CREB1 regulates KPNA2 by inhibiting mir-495-3p transcription to control melanoma progression

Specimens

From January 2021 to February 2022, after receiving written informed consent, 30 patients at Xiangyang First People’s Hospital Affiliated to Hubei Medical College with melanoma and surgical tumour samples and adjacent non-tumour tissues were recruited. The selected patients did not receive chemotherapy /or radiotherapy. This study was approved by the Ethics Committee of Xiangyang First People’s Hospital Affiliated to Hubei Medical College, and written informed consent was obtained from all patients.

Cell lines and culture

Procell Life Science and Technology Co. Ltd. (Wuhan, China) provided the HEMa-LP cells and melanoma cell lines, including A375, A2058, B16, and MUM2B cells used in this study. The cells were cultured in Dulbecco’s modified Eagle medium containing 1% penicillin‒streptomycin solution (P/S) and 10% FBS (DEME, Gibco, New York). Cells were cultured in a humidified incubator at 37 °C and 5% carbon dioxide.

Cell transfection

GeneChem (Shanghai, China) synthesized miR-495-3p mimics and its negative control NC mimics.

The full-length sequences of CREB1 and KPNA2 were obtained by POLYMERase chain reaction and cloned into the eukaryotic expression vector PC DNA3.1 for efficient expression of CREB1 and KPNA2. These plasmids (50 nM) were introduced into A375 and B16 cells using a liposome™3000 transfection agent (Invitrogen, California, USA).

qRT‒PCR

Total RNA of A375 and B16 cells was extracted with TRIzol reagent (Invitrogen), and PrimeScript RT Reagent Kit (Invitrogen) was used to reverse transcribe 1 µg of total RNA to complementary DNA (cDNA) in a final volume of 10 µL. MiRNAs were collected by mirVana microRNA Isolation kits (Invitrogen), and miR-495-3p levels were detected by using the TaqMan microRNA assay kit (Invitrogen). U6 RNA served as an endogenous control. Subsequently, CREB1 and KPNA2 gene levels were quantified by qRT‒PCR. GAPDH was used as the endogenous control for data analysis. The change in mRNA level was calculated by the 2−ΔΔCT method. The primers used are shown in Table 1.

EdU assay

A375 and B16 cells were added to a 96-well plate at 5 × 104 cells/well and incubated at 37 °C for 12 h. An EdU staining kit (Guangzhou RiboBio, China) was used for staining. Afterwards, EdU-labelled cells were analysed using a MoFlo Astrios (Beckman Coulter, Brea, CA, USA).

Transwell assay

The migration and invasion abilities of A375 and B16 cells were detected by Transwell assays. Cells (1 × 105/well) were added to the upper cavity of the inlay, and 600 µL culture medium containing 20% foetal bovine serum was added to the lower cavity. After culturing for 30 min, the migration and invasion of the cells stained with crystal violet were observed by microscopy.

Flow cytometry

The apoptosis rate was detected using an Annexin V-FITC/PI apoptosis detection kit (Beyotime, Nanjing, China, C1062S) based on previous research [14]. In detail, after exposure of 1.0 × 106 cells to 10 mL binding buffer and 1.25 mL Annexin V-fluorescein isothiocyanate, cells were incubated at room temperature for 15 min in the dark. Then, the cell suspension was centrifuged at 1000 ×g for 5 min, and the supernatant was removed; the cells were resuspended in 0.5 mL ice-cold 1× binding buffer. Finally, the cells were incubated with 10 mL propidium iodide and transferred to a fluorescent activated cell sorter tube (Fahrenheit, Munich, Germany). Fluorescence data analysis was performed with WinMDI 2.8 software.

Dual-luciferase reporter assay

Potential binding sites for CREB1 in the promoter of miR-495-3p, miR-495-3p and KPNA2 were predicted by using StarBase bioinformatics software. Plasmid construction and luciferase activity assays were performed as previously reported [5].

CHIP assay

Chips were analysed according to the instructions of EZ-Chiptm Chromatin Immunoprecipitation Kit (Sigma‒Aldrich, St. Louis, Missouri, USA). Chromatin fragments of 200 ~ 500 bp were immunoprecipitated with a specific antibody or negative control antibody. DNA released from the protein‒DNA complex was collected and purified with magnetic beads for RT‒qPCR analysis.

Statistical analysis

In this study, a mean ± standard deviation (SD) represents data from three independent trials. The t test was used for comparisons between two groups, and Tukey’s multiple comparison test was used for comparisons between multiple groups. P < 0.05 indicated a statistically significant difference.

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