Esophageal adenocarcinoma patients
In total 201 esophageal cancer patients (Her2 positive) were enrolled in first affiliated hospital of Xi`an Jiaotong University between March 2011 and March 2013. Every enrolled patient has received and signed the written consent. Protein and RNA were extracted from esophageal cancer and corresponding normal tissue samples obtained from each patient immediately; and remnant samples were paraffin-embedded. The main inclusion criteria were as follows: 1. Pathologically diagnosed as esophageal adenocarcinoma; 2. Receive surgery plus chemotherapy; 3. Did not receive any other treatment related to EAC elsewhere; 4. Older than 18-year-old and ability to take care of herself/himself. The main exclusion criteria were: 1. Diagnosed with other primary malignancies; 2. IV stage (based on 7th edition of UICC tumor staging
system); 3. Refuse to sing the informed consent. Two employees were in charge of documenting clinical and follow-up records of each patient. Besides, the corresponding author quality controlled every data and validated the data integrity. Our study has gained approval from the Ethics Committee of first affiliated hospital of Xi`an Jiaotong University. This study was performed in accordance with the Declaration of Helsinki.
RNA sequencing process was guided and supported by Genechen (Shanghai, China)
Total RNA was extracted from 4 EAC (Her2-positive) patients’ cancer tissues and corresponding normal tissues by means of TRIzol (Invitrogen, Carlsbad, CA). NA Clean XP Kit (Beckman Coulter, Kraemer Boulevard Brea, CA) and the RNase‐Free DNase Set (QIAGEN, GmbH, Germany) were used for the RNA purification. Illumina HiSeq 2000/2500 (Illumina Inc, San Diego, CA) was used for RNA-sequencing.
Normal esophageal epithelial cell line HEEC and adenocarcinoma cell lines OE-33 and SK-GT-4 were purchased from American Type Culture Collection (Virginia, USA). All kinds of cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a 37℃and 5% CO2 incubator. Culture medium were changed every day.
NR2F2-AS1 and miR-4429 knock down and overexpression plasmids were purchased from Genechem (Shanghai, China); miR-425-5p knock down and overexpression plasmids were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to assist in transfecting above plasmids into BIC-1 and SK-GT-4 cell lines.
Total RNA was extracted from cell lines, EAC patients’ cancer tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Canada) was used to isolate cytoplasmic and nuclear RNA. First-strand cDNA synthesis kit (Tiangen Biotech, Beijing, China) was used for reverse transcription to generate cDNA. SYBR® Premix Dimer Eraser kit (Takara Shiga, Japan) was used to perform qRT-PCR and test the expression of NR2F2-AS1 and IGF1R. β-actin was used as internal control. miScript microRNA RT PCR kit (Qiagen, Toronto, ON, Canada) was used for cDNA synthesis and qRT-PCR process for miR-4429 and miR-425-5p expression. U6 was used as the internal control. ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) was used to perform the qRT-PCR process. The expression level was calculated by 2-ΔΔCt method.
Total protein was extracted from cells using RIPA buffer (Sigma-Aldrich, Darmstadt, Germany) and was quantified by BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were transferred to PVDF after separated by SDS-PAGE. 5% skim milk was used for transferred PVDF membranes block for 1 hour at room temperature. IGF1Rβ (1:1000, CST, Shanghai, China) was incubated overnight at 4 ◦C, following secondary antibody(1:10000, Beyotime, Shanghai, China) Enhanced chemiluminescence (ECL, Beyotime, Shanghai, China) was used to quantify the protein expression level.
Dual luciferase reporter gene assay
Plasmids of pGL3-NR2F2-AS1 wild type (WT) or NR2F2-AS1 mutant type were co-transfected with the miR-425-5p/NC or miR-4429/NC mimic into SK-GT-4 cells for 48 hours with the assistance of Lipofectamine 2000 (Beyotime, Shanghai, China). Plasmids of pGL3-IGF1R wild type (WT) or IGF1R mutant type were co-transfected with the miR-425-5p/NC or miR-4429/NC mimic into SK-GT-4 cells as well. Then Dual-luciferase reporter system (Promega, Madison, WI, USA) was used to measure the firefly luciferase activity, which was normalized to renilla luciferase activity.
Biotin labeled miR-4429-WT/miR-4429-MUT or miR-425-5p-WT/miR-425-5p-MUT were synthesized by GeneCreate (Wuhan, China) and were transfected into SK-GT-4 cells, which were incubated with lysis buffer (Ambion, Austin, Texas, USA) for 15 minutes. Then, M-280 streptavidin beads (S3762, Sigma-Aldrich, St Louis, MO, USA) precoated with RNase-free bovine serum albumin (BSA) and yeast tRNA (TRNBAK-RO, Sigma-Aldrich, St Louis, MO, USA) were used to incubate with the cell lysates overnight at 4 °C. Trizol was then used to purify the bound RNA and qRT-PCR was used to test the NR2F2-AS1 enrichment.
GraphPad Prism 8.2 was used for plotting statistical graphs. R 3.3.1 was used for statistic analysis: WGCNA and Limma package were used for RNA sequencing analysis. t-test was used for two groups comparison; one-way ANOVA for multiple groups comparison and repeated measurement ANOVA for CCK-8 results analysis. P < 0.05 was indicative of statistically significant difference.