The study was approved by the Ethics Committee of Shaanxi Provincial People's Hospital and was performed according to the principles of the Declaration of Helsinki. All experiments were performed in accordance with relevant guidelines and regulations, and informed consent was obtained from the participants. Tumor tissue samples were obtained from complete staging of endometrial carcinoma (bilateral salpingo-oophorectomy and pelvic and para-aortic lymphadenectomy) with previously untreated EC and were divided into premenopausal (pre-ca group) and postmenopausal (post-ca group) groups according to whether the patients were menopausal or not. All samples were histologically classified and graded by a clinical pathologist according to WHO guidelines. Healthy endometrial tissues (N group) were collected as negative controls from patients undergoing hysterectomy or curettage procedures for benign problems, such as endometrial hyperplasia and uterine myoma, and did not have a diagnosis of any type of cancer or prior cancer history.
Patient set 1 used for next-generation sequencing consisted of samples from 6 individuals (Table S4): 3 premenopausal EC tissue samples and 3 premenopausal normal endometrial tissue samples. For validation of the sequencing results, RNA samples from a second series of tumor and healthy tissues were prepared to examine miRNA expression via quantitative real-time PCR (qRT-PCR). Patient set 2 consisted of 90 samples (20 cases in the pre-ca group, 40 cases in the post-ca group and 30 cases in the N group) (Table S5).
All samples were collected at the Department of Gynecology of Shaanxi Provincial People's Hospital, First Affiliated Hospital of Xi 'an Jiaotong University and Department of Female Tumor of Shaanxi Provincial Cancer Hospital from January 2017 to September 2019. Tissues were immediately snap-frozen in liquid nitrogen and then transferred to a -80°C freezer following surgery.
RNA Extraction, Small RNA Library Construction and Next-Generation Sequencing
Total RNA was extracted using a NAfast1000 Total RNA extraction kit (Piomeer, Xian, China) according to the manufacturer's instructions. RNA concentration and purity was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) at O.D. 260 nm. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent and Bioanalyzer 2100 System (Agilent Technologies, CA, USA) and RNA integrity number (RIN)>8.0 is the cutoff for RNA sequencing.
Sequencing libraries were generated according to the manufacturer's instructions using the NEBNext UltraTM small RNA Sample Library Prep Kit for Illumina (NEB, USA), and the index codes were added to the property sequence of each sample. Truseq PE Cluster KITV3-CBOT-HS (Illumina) was used to cluster the index-coded samples on the CBOT Cluster Generation System according to the manufacturer's instructions. After the cluster was generated, the library was prepared for sequencing, and reads were generated on the Illumina platform. Sequencing was performed at Biomarker Technologies (Beijing).
Bioinformatics and Data Processing
DEMs between premenopausal EC specimens and premenopausal normal endometrial tissue specimens were identified via the edgeR package. The resulting false discovery rate (FDR) was adjusted using the posterior probability of being DE (PPDE). An adjusted P-value<0.05 and a |FC|≥1.5 were the thresholds for significant differential expression.
Functional Enrichment Analysis of DEM Target Genes
The GO database is a structured standard biological annotation system containing three major branches, namely, the biological process, molecular function and cellular component branches. KEGG is a database resource to understand the advanced functions and utilities of biological systems. GO enrichment and KEGG pathway analyses of the target genes of DEMs were implemented by the clusterProfiler R package.
Detection of DEMs
The quantification of miRNAs was performed by a two-step reaction using an Evo M-MLV RT Kit for qPCR (AG, China, Hunan) and a SYBR Green Premix Pro Taq HS qPCR kit (Pioneer, China, Xi'an) for validating the expression of miRNAs(Table S6). qPCR was carried out using an ABI7500 fluorescence quantitative PCR instrument (ABI, USA). Cycling conditions were as follows: 95℃ for 5 min, followed by 40 cycles at [95°C for 20 sec, 50°C for 30 sec, 72°C for 30 sec, and 95°C for 15 sec], 50°C for 1 min, and 95°C for 30 s. All reactions were performed in triplicate. All samples were analyzed using the endogenous reference RNA RNU6B. The fold changes in miRNA expression were calculated using the 2-ΔΔCt method, where ΔΔCt=(ΔCt target-ΔCt control) Sample2-(ΔCt target-ΔCt control) Sample1.
The miRNA expression data were statistically analyzed using the Statistical Product and Service Solutions (SPSS) Statistical Package, Version 23.0 (Chicago, Illinois). All measurement data are expressed as the mean+s.d., and χ2 test was used for counting data. Student’s t-test or the Mann-Whitney U test was used to compare two groups, according to the result of the Shapiro-Wilk normality test. All statistical tests were two-sided, and P-values<0.05 were considered significant.