Bioinformatics analysis
The TargetScan website and the microRNA.org website were searched to screen for miRNAs highly related to Nestin [20, 21]. Data from both websites were collected and filtered based on free energy and score value. The filters were designed to predict potential targets, which were then analyzed.
Tissue samples
Fresh ESCC tissue samples and para-carcinoma tissue samples were collected from 3 patients with ESCC treated in First Affiliated Hospital, Sun Yet-San University in 2018. The Institutional Ethics Committee of the First Affiliated Hospital of Sun Yet-San University approved this study.
Genetic detection
Trizol (Invitrogen, U.S.A.) was applied to extract the total RNA from the ESCC tissues according to the manufacturer’s protocol. And then, the total RNA was qualified and quantified. For each sample, one microgram total RNA was prepared by library. The Agilent 2100 bioanalyzer was used to check the distribution of the size of the fragments for the library. Quantitative real-time polymerase chain reaction (qPCR, TaqMan Probe) was used to quantify the library.
The final PCR products were sequenced by the BGISEQ-500 platform (BGI-Shenzhen, China). The raw tags from the platform were processed according to the protocol, and the clean tags were obtained after filtering. The small RNA expression level was then calculated by using unique molecular identifiers[22]. Differential expression analysis was performed using the DEGseq to determine the significance of differences of expression [23].
Cell culture
ESCC cell lines of KYSE30, KYSE150, KYSE450 and a normal epithelial cell line were purchased from the Cell Bank (Shanghai, China). Cell lines were cultured according to the guideline.
Cell transfection and stable cell construction
In this study, pCDH-GFP + Puro lentiviral vectors were utilized for the stably transduced cell lines. All vectors used for the stably transduced cell lines were purchased from Hechuang Biotech Ltd. (Guangzhou, China). Nestin cDNA including 3’UTR was used in this study.
The KYSE30 and KYSE450 cell lines were transfected with different lentiviral vectors to establish blank expression vector-transfected stable cells (Blank vec group), miR-204-5p expression stable cells (miR-204-5p OE group), Nestin expression stable cells (Nestin OE group) and miRNA-204-5p expression plus Nestin expression stable cells (miR-204-5p OE + Nestin OE group), according to standard protocols. The stable cell lines were selected by measuring the expression level of GFP (Green fluorescent protein). In addition, the expression of miRNA-204-5p and Nestin in the stably transfected cells was verified by qPCR and Western blotting, respectively.
Total RNA extraction and qPCR
Total RNA from cultured cells or tissues was extracted by TRIzol reagent according to the manufacturer's instructions. For mRNA, 2 mg RNA was used for cDNA synthesis by High-Capacity RNA-to-cDNA™ Kit. For miRNA, 2 mg RNA was reverse transcribed using TaqMan miRNA assays (ABI, Forest City, CA, U.S.A.). The qPCR was performed by SYBR-based Roche Light-Cycler® 480II PCR instrument. Moreover, the relative expression levels of target genes was analyzed with 2− ΔΔCt method [24]. In this study, U6 and GAPDH were used as the internal references. The following primer pairs were used for qPCR: miR-204-5p forward: 5’-UUCCCUUUGUCAUCCUAUGCCU-3’, reverse: 5'-CTCAACTGGTGTCGTGGA-3'; Nestin: forward, 5’-TGCGGGCTACTGAAAAGTTC-3’; reverse, 5’-GGCTGAGGGACATCTTGAG-3’; U6 forward: 5'-CTCGCTTCGGCAGCACA-3', reverse: 5'-AACGCTTCACGAATTTGCGT-3'; GAPDH forward: 5'-GGGAAACTGTGGCGTGAT-3', reverse: 5'-GAGTGGGTGTCGCTGTTGA-3'.
Western blotting analysis
Total protein extracts were disposed according to the routine guideline. The membranes were blocked, incubated with rabbit polyclonal anti-Nestin antibody (19483-1-AP, Proteintech) overnight, and then treated with anti-mouse secondary antibody (Southern Biotech). An anti-GAPDH antibody (IPVH00010; MILLIPORE) was used as an internal control. Densitometric analyses of bands were performed by Image J (NIH Image, Bethesda, MD, U.S.A.), and then the quantification of protein abundance was performed by normalizing the densitometric data of target protein to that of GAPDH.
Luciferase reporter assay
The human wild-type Nestin 3’-UTR sequence or the mutated Nestin 3’-UTR sequence with the predicted target sites was amplified and subcloned into the pmirGLO Dual-Luciferasevector (HeChuang Biotech, Guangzhou, China). Kyse30 cells were seeded onto 24-well plates (5×105 cells/well) and co-transfected with luciferase reporter vectors (0.125 µg) and miR-204-5p mimic (50 nM) or mimic negative control (50 nM) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. miR-204-5p mimic and NC mimic were purchased from Sango (Shanghai, China). Finally, luciferase activity was measured by the Luciferase Assay System (Promega, U.S.A.) in a bioluminescence detector (Promeg, GloMax) and were normalized by the Renilla luciferase activity according to the manufacturer’s protocol.
Colony formation assay
Cell resuscitation and culture of stably transfected KYSE30 cells was performed according to the routine protocol. After trypsinization, the cells were diluted to 1×103 cell/ml and inoculated on the well plate. The cells were monitored daily until colony growth was observed. The number of colonies was calculated. The Enzyme-linked Spot Image Automatic Analyzer (AID, Germany) was used to scan and analyze the result.
Flow cytometric analysis
Apoptosis of the stably transfected cells was evaluated by flow cytometry (BD, Bioscience, U.S.A.) using an Annexin V-FITC Apoptosis Detection Kit (KGA106, Keygen, Jiangsu, China). CellQuest software was used to analyze the results.
TUNEL analysis
Cells were seeded onto a slide and fixed at 4℃ for 25 minutes. The slides were washed with phosphate buffer saline (PBS) twice for 5 minutes, and then treated with 70% ethanol at -20℃ overnight. At room temperature, 10 minutes, each sample was added by equipment buffer. The slides were incubated with TDT (terminal transferase) working solution, and then with 4', 6-diamidino-2-phenylindole (G3250, Promega, USA) according to standard protocols. The slides were then observed by fluorescence microscopy (MDI6000B, Leica, Germany). The nuclei of apoptotic cells exhibited green fluorescence, and the nuclei of all cells exhibited blue fluorescence.
Nude mice xenograft tumor model
Six-week-old male nude mice were adaptively fed for 3 days. And then, randomly, every 3 nude mice were grouped into 5 groups. Next, mice were treated with subcutaneous injection of wild type (WT) and stable transfected KYSE 30 or KYSE450 cells 3 times: 1) WT cells (NC group); 2) blank vector stable cells (Blank vec group); 3) miR-204-5p stable expression cells (miR-204-5p OE group); 4) Nestin stable expression cells (Nestin OE group); 5) miR-204-5p plus nestin stable expression cells (miR-204-5p OE + Nestin OE group). In all groups, trypsinized cells were resuspended in normal saline and 2×106 cells were injected each time.
The mice were observed, and tumor volume in each group was measured on day 4, 8, 12, 16, and 20 after injection. On day 21, the mice were killed by cervical dislocation, and tumor size and weight were measured. All animal experiments were conducted according to established guidelines for the use and care of laboratory animals, and the experiments were approved by the animal ethics committee of our institution.
Statistical analysis
SPSS software version 22 (IBM SPSS Inc. Chicago, IL, U.S.A.) was used to conduct the statistical analyses. Values of P < 0.05 were considered to be statistically significant.