10 endometrial carcinoma and 10 normal endometrial specimens were obtained from patients with surgical resection. No patients received any chemotherapy or radiotherapy before the surgery. Tissue samples were processed based on the ethical standards. We had obtained the informed consent form all the patients before enrollment. This research was approved by the Ethics Committee Beijing Friendship Hospital Affiliated to Capital Medical University.
RL-95-2, Ishikawa, HEC-1B, KLE and HHUA cells and hESCs (human endometrial stromal cells) and were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). DMEM added with 100 U/mL penicillin/streptomycin and 10% FBS. A 5% CO2 incubator at 37 °C was used to incubate the cells.
shRNAs for LINC00665 were constructed by Invitrogen (Carlsbad, CA, USA). 1 × 106 cells per well were grown into six-well plates overnight and infected with the vector. The efficacy of transfection was tested using PCR. HMGA1 siRNA (RiboBio, Guangzhou, China). Lipofectamine 3000 (Invitrogen, CA, USA) was utilized to do cell transfection.
Cell viability was assessed by CCK-8 assay. 2000 cells were seeded into 96-well plates. Absorption was tested using a CCK-8 kit (Dojindo, Japan) at different time points. OD values were evaluated at 490 nm by a spectrophotometer (BioTek, Winooski, VT, USA).
A Cell-Light EdU DNA Cell Proliferation Kit (KeyGEN BioTECH, China) was carried out to evaluate cell proliferation. Briefly, cells were grown into 96-well plates. Then, cells were treated with 50 μmol/L EdU for 2 hours. Then, cells were fixed using 4 % paraformaldehyde and then 1×Apollo reaction cocktail was used. Cell nuclei was stained using DAPI for 15 min. We captured the images using a fluorescence microscope (Nikon, Tokyo, Japan).
PI and FITC-labeled annexin V (BD Biosciences, New Jersey, USA) staining was used to assess cell apoptosis. Cells were washed twice using PBS and resuspended using 100 μL 1× binding buffer, and then incubated with 5μL FITC-annexin V and PI with no light. Then, 400 μL 1× binding buffer was used. Within 1 hour, cells were exposed to flow cytometry analysis.
Cell cycle assays
Cells cultured in six-well plates, were trypsinized for cell cycle analysis. Then, after cells were washed using PBS, 70% ice-cold ethanol was added to cells at -20°C for 2 hours for a whole night. Cell cycle detection kit and flow cytometry were used to determine cell cycle.
Wound healing assay
Cells were seeded into 6-well plates. To form wounded gaps, cell layers were scratched using a 200 μL tip. Cells were cultured in FBS-free medium with 20μg/mL mitomycin C. We photographed and analyzed the wounded gaps were at 0 and 24.
Cell invasion assays
To carry out cell invasion experiment, we used matrigel Transwell Cell Culture chambers (BD Biosciences, San Jose, CA, USA). Matrix was put into the upper chamber of the chamber. 5× 104 cells were incubated in 200 μL FBS-free culture medium and then, added to the upper chambers. Then, 600 μL culture medium containing 10% FBS was placed to the lower chambers. Then, cells were fixed using 4% paraformaldehyde. Cells in the lower chamber were stained using crystal violet and imaged under an Olympus fluorescence microscope.
TRIzol reagent (Takara, Tokyo, Japan) was used to extract total RNA. PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) was employed to do reverse transcription. Then, SYBR green (Takara, Tokyo, Japan) was conducted to do qRT-PCR on Bio-Rad CFX96 system to test the expression of LINC00665 and HMGA1 mRNA. 2−ΔΔCt method was carried out to analyze gene expression with primers listed in Table 1.
Protein was separated on 10% SDS polyacrylamide gels. Afterwards, proteins were transferred onto PVDF membranes. The membranes were blocked using 5% skimmed milk. Then, primary antibodies against HMGA1 and GAPDH (1:1000, CST, Boston, MA, USA) were used for a whole night. Next, the membranes were washed using TBST and incubated with the secondary antibodies (1:2000, CST, Boston, MA, USA) for 2 hours. Finally, we visualized the protein bands using the enhanced chemiluminescence reagent.
RIP assay was performed using Magna RIP Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Briefly, KLE cells were lysed using RIP lysis buffer. 100μL cell extract was incubated with magnetic beads conjugated to human anti-HMGA1 or normal mouse IgG.
Twelve 8-week-old female mice were bought from Shanghai Animal Laboratory Center. The mice were maintained in specific conditions with no pathogen. Then, mice were divided into 2 groups and injected with 5 × 106 KLE cells infected with LV-shLINC00665 or LV-NC, respectively. We measured tumor volume every week. 7 weeks later, the animals were sacrificed for future histopathological determination. Approval of this animal study was obtained from the Animal Research Ethics Committee of Beijing Friendship Hospital Affiliated to Capital Medical University.
Tumor tissues were fixed using 4 % paraformaldehyde and embedded in paraffin. H&E staining (Beijing Solarbio, Beijing, China) was carried out based on the manufacturer’s protocols. To carry out immunohistochemistry analysis, Ki-67 were detected in xenograft tumor tissues. The sections were deparaffinized, hydrated, and then antigen retrieved. Primary antibody (Ki-67, 1:500, Abcam, UK) was utilized and then a solution of anti-rabbit IgG was used for 15 min. Then, a 3,3-diaminobenzidine color kit was used. We captured the pictures using a light microscope.
Data were analyzed by SPSS 20.0 (Chicago, USA) and GraphPad Prism 7 (GraphPad, CA, USA). Statistical significance between various groups was analyzing using Student’s t tests or one-way ANOVA analysis. P < 0.05 indicated the statistical significance.