Cell Lines and COAD Tissues
Normal human colon mucosal epithelial cell line (CCD-18Co) and six COAD cell lines (SW480, HT29, LS174T, HCT116 and DLD-1) were procured from ATCC. The above cells were cultured in RPMI 1640 medium or DMEM (Invitrogen, USA) with 10% fetal bovine serum (Invitrogen, USA) and 1% Penicillin/Streptomycin (Sigma-Aldrich, USA) at 37 ºC incubator containing 5% CO2. 82 newly diagnosed patients with COAD in The Fifth Hospital of Wuhan were included in the present study. All the experiments were carried out according to the principles of The Fifth Hospital of Wuhan.
The design and construction of shRNAs and si-RNA for ACTA2-AS1 and BCL2L11, the synthesis of pcDNA 3.1 miR-4428 mimics vector, and the construction of a lentiviral vector overexpressing ACTA2-AS1 and BCL2L11 were separately conducted by Genechem (Shanghai, China). The transfection was carried out using Lipofectamine 2000 reagent (Invitrogen, USA) according to the guideline of manufacturer.
RNA Extract and Quantitative Real-Time PCR (qRT-PCR) Assay
Trizol reagent (TaKaRa, China) was used to extract total RNA according to the manufacturer’s instructions. The PrimeScript RT Master Mix (TaKaRa, China) was used to reverse-transcribe lncRNA and mRNA. The SYBR Premix Ex Taq II Kit (TaKaRa, China) was employed to carry out Real-time PCR. The relative RNA expression was normalized to the expression levels of U6 and GAPDH.
Cell Viability and Colony Assay
The Cell Counting Kit-8 (CCK-8) (Dojido, Japan) experiment was used to measure cell viability. COAD cells were seeded into 96-well plates at 37°C incubator with 5% CO2. After transfection, the OD 450 values were measured at 0, 24, 48, and 72 hours (h) via CCK-8 assay after incubation for 2 h at 37 °C. Then, the absorbance values were measured on a microplate reader at 450 nm. For colony formation assay, 2000 SW480 and HT29 cells were seeded in 6-well plates and cultured for seven to ten days at 37 °C incubator with 5% CO2. Then, the cells were washed twice with PBS (phosphate-buffered saline), fixed with 4% paraformaldehyde (Sinopharm Chemical, China) and stained with crystal violet (Sigma, USA) for 15 min respectively. The clone spots were counted observed under a microscope (Olympus, Japan) with 5 random view fields.
Apoptotic assay was performed using V-FITC Annexin and PI Apoptosis Detection Kit (Beyotime, China). SW480 and HT29 were fixed in 70% cooled ethanol and stained with Annexin V-FITC and PI for 20 minutes at room temperature according to the protocol, and then cell apoptosis was detected by flow cytometer.
Dual Luciferase Reporter Assay
The 3ʹ-UTR of BCL2L11 containing wild type (wt) and mutant type (mut) miR-4428 binding sequence was inserted downstream of the firefly luciferase gene in psi-CHECK2 vector to synthesis the BCL2L11-wt or ACTA2-AS1-wt and psi-CHECK2- BCL2L11-mut or ACTA2-AS1-mut plasmids, respectively. The wt and mut plasmids subsequently were co-transfected into HEK293T cells with negative control and miR-4428 mimics. After transfection, the relative luciferase activity was measured via the Dual-luciferase reporter Assay System.
Protein was extracted using Radioimmunoprecipitation assay (RIPA, Beyotime, China). Protein samples were separated through 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF (polyvinylidene fluoride) membranes (Thermo Fisher Scientific, USA). Membranes were blocked in 5% bovine serum albumin (BSA) for 2 h and then incubated with primary antibodies, including anti-Bcl-2, anti- Bax and GAPDH (Cell Signaling Technology, USA) overnight at 4 °C. Next day, after washed with PBST for three times, the membranes were incubated with the HRP‐conjugated secondary antibodies (Cell Signaling Technology, USA) at 37 °C for 1 h. Finally, the intensity of the bands was visualized by using enhanced chemiluminescence (ECL).
Tumor Xenografts in Nude Mice
BALB/c nude mice (16–20g, 5 weeks of age) were purchased from Shanghai Animal Laboratory Center (Shanghai, China). Cells transfected with sh-NC or sh-ACTA2-AS1 were digested with 0.25% trypsin, diluted in PBS, counted by trypan blue staining, adjusted to a concentration of 1.0 × 10^7cells/mL, and 0.1ml (1.0 × 10^6 cells) of this solution was injected hypodermically into the back flank of each mice. Tumor size was calculated every 7 days. Additionally, the present investigation was approved by the ethics committee of The Fifth Hospital of Wuhan.
Bioinformatics Analysis and Binding Sites Prediction
The Cancer Genome Atlas (TCGA, https://www.cureline.com/the-cancer-genome-atlas.html) database from Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/) was used to explore the expression of ACTA2-AS1 in COAD patients. The potential miR-4428 binding sites to 3ʹUTR of BCL2L11 was predicted by Starbase (http://starbase.sysu.edu.cn/) and the ACTA2-AS1 binding sites to the miR-4428 was predicted by lncBASE database (http://carolina.imis.athenainnovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted) to study the potential crossing network among ACTA2-AS1, miR-4428 and BCL2L11.
The data in in this study were presented as mean ± standard deviation (SD). The data were visualized through the GraphPad Prism 7.0 software. All statistical analysis was conducted via SPSS 20.0. p < 0.05 presented statistically significant.