(B) Luciferase activity was decreased 48 h following treatment with miR-183-5p mimics and Ezrin 3UTR-wt, suggesting that miR-183-5p regulated the expression of Ezrin (n=3)

(B) Luciferase activity was decreased 48 h following treatment with miR-183-5p mimics and Ezrin 3UTR-wt, suggesting that miR-183-5p regulated the expression of Ezrin (n=3). inhibitors, small interfering RNA targeting Ezrin or miR-183-5p inhibitors. Cell proliferation, cell cycle, apoptosis, migration and invasion were then evaluated using an MTT assay, flow cytometry, scrape test and Transwell assay, respectively. Compared with normal adjacent tissues, the expression of miR-183-5p was decreased in endometrial malignancy tissues, and the expression of Ezrin was significantly increased in endometrial malignancy tissues. The protein expression of Ezrin was correlated with the severity and poor prognosis of endometrial malignancy. Notably, the target prediction program and the luciferase reporter gene assay confirmed that miR-183-5p targeted and negatively regulated the expression of Ezrin. experiments revealed that this increased expression of miR-183-5p and decreased expression of Ezrin inhibited EMT, cell K-Ras-IN-1 proliferation, migration and invasion, but promoted cell apoptosis in Ishikawa cells. These results suggested that this upregulated expression of miR-183-5p promoted apoptosis and suppressed the EMT, proliferation, invasion and migration of human endometrial malignancy cells by downregulating Ezrin. luciferase (Takara Biotechnology Co., Ltd., Dalian, China) was used as the internal research for K-Ras-IN-1 transfection efficiency to adjust for the number of cells. miR-183-5p mimics and unfavorable control (NC) were co-transfected with luciferase reporter vectors into 293T cells (CRL-1415; Shanghai Xinyu Biotechnology Pharmacuetical Co., Ltd., Shanghai, China), and the luciferase activity was detected according to the methods provided by Promega. At 48 h post-transfection, the culture medium was discarded, and the cells were washed twice with PBS. Passive lysis buffer (100 luciferase activity was used as the relative luciferase activity. The experiment was independently repeated three times. Cell culture The five endometrial malignancy cell lines (Ishikawa, KLE, JEC, HEC-1-A, and HHUA cells) were purchased from Shanghai Fu Xiang Biotechnology Co., Ltd. (Shanghai, China) The cell lines were all cultured in Dulbecco’s altered Eagle’s medium (DMEM)-F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in a 5% CO2 incubator at 37C. The cells were passaged every 3C4 days, and the fourth generation cells were utilized for the experiments. RT-qPCR analysis was performed to determine expression of miR-183-5p in the five endometrial cell lines NBN to identify the cell collection with the highest expression for the subsequent experiments. Cell transfection and grouping The cells were assigned into the blank group (no transfection), the unfavorable control of miR-183-5p (NC) group, the miR-183-5p mimic group (transfected with miR-183-5p mimics), the miR-183-5p inhibitor group (transfected with miR-371-5p inhibitors; GenePharma Biological Co., Ltd. Shanghai, China), the small interfering RNA (si)Ezrin group (transfected with siEzrin from GenePharma Biological Co., Ltd.) and the miR-183-5p inhibitor + siEzrin group (transfected with miR-183-5p inhibitors and siEzrin). The cells were seeded into a 50 ml culture flask and were cultured in total medium to 70C80% density. Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and DNA were prepared in a sterile Eppendorf tube, and 5 l of Lipofectamine 2000 and 100 l of serum-free medium were incubated at room heat for 5 min. siRNA (50 nmol) and 100 l of serum-free medium were incubated at room heat for 20 min. The cells in the culture flask were washed. Serum-free medium (without antibiotics) K-Ras-IN-1 was added to the complex, which was then mixed, and the combination was added into the 50 ml culture flask for transfection. The flask was placed in an incubator made up of 5% CO2 at 37C for 6C8 h, and the reagent was then replaced with total culture medium. Finally, the cells were transfected for 48 h for further experiments. MTT assay When the Ishikawa cells of each group reached a density of ~80%, the cells were washed twice with PBS. The cells were detached with 0.25% trypsin and were then made into a single cell suspension. Following counting, the cells.