Between July 2020 and October 2022, a total of 32 pairs of CE and adjacent tissues were obtained from 32 female patients with EC (age range: 42-68) at the Third Affiliated Hospital of Guangzhou Medical University. Informed consent forms were signed by all patients or their families, and paracancerous tissues were utilized as control samples. None of the patients included in the research had undergone radiation or chemotherapy. The clinical characteristics of the study participants are listed in Table 1 . The experiment received approval from Guangzhou Medical University Hospital, with the approval number GD2019-036, in compliance with the principles outlined in the Helsinki Declaration (25). The evaluation and classification of the disease’s surgical staging were conducted based on the updated surgical staging criteria for endometrial cancer (EC) established by the International FIGO in 2009. A total of 28 instances of FIGO type III-IV and 14 instances of FIGO type I-II were gathered.

Sample GSE25405, which was split into two groups—non-cancerous endometrial tissue and endometrioid adenocarcinoma tissue—produced differently expressed miRNAs. Sample GSE63678, which was split into two groups—healthy receiver tissue and EC patient tissue—produced the differentially expressed mRNAs. The miRNAs/mRNAs that satisfied the following criteria: P < 0.05; and log2|fold change (FC)| ≥ 2; they were deemed differentially expressed and may be used for pathway enrichment analysis, heatmap creation, volcano plotting, or Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The binding sites of lncRNA/miRNA/mRNA were predicted using the lncBase v.3 and starBase databases. The GEO website provided the raw data (GSE25405 and GSE63678).

EC cell lines were maintained (37°C, 5% CO₂) in DMEM (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco), included HEC-1-A, HEC-1-B, RL95-2, Ishikawa, AN3 CA (American Type Culture Collection), JEC (YRGene), and human endometrial epithelial cells (hEECs; Procell, Wuhan, China). Using Lipofectamine^® 2000 (Invitrogen, Thermo Fisher Scientific, Inc.), target cells were transfected with miR-140-3p mimic/inhibitor, small interfering (si) RNA JPX, or overexpression (ov) PIK3CA plasmid, along with their corresponding negative controls (NC), for a duration of 4 hours. Table 2 shows the sequences for the siRNA-JPX, miR-140-3p mimic/inhibitor, and ov-PIK3CA that were manufactured by GenePharma Biotechnology Co, Ltd. (Shanghai, China).

Centrifugation was used to gather the cells after two PBS washes. The manufacturer’s instructions for the nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) directed resuspending 200 μL with an RNase inhibitor. Reagents containing PMSF were added after 15 minutes, and the mixture was centrifuged (16,000 × g; 10 minutes; 4°C). The supernatant was then collected for RT-qPCR analysis and total RNA extraction.

Biotinylated JPX (Bio-JPX) and the corresponding negative control (Bio-miR-NC) were synthesized by GenePharma. Cells were lysed, and the lysates incubated with the biotinylated RNA probes. Streptavidin-coated magnetic beads (Invitrogen) were added to capture the biotinylated RNA complexes. The pulled-down RNA was extracted and analyzed by RT-qPCR to determine the enrichment of miR-140-3p.

TRIzol reagent was used to extract total RNA from target cells. PrimeScript RT Kit (Takara Bio, Japan) was used to reverse transcribe 10 μL of DEPC water (Invitrogen) into cDNA from the precipitate after centrifugation at 12,000 × g (4°C; 10 min) and supernatant was adsorbed and disposed of. With an Applied Biosystems^® 7500 Real-Time PCR (California, USA) and the Takara SYBR^® Premix Ex TaqTM II kit, RT-qPCR analysis was carried out. The following temperatures were used for PCR experiments: 95°C for 10 minutes, 55°C for 2 minutes, and 72°C for 2 minutes. These were followed by 40 cycles of 95°C for 15 s and 60°C for 32 s. The sequences of the primer pairs are listed in Table 3 . The housekeeping genes GAPDH and U6 served as a reference for normalizing the target RNA levels. The 2^−ΔΔCt method (26), which calculates relative RNA levels, was used.

MiR-140-3p mimic, inhibitor, plasmids expressing wild-type (WT) or mutant (mut) JPX or PIK3CA, and pRL-SV40 reporter vector plasmid were used to transfect Ishikawa and JEC cells using Lipofectamine^® 2000. After 48 hours of incubation, the Dual-Luciferase Reporter Assay System (Promega) was used to assess the amount of luciferase activity in the transfected cells at 490 nm. The firefly luciferase data were normalized using the ratio of firefly to Renilla luciferase activity.

RIPA lysis buffer was used to lyse Ishikawa and JEC cells. A BCA protein assay kit (Solarbio) was used to estimate the protein contents in the lysate. Proteins that had been denatured were separated using 10% SDS-PAGE (Solarbio). The separated protein bands were then placed onto a PVDF membrane, blocked using 5% bovine serum albumin (Solarbio), and left to incubate at 4°C for an entire night using primary antibodies form Abcam (Cambridge, UK) against PIK3CA (1:2000; ab40776), JAK2 (1:3000; ab108596), p-JAK2 (1:1000; ab32101), STAT3 (1:1000; ab68153), and p-STAT3 (1:1000; ab267373). The sections were then treated with goat anti-rabbit antibody (1:5000, ab205718) for 2 hours at 25°C after being washed twice with TBST buffer (Solarbio) for 15 minutes. As a loading control, anti-GAPDH antibody (1:3000, ab181602) was used. The bound proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). Digital images of protein bands were captured using an imaging system (Bio-Rad, USA) and bands were quantified using ImageJ software (National Institutes of Health, USA).

Cell Counting Kit-8 (CCK-8) reagent (Solarbio; 10 μl) was applied at 0 and 72 hours to 96-well plates containing JEC 4 × 10³ and Ishikawa cells. After 60 minutes of dark incubation at 20°C, absorbance was measured at 450 nm with enzyme-labeled instrument (Thermo Fisher Scientific). Transfected cells were seeded onto 6-well plates for the colony formation test, and the plates were then incubated for two weeks. After being stained with 0.1% crystal violet (Solarbio) and treated with 4% paraformaldehyde, colonies were manually counted.

For flow cytometry (FCM), Ishikawa and JEC cells were incubated (15 min) with Annexin V-FITC, followed by the addition of propidium iodide (PI) for another 5 min. FCM (BD FACSCalibur, BD Biosciences, CA, USA) was used to analyze cell apoptosis. Incubation was performed in the dark at 4°C. For acridine orange (AO)/ethidium bromide (EB) staining, cells were stained (20 min) with a mixture of AO and EB (Sigma-Aldrich, MO, USA), visualized under a fluorescence microscope (Olympus, Tokyo, Japan).

Ishikawa and JEC cell suspensions were centrifuged (1,000 × g, 5 min) and immobilized in 70% ethyl alcohol at 4°C overnight. After washing with PBS, the cells were resuspended in PBS containing PI and RNase A (containing 0.2% Triton X-100). After 30 min of incubation at 4°C in the dark, FCM was used to analyze the cell cycle.

For the invasion assay, Matrigel^® coating (BD Biosciences) was first applied at 37°C for 6 h. Ishikawa and JEC cells (1 × 10⁵) were placed in the upper chamber (serum-free medium), and 10% FBS was added to the lower chamber. After 48 h of incubation, the upper chamber was gently rinsed twice with PBS and then fixed with absolute ethanol for 20 min. After natural air drying, cells were stained (20°C, 15 min) with crystal violet. In migration detection the Matrigel coating is omitted. The cells were counted under a light microscope (Olympus Corporation; magnification, 200×).

Data are presented as mean ± standard deviation. Statistical analyses were performed with GraphPad Prism software. Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test was used to compare groups. Pearson correlation analysis was used to analyze relationships between gene expression levels. Differences were considered significant at P < 0.05.

According to our findings, EC tissues had higher levels of lncRNA JPX expression than adjacent non-cancerous tissues ( Figure 1A ). Moreover, there was a correlation found between the expression of JPX and the prognosis of EC, with higher levels in advanced stage EC compared to early stage EC ( Figure 1B ). When compared to hEEC cells, JPX expression was elevated in vitro in six EC cell lines ( Figure 1C ), with Ishikawa and JEC cells exhibiting the highest levels. To better understand the molecular processes of JPX in EC cells, we have chosen these two cell lines. After constructing three siRNAs targeting JPX, we observed a significant downregulation of JPX expression in Ishikawa and JEC cells, especially with si-JPX-1, which showed the best inhibitory effect ( Figure 1D ). We selected si-JPX-1 as a JPX antagonist for further studies. We then found that inhibition of JPX expression reduced cell viability ( Figure 1E ) and colony formation ( Figure 1F ), promoted cell apoptosis ( Figures 1G, H ), and induced cell cycle arrest ( Figure 1I ), while decreasing cell migration and invasion abilities in Ishikawa and JEC cells ( Figure 1J ). Notably, lncRNA JPX was mainly expressed in the cytoplasm, consistent with the positive control GAPDH and opposite to the positive control U6 ( Figure 1K ).

We discovered 150 differentially expressed miRNAs by examining the GSE25405, a dataset ( Figure 2A ). Six putative target miRNAs of JPX were identified by combined analysis utilizing the Lncbase v.2 and starBase databases ( Figure 2B ): miR-140-3p, miR-193a-3p, miR-34a-5p, miR-449a, miR-449b-5p, and miR-92b-3p. Moreover, miR-140-3p was the only miRNA that was downregulated in EC tissues among these six miRNAs. MiR-140-3p expression was shown to be downregulated in cancer tissues ( Figure 2C ) and its expression level was linked to the prognosis of EC, with lower expression in advanced stage EC compared to early stage EC ( Figure 2D ). These findings were obtained by further RT-qPCR research. Compared to hEEC cells, six EC cell lines had lower miR-140-3p expression in vitro ( Figure 2E ). The levels of miR-140-3p and lncRNA JPX showed a negative linear connection, according to Pearson correlation analysis ( Figure 2F ). The connection between JPX and miR-140-3p was examined using dual-luciferase assays after the validation of miR-140-3p mimics and inhibitors ( Figure 2G ). In comparison to the WT-JPX + mimic NC group, the luciferase activity was lower in the co-transfection group of WT-JPX + mimic, according to the findings of the dual-luciferase evaluation. Furthermore, no discernible variations in luciferase activity were seen between the mut-JPX and mimic NC or mimic co-transfection groups ( Figures 2H, I ). Following site modification, RNA pull-down analysis revealed miR-140-3p enrichment in the bio-JPX group, with no discernible alterations when compared to the bio-miR-NC group ( Figure 2J ). Moreover, JPX expression inhibition increased the expression of miR-140-3p ( Figure 2K ), however JPX was not substantially affected by changes in miR-140-3p expression ( Figure 2L ).

We co-transfected inhibitor NC or inhibitor with si-JPX. The decrease of Ishikawa and JEC cell viability ( Figure 3A ), colony formation ( Figure 3B ), apoptosis ( Figures 3C, D ), cell cycle arrest ( Figure 3E ), and cell migration and invasion ( Figure 3F ) was reversed by the miR inhibitor, according to the results.

KEGG analysis revealed that the JAK/STAT pathway might be a critical pathway in EC ( Figure 4A ) and identified 23 mRNAs affecting the JAK/STAT pathway. The GSE63678 data set revealed 1165 differentially expressed mRNAs in EC tissues compared to the normal group ( Figure 4B ). We analyzed the hypothetical target of miR-140-3p using the starBase database and combined them with the 23 mRNAs affecting the JAK/STAT pathway to narrow the scope of the mRNA screening. The results predicted that four mRNAs (PRLR, STAT5B, FOXO1, PIK3CA) were potential targets ( Figure 4C ). Among them, GEO data analysis showed that PIK3CA was the only up-regulated mRNA, with increased expression in cancer tissues ( Figure 4D ) and higher expression in advanced stage EC than in early stage EC ( Figure 4E ). In vitro, PIK3CA expression was upregulated in six EC cell lines compared to hEEC cells ( Figure 4F ). PIK3CA expression was negatively regulated by miR-140-3p ( Figure 4G ) and decreased when JPX expression was inhibited ( Figure 4H ). PIK3CA expression and lncRNA JPX had a positive linear connection, according to Pearson correlation analysis ( Figure 4I ); miR-140-3p and PIK3CA expression had a negative linear association ( Figure 4J ). The luciferase activity was decreased in the WT-PIK3CA + mimic co-transfection group compared to the WT-PIK3CA + NC mimic group. In addition, no differences in luciferase activity between the mut-PIK3CA and either the NC or mimic co-transfection groups ( Figures 4K, L ). Furthermore, suppression of JPX expression in Ishikawa and JEC cells resulted in decreased PIK3CA protein levels and dephosphorylation of JAK2/STAT3 ( Figure 4M ). Under the influence of miR-140-3p inhibitor, this effect was reversed ( Figure 4N ). Thus, PIK3CA may be a critical factor in the lncRNA JPX/miR-140-3p-mediated regulation of the JAK2/STAT3 pathway.

After validating the efficacy of the PIK3CA overexpression vector ( Figures 5A, B ), we found that it reversed the si-JPX-induced reduction of PIK3CA protein levels and dephosphorylation of JAK2/STAT3 ( Figure 5C ). Subsequently, we observed that si-JPX-mediated reduction of Ishikawa and JEC cell viability ( Figure 5D ) and colony formation ( Figure 5E ), increased cell apoptosis ( Figures 5F, G ), cell cycle arrest ( Figure 5H ), and decreased cell migration and invasion abilities ( Figure 5I ) were all reversed by PIK3CA overexpression.