incubation at 37°C, the digested fragments were recovered by agarose gel electrophoresis. The products were transformed into DH5α competent cells after ligation at 16°C, and the positive colonies were selected for PCR and sequencing identification. The wild-type plasmid carrying the target sequence was named c-Bak1-WT. Reverse primers were designed for the end of the mutated sequence of the binding site (5'-3') between miR-2779-x and Bak1. A mutant plasmid carrying the target sequence was established by the above method and named c-Bak1-MUT.
MiR-2779-x mimics, c-Bak1-WT, c-Bak1-MUT and Lipo 6000™ transfection reagents were diluted in 5 µL OPTI-MEM and transfected into 293T cells after incubation for 5 min at room temperature. There were three replicates in each group, and the culture medium was replaced 6 h after transfection. The fluorescence values of Renilla luciferase (Ruc) and firefly luciferase (Luc) activity were determined 48 h after transfection. The Ruc/Luc ratio was used to correct the luciferin values, and the relative luciferin values were calculated.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (8.0.2). The data are presented as the mean ± standard deviation (SD). The differences were analysed using Student’s t test or one-way ANOVA. Differences with P ≤ 0.05 were considered statistically significant (*), and those with P ≤ 0.01 were considered extremely significant.