Transmission electron microscopy (TEM)
The study was approved by the Institutional Review Board of Anhui Medical University (No: 20180023). A written informed consent was obtained from all patients prior to sample collection and storage. EC tissues were obtained from the EC patients with total hysterectomy with bilateral salpingo-oophorectomy. Normal endometrium were obtained from biopsies taken for the investigation of reproductive problems. All patients did not undergo chemotherapy, radiotherapy, biotherapy, or any other operation prior to surgery. Endometrial tissues and EC cells were fixed using 2% glutaraldehyde, rinsed with 100 mM cacodylate buffer (pH 7.4), fixed with 1% OsO4 in cacodylate buffer, dehydrated with gradient ethanol (50%, 70%, 80%, 90%, 95%, 100%) at 25 °C, and embedded in Eponate. Ultra-thin sections exposure to double staining with uranyl acetate and 1% lead citrate, and images were captured using a transmission electron microscope (HT7700, Hitachi High Technologies, Japan).
Briefly, the total RNA was isolated from EC and normal tissues using TRIzol reagent (Takara Bio, Japan). On the denaturing agarose gel, electrophoresis were used to validate RNA integrity and DNA contamination. Using pretreated RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA), RNA-seq libraries were constructed. The libraries were denatured as single-stranded DNA, bound to the surface of Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq™ 4000 Sequencer (Illumina, San Diego, CA, USA).
Cells were lysed using the protein extraction solution (Beyotime). Protein concentration was calculated using the BCA assay kit (Sino Biotech, China). Cell lysates were obtained using centrifugation speeds of 12000rpm at 4 °C for 30 min, and they were transferred onto polyvinylidene difluoride membranes (Millipore Corp, Billerica, MA, USA). After blocking the membranes, nitrocellulose blots were incubated for 6 h with primary antibodies diluted in a primary antibody dilution buffer (Beyotime). The primary antibodies recognizing LC3 (ab192890, Abcam), Beclin1 (ab207612, Abcam), P62 (18420-1-AP, Proteintech), NLRC5 (DF13672, Affinity), human leukocyte antigen A (HLA-A, ab52922, Abcam), low molecular mass polypeptides 2 (LMP2, ab184172, Abcam), transporter associated with antigen processing 1(TAP1, ab83817, Abcam), beta-2-microglobulin (β2-M, ab75853, Abcam), and β-actin (GB12001, Servicebio) were used at 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, and 1:5000 dilutions, respectively. The membranes were incubated in Tris-buffered saline with 0.1% Tween 20 (TBST, Boster Bio, China) containing 5% skim milk at 37 °C for 4 h, and subsequently, they were incubated with specific primary antibodies at 4 °C overnight. Next, the membranes were washed thrice with TBST, and they were incubated with -conjugated secondary antibodies (Thermo Fisher Scientific, 1:10000) at 37 °C for 1 h. After washing thrice with TBST, the proteins were visualized using an ECL chemiluminescent kit (ECL-plus, Thermo Fisher Scientific). The protein levels of LC3-II, which represent the protein levels of LC3, were measured. All experiments were performed in triplicate, and they were repeated at least thrice.
RNA extraction and qRT-PCR
Total RNA was collected from cultured cells using the TRIzol reagent (Takara Bio, Japan), following the manufacturer’s instructions. First-strand cDNA was synthesized using a Thermoscript RT-PCR synthesis kit (Fermentas, USA). qRT-PCR analyses for the mRNAs of LC3, Beclin1, P62, NLRC5, HLA-A, LMP2, TAP1, β2-M, and β-actin were performed using Thermoscript qRT-PCR kits (Fermentas) in an ABI Prizm step-one plus real time PCR System (Applied Biosystems, USA). The β-actin mRNA level was used as an internal control. Relative expression levels were calculated based on the standard 2-ΔΔCt method. All experiments were performed in triplicate, and they were repeated at least thrice. qRT-PCR primers are listed in Table 1.
The human EC cell lines HEC-1A, AN3CA, and Ishikawa cells were obtained from American Type Culture Collection, and they were cultured in RPMI-1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) under 5% CO2 at 37 °C.The LC3 plasmid, LC3 plasmid (GFP) + NLRC5 plasmid (RFP, LC3+NLRC5), shRNA-LC3 (GFP), shRNA-NLRC5 (RFP) were constructed, and the RNA sequences of LC3 plasmid, shRNA-LC3, NLRC5 plasmid, and shRNA-NLRC5 are presented in Table 2. LC3 plasmid, shRNA-LC3, NLRC5 plasmid, and shRNA-NLRC5stably expressing HEC-1A, AN3CA, Ishikawa cells were cultured in RPMI-1640 containing 10% FBS and 2 μg/mL (Sigma, USA) under 5% CO2 at 37 °C.Western blotting and quantitative reverse transcription PCR (qRT-PCR)were performed to validate the stable expression of HEC-1A, AN3CA, and Ishikawa cellsin LC3 plasmid, shRNA-LC3, NLRC5 plasmid, and shRNA-NLRC5.
Treatment with the autophagy agonist, rapamycin, or autophagy inhibitor, chloroquine (CQ)
Rapamycin (100 nM, Sigma) and CQ (20 mM, Sigma) were dissolved in dimethyl sulfoxide (Sigma) according to the methods described in previous studies [20, 21]. HEC-1A, AN3CA, and Ishikawa cells were seeded overnight in culture dishes, and they were treated with rapamycin or CQ for 48 h.
HEC-1A, AN3CA, Ishikawa, and their shRNA-NLRC5 cells were fixed in 4% paraformaldehyde (Sigma) for 30 min, washed with phosphate buffered saline (PBS), and then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific) for 20 min. Subsequently, primary antibodies, anti-NLRC5 (DF13672, Affinity) and anti-LC3 (GB11124, Servicebio) were added, and they were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and each section was washed thrice with immunohistochemical washing solution (Beyotime, Shanghai, China) at room temperature for 5 min. Next, secondary antibodies were added, and the tissues were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and each section was washed thrice with immunohistochemical washing solution at room temperature for 5 min. Finally, the immunofluorescence blocking solution (Sigma) was added, sections were mounted, and images were captured using a confocal laser-scanning microscope (Nikon, Eclipse Ti-U, Japan). The double immunofluorescence staining of NLRC5 (DF13672, Affinity) and MHC I (ab281903, Abcam, Cambridge, MA, USA) in NLRC5 HEC-1A cell was operated as above described.
The interactions between LC3 and NLRC5 in HEC-1A, AN3CA, and Ishikawa cells were investigated using CO-IP. Briefly, anti-LC3-labeled beads were co-incubated with HEC-1A, AN3CA, and Ishikawa cells. After 6 h of incubation, the beads were washed twice with TBST, and subsequently, they were eluted with 2XNRSB buffer (Thermo Fisher Scientific) at 95 °C. The eluates were analyzed using western blotting with antibodies against LC3 () and NLRC5 (sc-515668, Santa Cruz, USA).
Glutathione S-transferase (GST) pull-down assay
The interactions between LC3 and NLRC5 were further determined using a GST pull-down assay. The plasmids for GS-NLRC5 and His-LC3 were transfected into Escherichia coli. GST, GST fusion proteins, and NLRC5 were constructed and purified with glutathione Sepharose 4B beads (Sigma). The His-LC3 fusion protein was purified, and it was collected using Ni-NTA beads (Sigma). His-LC3 protein was rotated with GST and GST-NLRC5 at 4 °C for 4 h, and subsequently, the mixture was added to Ni-NTA beads at 4 °C for an additional 4 h. After centrifugation speeds of 3000rpm at 4 °C for 2 min and three washes, the beads were eluted with 30 μL of 1×SDS-PAGE loading buffer (Thermo Fisher Scientific), and subsequently, they were boiled for 10 min and analyzed using western blotting.
Tissue microarrays consisting EC endometrial tissues and thirty-six normal endometrial tissues were examined in the study. Clinical information of EC patients include age, body mass index (BMI), menopausal status, histological subtype, tumor stage, tumor grade, myometrial invasion, and lymph node metastasis. Tumor stage was determined according to the 2009 International Federation of Gynecology and Obstetrics (FIGO) criteria. Histological subtype and tumor grade were assessed according to the World Health Organization guidelines. Clinical information of thirty-six normal endometrial include age and BMI.
Immunohistochemistry assay was performed as previously described to measure the expression levels of LC3 and NLRC5. Briefly, tissue microarray sections were dewaxed and dehydrated in xylene and alcohol respectively, and antigen recovery was accomplished by microwaving in citric saline for 15 min. Subsequently, the sections were deparaffinized and deal with 0.3% hydrogen peroxide for 15 min to suppress endogenous peroxidase activity. Furthermore, the sections were blocked with 2% bovine serum albumin, then incubation with primary rabbit monoclonal antibodies against LC3 (GB11124, Servicebio, diluted 1:500) and NLRC5 (DF13672, Affinity, diluted 1:100) at 4 °C for 16 h. After rinsing, microarray sections were incubated with a biotinylated secondary antibody (G1210-2-A, Servicebio) at 20-25°C temperature for 60 minutes. LC3 and NLRC5 expression levels were visualized using 3, 3¢-diaminobenzidine tetrahydrochloride staining. Slides were counterstained with hematoxylin before dehydration and mounting. Quantitative analyses were conducted for five random fields at ×200 magnification for each endometrial tissue slice. All sections were observed under the Axioscope A1 microscope (Carl Zeiss, Germany) and photographed. The background light of each photo was consistent. Dark brown staining indicated a positive reaction. The intensity of dark brown staining was analyzed using Image-Pro plus 6.0 (Media Cybernetics, Inc.).
To analyze the CD8+ T cell among CD45+ cells from the of mice, CD8+ T cells were collected and concentrated, and they were incubated with 5 µL of specific fluorescent CD8 antibody (100705, Biolegend, USA) and specific fluorescent CD45 antibody (368507, Biolegend) for 20 min. Saline (500 µL) was added separately into the cell solution, which was followed by centrifugation at 5000 rpm for 5 min. The supernatant was removed, and the collected cells at the bottom were dispersed into 300 µL of saline. Finally, the effective separation rate was analyzed by counting 1.2 × 104 cells per sample using an LSR II flow cytometer (BD Biosciences, CA, USA) and Cell Quest software (BD Biosciences).
CD8+ T cell proliferation assay
CD8 T+ cell was purified from peripheral blood mononuclear cells (PBMCs) of healthy female volunteers. Cell purity was checked. CD8+ T cellwas labeled with 1μM CFSE (S8269, selleck, USA) and stimulated with CD3/CD28 antibody and co-cultured with vector transfected, LC3plasmid, and LC3 + NLRC5 HEC-1A cells, in 96-well plates at a ratio of 5: 1 for 3 days. CD8+ T cell was collected, and the dilution of intracellular CFSE caused by proliferation was calculated using flow cytometry.
Enzyme-linked immune sorbent assay (ELISA)
The expression levels of CD8+ T cell secreted interferon gamma (IFN-γ), , and interleukin-2 (IL-2) from the of mice according to manufacturer’s instructions. Samples were run in duplicates. Inter-assay coefficients of variation were calculated using the results obtained in 10 different assays performed at different time points using different plates. Intra-assay coefficients of variation were calculated for 10 replicates of the sample in the same plate.
In vivo xenograft experiments
six-week-old female BALB/C mice were purchased from Hangzhou Ziyuan Laboratory Animal Science and Technology Co. Ltd, and they were reared in an SPF environment. Following this, 5 × 106 HEC-1A cells transfected with LC3 (n=5), LC3+NLRC5 (n=5), shRNA-LC3 (n=5), or vector transfected (n=5) and suspended in RPMI-1640 medium (Invitrogen, USA) were left armpit subcutaneously injected. The tumor volume was measured twice a week until the tumor could be visually observed. After 4 weeks, the mice were euthanized, and the implanted tumors were removed, measured, and photographed. The tumors were weighed, and the volumes were calculated using the following formula: tumor volume (mm3)=(ab2)/2 [a: the longest axis (mm), b: the shortest axis (mm)]. CD8+ T cell among CD45+ cells from the of mice was detected by flow cytometry. The expression levels of CD8+ T cell secreted IFN-γ, , and IL-2 from the of mice were validated using an ELISA kit (R&D Systems, USA), according to manufacturer’s instructions. Immunohistochemistry was performed todetect the CD8+ level from the tumor.All animal feeding and in vivo experimental procedures were conducted in accordance with the Regulations on Laboratory Animal Management of the Animal Experimental Department of Anhui Medical University, and the animal experiment was approved by the Ethics Review Committee of the department of Laboratory Animal Science of Anhui Medical University (No: LLSC201800855).
All data were analyzed using the SPSS 23.0 software (SPSS Inc., Chicago, USA). Data are expressed as mean ± SEM. Statistical analyses were performed using analysis of variance. Significant differences and variance between groups were identified using using Student t-test and F-test respectively. Correlation analysis was performed using Pearson’s correlation. The median LC3 and NLRC5 expression was used as a cut-off value for grouping. The survival curves were measured by the Kaplan-Meier method. P<0.05 was considered statistically significant.