Human endometrial endothelial cell (HEEC) and human endometrial cancer cells (Ishikawa, RL95-2 and KLE) were purchased from the American Type Culture Collection (ATCC, USA) or National Infrastructure of Cell Line Resource (Beijing, China). Cells were incubated in DMEM (Gibco, USA) contained 10% fetal bovine serum (FBS; PAN biotech, Germany) and 1% penicillin/ streptomycin (Solarbio, China) at 37°C and 5% CO2.
Total RNA was extracted using Neurozol reagent (MACHEREY-NAGEL, Germany) and cDNA was generated using reverse transcription reagent kit (PROMEGA, USA). Real time PCR was performed using SYBR Green PCR kit (TaKara, China). U6 and GAPDH are internal controls. The qPCR analysis was then performed on an ABI 7500 Real-time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA) according to the instructions supplied by the manufacturer. The relative expression levels of the genes were calculated by comparing to U6 or GAPDH using 2−ΔΔCT method. The primers were used as follows:
miR-217 FORWARD: CGCGTACTGCATCAGGAACTG;
miR-217 REVERSE: AGTGCAGGGTCCGAGGTATT;
miR-217-5p RT (anti-miR-217) Primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCCAAT; U6 FORWARD: CTCGCTTCGGCAGCACA;
U6 REVERSE: AACGCTTCACGAATTTGCGT;
circ_0023404 FORWARD: ACCGTGGCCATGAAGCTATG;
circ_0023404 REVERSE: GGTCACCATATTGTAGGAGCGT;
GAPDH FORWARD: AGAAGGCTGGGGCTCATTTG;
GAPDH REVERSE: AGGGGCCATCCACAGTCTTC;
MAPK1 FORWARD: CAGTTCTTGACCCCTGGTCC;
MAPK1 REVERSE: GTACATACTGCCGCAGGTCA.
si-NC (negative control) sequence: UUCUCCGAACGUGUCACGUTT, si-circRNA (si-hsa_circ_0023404 #1-3; #3 sequence: GGUUCCUGCUAAUCUAUAATT, miR-217 or anti-miR-217 were synthesized by GenePharma(Shanghai, China). They were transfected in Ishikawa cells using Lipofectamine 3000 Reagent (Life Technologies, USA) and then culture in at 37°C and 5% CO2 for 48-72 hrs.
Detection of cell proliferation by CCK-8 assay
Ishikawa cells were plated at 2×10E3 cells/well in 96-well plates and grown in medium containing 10% FBS for 24 hrs. After transfection with siRNA , 10 μl of cell count kit-8 (CCK-8, CK04, Dojindo, Japan) was added into each well and cells were incubated for 2 hrs in a 5% CO2 incubator at 37°C. The absorbance of each well at 450 nm was read in GloMax™ 96 MICROPLATE (Promega, USA).
Colony formation assay
Ishikawa cells were transfected with siRNA for 48 hrs and trypsinized and dispensed into 6-well plates with a density of 800 cells/well. When the number of cells in a colony is more than 50, 10% formaldehyde was employed to fix colonies for 10 min and 0.5% crystal violet was adopted to stain colonies for 5 min. Images were photographed and the number of colonies was calculated by ImageJ.
Transwell migration and invasion assay
For migration assay, transfected Ishikawa cells (1×10E5 cells) were suspended in 200ul serum-free medium and then seeded on the top chamber. Medium contained 10% FBS was added into the lower chamber. After 24 hrs of incubation, cells on the lower surface of the lower chamber were fixed with 4% PFA and stained with 0.1% crystal. Cell were counted from five randomly selected microscopic fields. For invasion assay, Transwell inserts (Fisher Scientific, USA) were coated with Matrigel (BD, USA). After 24 hrs incubation, cells on the upper surface of the Transwell membrane were gently removed, and cells on the lower surface of the Transwell membrane were fixed and stained with crystal violet, counted from five randomly selected microscopic fields.
Cells were collected and lysed with RIPA buffer (Beyotime, China). Equal amount of protein was separated on SDS-PAGE and transferred to PVDF (Millipore, USA). Then, the membranes were incubated with the primary antibodies anti-MARK1 (Cat:125403, Novopro, China) and anti-actin (Simga, USA). ECL substrates were used to visualize protein bands (Millipore, USA).
All experiments were replicated thrice and all data was expressed as mean + standard deviation (SD). The software GraphPad 8.0 were used to carry out all statistical analyzes. Student's t-test and one-way ANOVA followed by Bonferroni 's post hoc test were utilized to analyze 2 or multiple groups, respectively. * means p < 0.05; ** means p < 0.01; *** means p < 0.001.