Ethical statement
This study involved in human LUAD tumor tissues and normal tissues. Ethics Committee of Guangzhou First People’s Hospital approved this study. Written informed consent was signed by all the patients.
Data retrieval from database
For data set screening, we searched lung adenocarcinoma data in TCGA database (https://cancergenome.nih.gov/) and Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. Datasets that with normal lung tissue data were also downloaded. RNA sequencing data and corresponding clinical data were downloaded from TCGA database and GSE118370 gene set. The platform of GSE118370 was GPL570 ([HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). Differentially expressed lncRNAs and co-expressed genes were identified from 497 cases of LUAD tumor tissues and 54 cases of normal tissues. The overall survival was assessed using Kaplan-Meier Survival curve with log rank test. The correlations between WASIR2 and miR-374b-5p as well as MEX3A were assessed using Pearson correlation coefficient. Package “survivalROC” was used to analyze the summary receiver operating characteristics (sROC) of WASIR2, miR-374b-5p and MEX3A in LUAD in R software .
Cell culture
The human embryonic kidney cell line 293T, normal human lung epithelial cell line (16HBE) and human lung adenocarcinoma cell lines (A549 and NCI-H226) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). theNCI-H226 cells were cultured using RMPI-1640 medium supplemented with 10% fetal bovine serum. 293T, 16HBE and A549 cells were cultured using DMEM medium supplemented with 10% fetal bovine serum.
Cell line construction
To construct WASIR2 stably expressed A549 cells, the sequence of WASIR2 was cloned into the pcDNA3.1 vector. Tumor cells transfected with empty vector (EV) was used as control. NCI-H226 cells were transfected with short hair RNAs (shRNA) to construct WASIR2 knockdown cell lines. Lentiviruses were constructed by transfecting target plasmid and virus packaging plasmids (VSVG, Delta8.9) into 293T cells using Lipo2000 (11668-019, Invitrogen, USA). Lentivirus supernatants were collected after 48h and infected lung cancer cells with the help of polybrene. After 48h, the cells were screened with puromycin at the concentration of 5 µg/ml to obtain stable WASIR2 knockdown or overexpression cell lines.
The miR-374b-5p mimics, miR-374b-5p inhibitors and corresponding negative controls (NC mimics and NC inhibitors) were purchased from RiBio (Guangzhou, China). The transfection of above experiments were carried out using Lipofectamine 2000 according to the manufacturer’s instruction.
RNA extraction and real time quantitative PCR (qRT-PCR)
Total RNA was extracted from human tissues and cell lines using TRIzol reagent (15596-026, Invitrogen, USA) as previously described[15]. cDNA was synthesized using a reverse transcription kit (RR036A, Takara, Japan). Then the expression of WASIR2 and MEX3A were detected using a SYBR Green Real-Time PCR Kit (Qiagen, Germany). GAPDH and U6 were used as endogenous controls. The relative expression levels of indicated genes were calculated using 2-ΔΔCT method, followed by normalization to GAPDH or U6. The premiers used were as follows, 5’-AGCAAGAGCGAAACTCCTT-3’ (F) and
5’-GCGCCCCTAGATGAGTG-3’ (R) for WASIR2;
5’-TGCGCATATAATACAACCTGC -3’ (F)
5’-TCCTCCTCTCCTTCCTTCTC-3’ (R) for miR-374b-5p; 5’-GCCTATGTGAGCCATCCT-3’ (F)
5’-TCTTCTGCCTTTGTGGGT-3’ (R) for MEX3A; 5’-CTCCTCCTGTTCGACAGTCAGC-3’ (F) and 5’-CCCAATACGACCAAATCCGTT-3’ (R) for GAPDH; 5’-TTATGGGTCCTAGCCTGAC-3’ (F) and 5’-CACTATTGCGGGCTGC-3’ (R) for U6.
CCK8 assay
The cell proliferation capability of indicated tumor cells was detected using CCK8 assay. Briefly, 2 x 104 cells were seeded in 96-well plates and incubated for 24, 48 and 72 h, respectively. CCK8 (K1018, APExBIO, USA) solution was added and incubated for 2h. And the absorbance was detected by a microplate reader at 450 nm. All the experiments were repeated at least three times.
Western blot
The cells were lysed with RIPA lysis solution (BL504A, Biosharp Biotechnology, Shanghai, China). The proteins were separated in SDS gel and transferred to nitrocellulose membrane. The membrane was incubated with 5% skim milk for 1 hour and then incubated with primary antibody at 4℃ overnight. Then HRP secondary antibody was incubated with membrane at room temperature for 1h. Finally, ultrasensitive ECL chemiluminescence substrate (BL520A, Biosharp, Shanghai, China) and Amershan Iamger600 imaging system were used for imaging. Anti-MEX3A antibody (ab79046, Abcam, England) and anti-β-actin antibody (ab8227, Abcam, England) were purchased from Abcam.
Migration and invasion
Cell migration and invasion were assessed using transwell assays. For the invasion assay, the upper chambers were coated with Matrigel (356234, BD, USA). 4x105 cells were suspended in serum free culture medium and placed in the upper chambers. After being incubated for 48h, the chambers were fixed with 4% paraformaldehyde for 15 mins and stained with 0.1% crystal violet. After removing cells on the upper surface of the insert, stained cells were washed with PBS and observed under a microscope. The numbers of migrated and invaded cells were assessed by counting the mean numbers in 5 random visual regions
Subcellular fractionation
Fractionated RNA from cytoplasm and nuclei were collected using a PARISTM kit (AM1921, Invitrogen, USA). Then the relative expression of WASIR2 in the nuclei and cytoplasm were assessed using qRT-PCR. U6 and GAPDH were utilized as normalization controls for nuclei and cytoplasm fractionation, respectively.
Dual Luciferase reporter assay
WASIR2 or MEX3A gene contained wild type (WT) or mutated (MUT) version of predicted miR-374b-5p binding sites were purchased from GeneChem. A dual-luciferase reporter assay system (Promega, WI, USA) was applied to measure the luciferase activity of indicated tumor cells.
Statistical analysis
Statistical analyses were assessed using GraphPad Prism, R software or Perl software. The comparison between two groups was calculated using student’s t test. The overall survival was assessed using Kaplan-Meier survival curve with log-rank test. One-way ANOVA was applied to analyze differences among two or more groups. Pearson correlation analysis was used to assess correlations between WASIR2, miR-374b-5p and MEX3A. A two-tailed P value < 0.05 was accepted as statistically significant.