A total of 40 paired cancer-adjacent and UCEC tissue samples were obtained from January 2018 to December 2019 and frozen for further analysis. All volunteers (mean age, 57.78) provided written informed consent and the present study was approved (approval no. 2021PS723K) by the Ethics Committee of Shengjing Hospital of China Medical University (Shengjing, China) and in accordance with the Declaration of Helsinki. The inclusion and exclusion criteria were as follows: The patients with UCEC included in this study were diagnosed by histopathological detection. The tumor tissues and their adjacent normal tissues were collected by surgical resection. None of these patients received radiotherapy or chemotherapy before the surgery. The patients that had undergone non-curative resection, cancer recurrence, severe injury of vital organs or had a history of autoimmune diseases were excluded. The clinicopathological characterisitics of the patients with UCEC included in the present study are provided in Table I.

Human endometrial cancer cell lines Ishikawa (cat. no. iCell-h113), RL95-2 (cat. no. iCell-h182), HEC-1-B (cat. no. iCell-h084), AN3CA (cat. no. icell-h018), and HEC-1-A (cat. no. iCell-h083) were purchased from iCell Bioscience, Inc. Ishikawa, AN3CA, and HEC-1-B cells were grown in MEM medium (Beijing Solarbio Science & Technology Co., Ltd.). HEC-1-A cells were cultured in McCoy's 5A medium (Procell Life Science & Technology Co., Ltd.). RL95-2 cells were cultured in DMEM/F12 medium (Procell Life Science & Technology Co., Ltd.). All the culture media were supplemented with 10% FBS (Sangon Biotech Co., Ltd.) and cells were cultured in a CO₂ incubator at 37°C.

Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/) was used to analyze the different expression of genes in various human cancers. There was a binding relationship between BLACAT2 and hsa-miR-378a-3p, as predicted by starBase (https://starbase.sysu.edu.cn/agoClipRNA.php?source=lncRNA). Furthermore, 49 target genes common to miRDB (http://www.mirdb.org/), TargetScan (https://www.targetscan.org/vert_72/) and starBase were identified. Jasper database (https://jaspar.genereg.net/) was utilized to show the association between YY1 and BLACAT2 promotor region.

The helper plasmids pSPAX2 and pMD2.G were obtained from Hunan Fenghui Biotechnology Co., Ltd. For lentivirus packaging, 293T cells (cat. no. ZQ0033; https://www.zqxzbio.com/Index/p_more/pid/486.html; Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.) were cultured to a confluence of ~70% and then transfected with pSPAX2 (3 µg), pMD2.G (2.2 µg), and the plasmids with overexpressed BLACAT2/Negative control (5 µg) or shRNA against BLACAT2/YY1/Negative control (5 µg) using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) at 37°C according to the manufacturer's protocols. At 6 h post transfection, the culture medium of the cells was changed into fresh DMEM medium (Wuhan Servicebio Technology Co. Ltd.) with 10% FBS (37°C and 5% CO₂). At 48 h post trasfection, the cell supernatant was harvested as packaged lentivirus and filtered through a 0.45-µm pore size filter.

To establish the stable UCEC cell line with overexpressed BLACAT2 or knocked down BLACAT2, the AN3CA and HEC-1-A cells were selected and infected with the corresponsing lentiviruses at a multiplicity of infection (MOI) of 10. The puromycin (0.9 µg/ml) was used for stable cell line selection, while 0.3 µg/ml of puromycin was for maintenance.

To clarify the function of miR-378a-3p in UCEC, HEC-1-A cells were transfected with miR-378a-3p mimics (100 pmol) or negative control (NC; 100 pmol) mimics using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

To investigate the role of YY1 in the regulation of BLACAT2 underlying UCEC development, AN3CA cells with stably overexpressed BLACAT2 were infected with lentivirus encoding shRNA against YY1 (Lv-shYY1) at a MOI of 10.

The shRNAs against BLACAT2, YY1 and NC were synthesized by General Biological System and inserted into the lentiviral vector. The sequence information used in the present study was as follows: shBLACAT2#1, 5′-GGTTAAGACATTTCTACAAAT-3′; shBLACAT2#2, 5′-GAGTGGAGAGACTCAGCTACC-3′; shYY1, 5′-CGACGACTACATTGAACAAAC-3′; shNC, 5′-TTCTCCGAACGTGTCACGT-3′; miR-378a-3p mimics sense, 5′-ACUGGACUUGGAGUCAGAAGGC-3′; NC mimics sense, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′.

All cells after transfection were cultured at 37°C and 5% CO₂. Overexpression/knockdown efficiency was assessed using reverse transcription-quantitative PCR (RT-qPCR).

UCEC cells (6×10⁵/well; 6-well plate) or tumor tissues were lysed in TRIpure (BioTeke Corporation) to isolate the total RNAs. The miRNAs were reverse transcripted into cDNA using the miRNA First Strand cDNA Synthesis Kit according to the manufacturer's instructions (Sangon Biotech Co., Ltd.). LncRNA and mRNA were synthesized into cDNA via reverse transcription PCR. The thermocycling conditions for reverse transcription PCR were as follows: Step 1, a total of 12.5 µl reaction was prepared by adding 1 µg RNA, 1 µl oligo (dT)₁₅, 1 µl random primers, and ddH₂O. The reaction was incubated at 70°C for 5 min and then placed on ice for 2 min; step 2, 2 µl dNTP, 4 µl 5X buffer, 0.5 µl Rnase inhibitor, and 1 µl Super M-MLV reverse transcriptase (BioTeke Corporation) were added into the reaction. The total reaction samples were then incubated at 25°C for 10 min, 40°C for 50 min, and 80°C for 10 min.

The cDNAs were amplified and detected using 2X Power Taq PCR MasterMix (BioTeke Corporation) and SYBR Green fluorescence (SYBR Green qPCR kit; Sigma-Aldrich; Merck KGaA) on an Exicycler^™ 96 Real-Time System (Bioneer Corporation). The thermocycling conditions for qPCR were as the following: Step 1, 94°C for 5 min; step 2, 94°C for 15 sec; step 3, 60°C for 25 sec; step 4, 72°C for 30 sec; scan and step 2, 40 cycles; step 5, 72°C for 5 min 30 sec; step 6, 40°C for 2 min 30 sec; step 7, melting 60–94°C every 1.0°C for 1 sec; and step 8, 25°C for 1–2 min. The mRNA levels of the target genes were quantified using the 2^−ΔΔCq method (36). β-actin was the reference for the quantification of BLACAT2 and YY1. The reference gene U6 was used for miR-378a-3p quantification. Primers for the genes assessed were as follows: BLACAT2 forward, 5′-GAGGAGGAGAAGCAATCA-3′ and reverse, 5′-GGAGCCCATCCATTAGAG-3′; YY1 forward, 5′-ACCCACGGTCCCAGAGT-3′ and reverse, 5′-AAAGCGTTTCCCACAGC-3′; miR-378a-3p forward, 5′-ACTGGACTTGGAGTCAGAAGGC-3′. All primers were synthesized by Genscript.

Proteins samples in cells or tumor tissues were extracted using cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology). Protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology). Subsequently, 20–40 µg proteins in each lane were separated on the SDS-PAGE gel (11% or 8%). The 11 or 8% gel was respectively selected for protein separation according the molecular weight of the target proteins. The proteins were then transferred onto PVDF membranes and blocked with a 5% skimmed milk solution at room temperature for 1 h. The membranes were incubated with the primary antibodies against MMP2 (1:500; cat. no. 10373-2-AP; ProteinTech Group, Inc.), MMP9 (1:1,000; cat. no. 10375-2-AP; ProteinTech Group, Inc.), cyclin D1 (1:500; cat. no. A19038; ABclonal Biotech Co., Ltd.), cyclin E (1:1,000; cat. no. A14225; ABclonal Biotech Co., Ltd.), phosphorylated (p)-MEK1/2 (1:500; cat. no. AP0209; ABclonal Biotech Co., Ltd.), MEK1/2 (1:500; cat. no. AF6385; Affinity Biosciences, Ltd.), p-ERK1/2 (1:500; cat. no. AF1015; Affinity Biosciences, Ltd.) or ERK1/2 (1:500; cat. no. AF0155; Affinity Biosciences, Ltd.) overnight at 4°C. This was followed by a 40-min incubation with the HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:10,000; cat. no. A0208 and A0216, respectively; Beyotime Institute of Biotechnology) at 37°C. Enhanced chemiluminescence (ECL)-detecting reagent (Beyotime Institute of Biotechnology) was then added onto the membrane, before the protein bands were visualized in WD-9413B gel-imaging system (Beijing LIUYI Biotechnology Co., Ltd.) and analyzed using Gel-Pro-Analyzer 4.0 software (Media Cybernetics, USA).

293T cells (2×10⁵/well; 12-well plate) were used for dual-luciferase reporter assay. To assess the potential association between miR-378a-3p and BLACAT2 or YY1, the wild-type BLACAT2 or 3′UTR of YY1 mRNA containing a putative binding site for miR-378a-3p and their corresponding mutant sequences were amplified and cloned into the pmirGLO plasmid (General Biological System). Vectors containing the wild-type or mutant sequence were co-transfected with miR-378a-3p mimics or NC mimics into 293T cells using Lipofectamine^® 3000 at 37°C using the established protocol. At 6 h post transfection, the culture medium was replaced by fresh DMEM (Wuhan Servicebio Technology Co., Ltd.) containing 10% FBS (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.). In total, 48 h after transfection, the activity of firefly luciferase in cells was detected using a Dual-Luciferase Reporter Gene Assay Kit (Nanjing KeyGen Biotech Co., Ltd.), which was normalized to that of Renilla luciferase.

Similarly, to assess the binding of YY1 to BLACAT2 promoter, three regions (−500+5, −1,000+5 and −1,500+5) in the BLACAT2 promoter or the YY1 coding sequence were inserted into the pmirGLO vector. Vectors encoding each of the BLACAT2 promoters were co-transfected with the vector encoding YY1 into 293T cells at 37°C using the established protocol. The luciferase activity was detected at 48 h post transfection.

Cells (3×10³/well)were seeded into a 96-well plate and cultured under standard conditions. Cell viability was measured at 0, 24, 48 and 72 h using the CCK-8 (Sigma-Aldrich; Merck KGaA) solution. In total, 800 µl CCK-8 was added into each well, before the cells were cultured for 1 h. The optical density of each well was then detected at 450 nm using a microplate reader (800Ts; BioTek Instruments, Inc.).

Cell cycle distribution was revealed using a Cell Cycle and Apoptosis Detection kit (cat. no. C1052; Beyotime Institute of Biotechnology). After the fixing with cold 70% ethanol for 12 h at 4°C, the cells were harvested by centrifugation at 4°C for 3 min with a speed of 420 × g, and 500 µl dye buffer was added. Next, the cells were incubated with 25 µl of propidium iodide staining solution and 10 µl RNase A for 30 min at 37°C. The percentage of the cells in G₁, S, and G2 phases were analyzed using a flow cytometer (NovoCyte; ACEA Bioscience, Inc.) and quantified using the software NovoExpress (version 1.5.0; Agilent Technologies, Inc.).

Cells were seeded into a six-well plate at a density of 5×10⁵/well and grown to a confluence of >95% at the start of the assay. The cells were then cultured in serum-free medium containing 1 µg/ml mitomycin C (Sigma-Aldrich; Merck KGaA) for 1 h. Subsequently, a 200-µl pipette tip was used to make a straight vertical scratch down through the cell monolayers. Cell debris were then removed and an image was recorded using an inverted phase-contrast microscope (IX53; Olympus Corporation). The cells were thereafter cultured in fresh serum-free medium at 37°C and 5% CO₂ for 24 h. The images of wounds at 0 and 24 h were recorded, before the migration rate was calculated as: (Wound distance at 0 h-wound distance at 24 h)/wound distance at 0 h ×100%.

Cell invasion in AN3CA and HEC-1-A cells was assessed using Transwell assays with a 24-well Transwell chamber (Corning, Inc.). The chamber was pre-coated with Matrigel at 37°C for 2 h (Corning, Inc.), which wasdiluted with serum-free mediumat a ratio of 1:3. The cells were then seeded into the pre-coated upper chamber in their serum-free culture medium at a density of 1.5×10⁴ cells/well. In total, 800 µl culture medium (MEM for AN3CA cells and McCoy's 5A for HEC-1-A) containing 10% FBS (Biological Iundustries) was added into the lower chamber. After 24 h of incubation at 37°C, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then stained with 0.1% crystal violet solution at room temperature for a further 5 min. A total of five fields of view were randomly selected for each chamber and the number of cells were counted under an inverted phase-contrast microscope (IX53; Olympus Corporation).

MMP-2 and MMP-9 activity in AN3CA and HEC-1-A cells were determined using gelatin zymography as previously described (37). Total protein concentration in the culture supernatants was quantified with a BCA kit (Beyotime Insitute of Biotechnology). A total of 10 mg/ml gelatin (Sigma-Aldrich; Merck KGaA) was added to a 10% acrylamide separating gel. The proteins in the supernatants were separated using SDS-PAGE. The SDS-PAGE gel was washed twice for 40 min in an elution solution (2.5% Triton X-100, 50 mmol/l Tris-HCl, 5 mmol/l CaCl₂ and 1 µmol/l ZnCl₂; pH 7.6). The gel was then incubated in the developing buffer (50 mmol/l Tris-HCl, 5 mmol/l CaCl₂, 1 µmol/l ZnCl₂, 0.02% Brij and 0.2 mol/l NaCl) at 37°C for 40 h. Following incubation, the gel was stained with a staining solution (0.5% Coomassie blue R250, 30% methanol and 10% glacial acetic acid; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 3 h. Digestion bands were visualized using a Gel imaging system (WD-9413B; Beijing LIUYI Biotechnology Co., Ltd.) and analyzed with a microplate reader (ELX-800; BioTeke Corporation).

The ChIP assay was conducted using a ChIP assay kit (cat. no. WLA106a; Wanleibio, Co., Ltd.) according to the manufacturer's protocol. Briefly, HEC-1-A cells were formaldehyde-crosslinked and then lysed using lysis buffer from Wanleibio, Co., Ltd. (cat. no. WLA106a). The chromatin of HEC-1-A cells was fragmented by sonication and collected, followed by the incubation with PBS-washed ProteinA+G beads (60 µl) on a rotator at 4°C for 1–2 h. Following centrifugation with a speed of 625 × g at room temperature for 1 min, the cell supernatants were collected and 20 µl was used as the Input. The chromatin fragments (100 µl/group) were then incubated with the anti-YY1 antibody (2 µg, cat. no. 66281-1-Ig; ProteinTech Group, Inc.) on a rotator at 4°C overnight. The PBS-washed ProteinA+G beads (60 µl) were next added and maintained on a rotator at 4°C for 1–2 h. IgG (1 µg; ChIP assay kit; Wanleibio, Co., Ltd.) was used as a negative control antibody. The protein-DNA complexes were then washed using the wash buffer from Wanleibio, Co., Ltd. (cat. no. WLA106a) on a rotator at 4°C for 10 min/time. The supernatant was removed using centrifugation at a speed of 625 × g at room temperature for 1 min. A total of 100 µl of elution buffer was added and incubated overnight at 65°C to reverse cross-links. The DNA specimens released from the protein-DNA complexes were purified and analyzed using ChIP followed by PCR (ChIP-PCR; BioTeke Corporation) to amplify the promoter region of BLACAT2. The primer pair for the BLACAT2 promoter was as follows: Forward, 5′-AGTCTCATTCTGTCGCCT-3′ and reverse, 5′-TGTAATCCCAGCACTGTG-3′. PCR products were detected using electrophoresis on 2% agarose gel (Biowest).

For the present study, 4-week-old female BALB/c nude mice weighing 16±1 g (n=6 per group, with a total of 24 mice) were purchased from Huafukang Biosciences and adaptively housed for a week under a controlled environment (humidity, 45–55%; temperature, 22±1°C; and a 12-h day/night cycle), with free access to water and food. The mice were then randomly divided into four groups: Lv-NC (AN3CA), Lv-BLACAT2 (AN3CA), Lv-shNC (HEC-1-A), and Lv-shBLACAT2 (HEC-1-A) groups. AN3CA cells with stably overexpressed BLACAT2/NC and HEC-1-A cells with stably knocked down BLACAT2/NC (1×10⁷ cells/100 µl) were subcutaneously injected into the right flank of the nude mice. The tumor size was measured every 4 days. A total of 4 weeks later, all mice were sacrificed by CO₂ asphyxiation. The flow displacement rate of CO₂ was 40% of the chamber volume per min. Death was confirmed by the cessation of movement, breathing and heartbeat. The tumor tissues were then separated and weighed, before they were fixed in 4% paraformaldehyde for further analysis. The tumor volumes were calculated according to the following formula: Volume=length × width² ×0.5. The animal experiments were approved by the Ethics Committee of Shengjing Hospital of China Medical University (approval no. 2021PS741K). A tumor size of 20 mm at the largest diameter was permitted by the Ethics Committee. After 4 weeks, the maximum tumor diameter observed was not exceeded.

The tumor tissues fixed in 4% paraformaldehyde (at room temperature for 24 h) were embedded in paraffin and then sectioned into 5-µm slices. After dewaxing, rehydration, and antigen retrieval, the tumor slices were incubated with 3% H₂O₂ at room temperature for 15 min. Following 15 min of blocking with goat serum (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature, tumor slices were incubated with a anti-Ki67 antibody (1:100 dilution; cat. no. A2094; ABclonal Biotech Co., Ltd.) overnight at 4°C. Subsequently, the sections were incubated with an HRP-conjugated secondary antibody (cat. no. 31460; Thermo Fisher Scientific, Inc.) at 37°C for 1 h. The sections were then stained with diaminobenzidine (DAB; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 5 min. Following DAB visualization, the sections were immersed into water and then re-stained using hematoxylin at room temperature for 3 min. The Ki67-positive cells were finally observed under a light microscope (BX53; Olympus Corporation).

Data were analyzed using the GraphPad Prism 8.0 software (GraphPad Software, Inc.) and the results are presented as the mean ± standard deviation. Statistical analyses were performed using one-way ANOVA followed by Tukey's multiple comparisons test, two-way ANOVA followed by Sidak's multiple comparisons test or unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

BLACAT2 was found to be upregulated in cervical cancer, ovarian cancer and UCEC, whereas its expression was not found to be significantly different in breast cancer (Fig. 1A). Although carcinogenic functions have been previously identified in cervical cancer (38) and ovarian cancer (39), the role of BLACAT2 in UCEC remains unclear. BLACAT2 expression was therefore detected in clinical samples using RT-qPCR. BLACAT2 expression was found to be significantly increased in UCEC tumors compared with that in the adjacent normal tissues (P<0.01; Fig. 1B). Furthermore, RT-qPCR confirmed that BLACAT2 is expressed in human endometrial cancer cell lines Ishikawa, RL95-2, HEC-1-B, AN3CA and HEC-1-A (Fig. 1C).

As revealed in Fig. 1C, AN3CA cells exhibited the lowest expression of BLACAT2 than that in the other UCEC cell lines, while HEC-1-A cells exhibited the highest expression of BLACAT2. Thus, AN3CA cells were selected for BLACAT2 overexpression, whereas HEC-1-A cells were selected for BLACAT2 knockdown. As the results of RT-qPCR revealed, the expression of BLACAT2 in AN3CA cells was higher when compared to the Lv-NC group, whereas that in HEC-1-A cells was significantly lower when compared to the Lv-shNC group (P<0.01; Fig. 2A). Cell viability was then detected using CCK-8 assay. Notably, BLACAT2 overexpression enhanced UCEC cell viability, whereas BLACAT2 knockdown reduced cell viability (P<0.01; Fig. 2B). Cell cycle progression was next investigated in these cells. BLACAT2 overexpression decreased cell accumulation in the G₁ phase and increased the number of cells in the S and G₂ phases. By contrast, there were more HEC-1-A cells in the G₁ phase and fewer cells in the S and G₂ phases following BLACAT2 knockdown (P<0.05; Fig. 2C). The expression of cyclin D1 and cyclin E, which are essential regulators of G₁/S transition during cell cycle progression (40), were found to be upregulated in UCEC cells following BLACAT2 overexpression. However, their protein expression levels were significantly reduced by BLACAT2 knockdown (P<0.01; Fig. 2D and E). Therefore, these data indicated that BLACAT2 overexpression promotes the proliferation of UCEC cells.

The migration and invasion of AN3CA and HEC-1-A cells were subsequently assessed by wound healing and Transwell assays. BLACAT2 overexpression was observed to improve the migration rate of UCEC cells, whilst knockdown of BLACAT2 expression significantly inhibited the migration of these tumor cells (P<0.05; Fig. 3A). BLACAT2 appeared to mediate similar effects on UCEC cell invasion (P<0.01; Fig. 3B). MMP9 and MMP2 are proteins that can potentiate cancer cell invasion (41). Therefore, the present study next examined the protein expression levels of MMP9 and MMP2 in cells, and determined that BLACAT2 overexpression increased the expression of MMP9 and MMP2. By contrast, BLACAT2 knockdown inhibited their protein expression levels in UCEC cells (P<0.01; Fig. 3C). In addition, gelatin zymography assay revealed that BLACAT2 overexpression enhanced the activity of MMP9 and MMP2, whilst BLACAT2 knockdown weakened their activities (P<0.01; Fig. 3D). These findings indicated that BLACAT2 serves an important role in the migration and invasion of UCEC cells.

The ERK signaling pathway has been reported to be aberrantly activated in cancer, which can promote cell proliferation and invasion (42). Therefore, the activation status of ERK and MEK in UCEC cells was assessed using western blotting (Fig. 4A and C). BLACAT2 overexpression was found to increase the phosphorylation of MEK1/2 and ERK1/2 without altering the expression of their corresponding total protein in UCEC cells. The increased ratio of p-MEK1/2 or p-ERK1/2 to total MEK1/2 or total ERK1/2, respectively, suggested that the MEK/ERK pathway was activated by BLACAT2 overexpression (P<0.05; Fig. 4B). By contrast, BLACAT2 knockdown significantly suppressed the activation of this pathway in UCEC cells (P<0.01; Fig. 4C and D). These results indicated that BLACAT2 can regulate MEK/ERK signaling in UCEC cells.

The effect of BLACAT2 on UCEC progression was next studied in vivo. AN3CA cells with stably overexpressed BLACAT2/NC and HEC-1-A cells with stably knocked down BLACAT2/NC, were injected into nude mice. The tumor volumes and weights of the nude mice injected with cells overexpressing BLACAT2 were found to be increased compared with those of nude mice injected with cells expressing Lv-NC. However, BLACAT2 knockdown suppressed tumor growth in nude mice (P<0.05; Fig. 5A-C). The expression of Ki-67 using immunohitochemical (IHC) staining indicated that BLACAT2 overexpression promoted, whereas BLACAT2 knockdown suppressed UCEC tumor cell proliferation (Fig. 5D). Furthermore, the protein expression levels of cyclin D1, MMP2 and phosphorylated ERK1/2 were also increased by BLACAT2 overexpression but inhibited by BLACAT2 knockdown (P<0.01; Fig. 5E and F). These observations indicated that BLACAT2 promoted UCEC tumor growth and activation of MEK/ERK signaling in vivo.

The predictive binding among BLACAT2, miR-378a-3p, and YY1 is presented in Fig. 6A. The putative binding sites of BLACAT2 to miR-378a-3p and miR-378a-3p to YY1 3′UTR are shown in Fig. 6B. BLACAT2 overexpression was demonstrated to reduce miR-378a-3p expression, but to increase that of YY1 in UCEC cells, whilst BLACAT2 knockdown exerted the opposite effects (P<0.01; Fig. 6C). BLACAT2 overexpression also led to reduced miR-378a-3p expression and increased YY1 expression in UCEC tumors, whilst BLACAT2 knockdown enhanced miR-378a-3p expression and reduced YY1 expression (P<0.01; Fig. S1). The potential targeted association between miR-378a-3p and BLACAT2 or YY1 was next assessed using dual-luciferase reporter assays. The relative luciferase activity in 293T cells co-transfected with vectors containing wild-type BLACAT2 3′UTR and miR-378a-3p was reduced, suggesting that miR-378a-3p can bind to BLACAT2. miR-378a-3p was also found to bind to the 3′UTR of YY1 (P<0.01; Fig. 6D). In addition, Fig. 6E revealed the possible binding sites of YY1 in the BLACAT2 promoter region. The increased relative luciferase activity in 293T cells co-transfected with the YY1 and BLACAT2 promoter suggested that YY1, as a transcription factor, may bind to the BLACAT2 promoter (Fig. 6E). This was further verified using ChIP assay (Fig. 6F).

To study the function of miR-378a-3p in UCEC cells, HEC-1-A cells were transfected with either miR-378a-3p mimics or NC mimics. The transfection efficiency was validated and is presented in Fig. S2. The mRNA expression level of YY1 in HEC-1-A cells was significantly decreased following miR-378a-3p overexpressin (P<0.01; Fig. 7A). miR-378a-3p overexpression also significantly suppressed the viability and invasion of UCEC cells (P<0.01; Fig. 7B and C). The expression of cyclin D1, MMP2, and ERK1/2 phosphorylation was found to be inhibited by miR-378a-3p overexpression (P<0.01; Fig. 7D), suggesting that miR-378a-3p can suppress cell proliferation, invasion, and ERK signaling in UCEC cells.

To investigate whether YY1 is involved in the regulation of BLACAT2 and therefore UCEC development, Lv-shYY1 was used to infect AN3CA cells, and YY1 expression was knocked down effectively in AN3CA cells after the infection (P<0.01; Fig. 8A). AN3CA cells with stably overexpressed BLACAT2 were then infected with Lv-shYY1 or Lv-shNC. CCK-8 assay revealed that in BLACAT2-overexpressed UCEC cells, YY1 downregulation significantly reduced cell viability (P<0.01; Fig. 8B). In addition, YY1 knockdown abrogated the positive effects of BLACAT2 on UCEC cell migration and invasion (P<0.05; Fig. 8C and D). The protein expression levels of cyclin D1, MMP2 and ERK1/2 phosphorylation were also significantly supressed by YY1 inhibition (P<0.01; Fig. 8E and F). These results indicated that YY1 is involved in the regulation of BLACAT2 expression upstream of UCEC cell proliferation, migration, invasion, and MER/ERK signaling.