Long non‑coding RNA BLACAT2/miR‑378a‑3p/YY1 feedback loop promotes the proliferation, migration and invasion of uterine corpus endometrial carcinoma
- Authors:
- Published online on: April 13, 2023 https://doi.org/10.3892/or.2023.8544
- Article Number: 108
-
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Uterine corpus endometrial carcinoma (UCEC) is a malignancy that originates from the endometrium and is frequently symptomatic at advanced stage (1,2). It is one of the most common gynecological malignancies worldwide with increasing incidence (1,2). Patients with advanced disease present with abnormal bleeding in the vagina or pelvic organs (3). For women with localized disease on diagnosis, the 5-year survival rate is >90%, but the 5-year overall survival rate decreases to <20% in patients with distal metastases (4). Therefore, identifying novel therapeutic targets for UCEC is particularly important for treating patients with UCEC.
Long non-coding RNAs (lncRNAs) belong to a class of non-coding RNAs that are typically >200 nucleotides in length and have little or no protein-coding potential (5). LncRNAs reverse the inhibitory effects of microRNAs (miRNAs or miRs) on mRNAs by functioning as miRNA sponges (6). To date, numerous lncRNAs have been reported to be aberrantly expressed in various cancers, where lncRNAs serve pivotal regulatory roles in tumorigenesis and metastasis (7,8). Bladder cancer-associated transcript 2 (BLACAT2) was first identified to be an oncogene in bladder cancer, which increases tumor lymphangiogenesis and lymphatic metastasis (9,10). It has been previously reported that the expression of BLACAT2 is upregulated in several malignancies, including hepatocellular carcinoma, cervical cancer, and nasopharyngeal carcinoma (11–19), which suggests that BLACAT2 is closely associated with cancer progression. In fact, BLACAT2 has been demonstrated to aggravate hepatocellular carcinoma via sponging miR-3619-5p, and plays a functional role in modulating radiotherapy resistance in cervical cancer (11,12). Previous RNA-sequencing analysis indicated that BLACAT2 expression is highly upregulated in the endometrium of patients with UCEC compared to adjacent normal controls (20). In addition, another previous study revealed that BLACAT2 is a possible oncogenic lncRNA in endometrial cancer (21). However, the regulatory mechanism underlying the role of BLACAT2 remains unclear.
Transcription factor Yin Yang-1 (YY1) is a zinc finger protein belonging to the GLI-Kruppel family (22). As a DNA-binding protein, it serves dual functions and has been documented to regulate cell differentiation, proliferation, apoptosis and cell division (23). YY1 can either function as an activator or repressor of gene transcription, depending on the context of YY1-binding proteins in proximity (24). Specifically, YY1 can bind to the promoter region of LINC00673, thereby increasing its transcription in cis (25). Numerous studies have demonstrated the upregulation of YY1 in ovarian, prostate, esophageal, and melanoma cancer (26–29). In addition, YY1 expression has also been demonstrated to be upregulated in human endometrioid endometrial adenocarcinoma cell lines and tumor tissues. YY1 knockdown can inhibit cell proliferation and migration in endometrioid endometrial carcinoma (EEC), whereas YY1 overexpression was found to promote EEC cell proliferation (30).
MicroRNAs (miRNAs or miRs) form another family of highly conserved non-coding RNAs, which are 20–25 nucleotides in length (31,32). They typically mediate translational repression or mRNA transcript degradation by targeting the 3′-untranslated region (UTR) of downstream mRNAs (31,32). An increasing number of miRNAs have been observed to regulate the progression of UCEC (33,34). In particular, miR-378a-3p has been recognized as a potential miRNA that can bind to BLACAT2 according to bioinformatic analysis. However, its function in UCEC remains unreported. Recently, BLACAT2 was revealed to bind to miR-378a-3p in bladder cancer cells, where it regulated cell viability, migration and invasion (35).
Based on previous studies and findings, it was hypothesized that BLACAT2 may serve an important biological function in UCEC by regulating miR-378a-3p and/or YY1. Therefore, the present study explored the possible functions of BLACAT2, YY1 and miR-378a-3p, in addition to their associations with UCEC progression.
Materials and methods
Clinical specimens
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.
Cell lines and culture conditions
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.
Database analysis
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.
Lentivirus packaging
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.
Cell treatments
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).
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.
Western blot analysis
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).
Dual-luciferase reporter assay
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.
Cell counting kit-8 (CCK-8) assay
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.).
Flow cytometric analysis
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.).
Wound healing assay
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%.
Transwell assay
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).
Gelatin zymography assay
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).
Chromatin immunoprecipitation (ChIP)
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).
In vivo tumorigenesis model
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.
Immunohistochemistry
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).
Statistical analysis
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.
Results
Expression of BLACAT2 in UCEC
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).
BLACAT2 overexpression promotes the proliferation of UCEC cells
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.
BLACAT2 overexpression facilitates UCEC cell migration and invasion
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.
BLACAT2 overexpression enhances MER/ERK signaling in 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.
BLACAT2 promotes UCEC tumor growth in vivo
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.
Association among BLACAT2, miR-378a-3p, and YY1
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).
miR-378a-3p overexpression inhibits UCEC cell proliferation, invasion and the ERK pathway
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.
YY1 knockdown reverses BLACAT2 overexpression-mediated UCEC development
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.
Discussion
In the present study, BLACAT2 overexpression was found to promote UCEC cell proliferation, migration, and invasion. The tumor-promoting effects of BLACAT2 in UCEC are likely to be mediated by a miR-378a-3p/YY1 axis. This BLACAT2/miR-378a-3p/YY1 axis in UCEC development is also likely to involve the MEK/ERK signaling pathway. In addition, YY1 may bind to the promoter of BLACAT2 to regulate its expression.
LncRNAs are expressed in a tissue- and cell-type-specific manner, where they have been demonstrated to serve vital roles in multiple cancers (43,44). A previous study identified seven lncRNAs to be potential prognostic factors in UCEC according to RNA-sequencing and expression profiling coupled with analysis of corresponding clinical data of patients with UCEC from The Cancer Genome Atlas database (45). Illumina paired-end RNA sequencing demonstrated that BLACAT2 expression is upregulated in endometrial, ovarian and cervical cancer (20). BLACAT2 has been found to be differently expressed in UCEC tumor and normal tissue, but its function and mechanism in UCEC remain poorly understood. In the present study, BLACAT2 overexpression was found to suppress miR-378a-3p expression, whilst promoting YY1 expression, UCEC cell viability, cell cycle progression, cell migration and invasion. By contrast, BLACAT2 knockdown exerted the opposite effects on all of the aforementioned physiological processes. As part of the cyclin family of proteins, cyclin D1 and cyclin E are important regulators of cancer cell proliferation, the downregulation of which was associated with cell cycle arrest (46,47). By contrast, the migration and invasion of cancer cells are mediated by metalloproteinases, MMP2 and MMP9 (48,49). The present study revealed increased cyclin D1, cyclin E, MMP2, and MMP9 in UCEC cells following BLACAT2 overexpression, whereas they were all reduced by BLACAT2 knockdown. This suggests that BLACAT2 overexpression can promote UCEC cell proliferation, migration, and invasion. In addition, it was found that the BLACAT2-mediated UCEC cell proliferation was associated with increases in p-MEK/MEK and p-ERK/ERK ratios, suggesting that BLACAT2 overexpression can also activate MEK/ERK signaling in UCEC cells. The tumor-promoting effects of the activated MEK/ERK pathway have been demonstrated in previous studies (50,51). Therefore, the results from the present study revealed a potential novel signaling pathway regulated by BLACAT2 in UCEC.
LncRNAs typically function as competing endogenous RNAs with other RNA transcripts for the same miRNAs (52). LncRNAs can function as miRNA sponges and block the regulatory effects of miRNAs on mRNAs (53). The regulation of transcription is a complex process that involves interactions among different types of signaling components, including mRNA, lncRNA and miRNA. Liu et al (54) previously proposed a novel method for constructing genome-wide interactive networks and found the cross-regulation of lncRNA, miRNA and mRNA, called the ‘mRNA-lncRNA-miRNA triplet’. miR-378a-3p was predicted to bind to BLACAT2 using bioinformatic analysis, which was then confirmed using a dual-luciferase report assay. The interaction of BLACAT2 with miR-378a-3p was also found in a previous study by Cui et al (35), which demonstrated that BLACAT2 can regulate cancer development by sponging miR-378a-3p. Previous studies demonstrated that tumor promotion may involve miR-378a-3p inhibition (55,56). In the present study, miR-378a-3p overexpression resulted in the suppression of YY1 expression, UCEC cell proliferation and invasion, suggesting the antitumor role of miR-378a-3p in UCEC. Hu et al (16) previously showed that BLACAT2 can serve as an oncogene by modulating the miR-193b-5p/methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit axis (16). In the present study, the function of BLACAT2 in UCEC progression may be achieved by the regulation of miR-378a-3p and YY1 downstream, which further affected the MEK/ERK pathway. Therefore, the differential roles of BLACAT2 in human cancers may depend on its different downstream miRNAs and target mRNAs.
YY1 was shown to be a potential target of miR-378a-3p. YY1 was previously found to be overexpressed in tumors and identified to be an oncogenic factor. This was potentially mediated by the recruitment of enhancer of zeste homolog 2 and the activation of histone H3K27me3 in the adenomatous polyposis coli gene promoter region in EEC, which silences the expression of this gene. In addition, YY1 depletion was found to suppress EEC cell proliferation and migration, whilst YY1 overexpression could promote these processes (30). YY1 can also function as a positive regulator of AKT1, a member of the PI3K/AKT/mTOR signaling pathway activated in endometrial tumors (57). These previous studies suggest that YY1 is an important regulator of endometrial carcinoma progression. Consistently, the present study showed that YY1 knockdown abolished the BLACAT2 overexpression-induced UCEC cell proliferation, invasion and activation of MEK/ERK signaling, suggesting that YY1 was involved in the regulation of BLACAT2 and therefore UCEC progression. In gastric cancer cells, YY1 knockdown resulted in the inhibition of ERK/MAPK signaling, a major oncogenic pathway (58). Therefore, the present study revealed that the induction of YY1 expression may activate the ERK pathway to promote UCEC tumor growth.
The direct binding of YY1 to the gene promoter of BLACAT2 was identified using dual-luciferase reporter and ChIP assays in the present study, suggesting that YY1 can regulate BLACAT2 transcription. The regulatory relationship among BLACAT2, miR-378a-3p, and YY1 appears to form a feedback loop instead of an axis. Therefore, BLACAT2 not only controls UCEC development by regulating the miR-378a-3p/YY1 axis, but is also under the control of the transcription factor YY1. Based on these findings, BLACAT2 may bind to miR-378a-3p and function as a sponge of this miRNA, resulting in the abolishment of the inhibitory effects of miR-378a-3p on YY1 in UCEC. However, the regulatory mechanism of YY1 in BLACAT2 expression requires further investigation. In addition, the association of the BLACAT2/miR-378a-3p/YY1 loop with the ERK signaling pathway in UCEC cells warrants further exploration in the future.
Findings from the present study suggest the potential of BLACAT2 as a biomarker in UCEC. However, the present study also has various limitations. It is essential to enroll an increased number of cancer cases in future studies. In addition, the majority of lncRNAs can regulate >1 target gene, but only the preliminary regulatory mechanism of the effect of BLACAT2 was revealed in the present study. The specific mechanisms involved in the effect of BLACAT2 in UCEC require further investigation. Furthering the understanding of the function and potential molecular mechanisms of BLACAT2 will facilitate the development of lncRNA-based therapeutics in the clinic.
In conclusion, in the present study it was revealed that BLACAT2 regulated the progression of UCEC through the miR-378a-3p/YY1 axis. However, downstream YY1 can bind to BLACAT2 promoter to promote its expression as a transcription factor. In addition, MEK/ERK is involved downsteam of the BLACAT2/miR-378a-3p/YY1 loop in UCEC progression. The present study therefore provides novel therapeutic targets for UCEC and insights into UCEC tumorigenesis.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the Public Health Research and Development Project of Shenyang (Medical Technology Improvement Project of Common Multiple Diseases), grant no. 22-321-33-15.
Availability of data and materials
The data used to support the findings of this study are included in the manuscript.
Authors' contributions
CZ and QY conceived and designed the experiments. CZ and RW performed the experiments. CZ, RW and ML analyzed the data, and performed the bioinformatics analysis. CZ wrote the manuscript. QY helped edit the manuscript. CZ and QY confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The research protocol was approved (approval no. 2021PS723K) by the Ethics Committee of Shengjing Hospital of China Medical University (Shenyang, China) and was in accordance with the Declaration of Helsinki. The informed consent was obtained from all volunteers. Animal experimental procedures were approved (approval no. 2021PS741K) by the Animal Ethics Committee of Shengjing Hospital of China Medical University.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
lncRNAs |
long non-coding RNAs |
|
miRNAs |
microRNAs |
|
YY1 |
transcription factor Yin Yang-1 |
|
UCEC |
uterine corpus endometrial carcinoma |
|
RT-qPCR |
reverse transcription-quantitative polymerase chain reaction |
|
ChIP |
chromatin immunoprecipitation |
|
ERK |
extracellular signal-regulated kinase |
References
|
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Morice P, Leary A, Creutzberg C, Abu-Rustum N and Darai E: Endometrial cancer. Lancet. 387:1094–1108. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Aoki Y, Kanao H, Wang X, Yunokawa M, Omatsu K, Fusegi A and Takeshima N: Adjuvant treatment of endometrial cancer today. Jpn J Clin Oncol. 50:753–765. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Siegel R, Ma J, Zou Z and Jemal A: Cancer statistics, 2014. CA Cancer J Clin. 64:9–29. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Slavoff SA, Mitchell AJ, Schwaid AG, Cabili MN, Ma J, Levin JZ, Karger AD, Budnik BA, Rinn JL and Saghatelian A: Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat Chem Biol. 9:59–64. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A and Bozzoni I: A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell. 147:358–369. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Bhan A and Mandal SS: Long noncoding RNAs: Emerging stars in gene regulation, epigenetics and human disease. ChemMedChem. 9:1932–1956. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Bhan A, Soleimani M and Mandal SS: Long noncoding RNA and cancer: A new paradigm. Cancer Res. 77:3965–3981. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Seitz AK, Christensen LL, Christensen E, Faarkrog K, Ostenfeld MS, Hedegaard J, Nordentoft I, Nielsen MM, Palmfeldt J, Thomson M, et al: Profiling of long non-coding RNAs identifies LINC00958 and LINC01296 as candidate oncogenes in bladder cancer. Sci Rep. 7:3952017. View Article : Google Scholar : PubMed/NCBI | |
|
He W, Zhong G, Jiang N, Wang B, Fan X, Chen C, Chen X, Huang J and Lin T: Long noncoding RNA BLACAT2 promotes bladder cancer-associated lymphangiogenesis and lymphatic metastasis. J Clin Invest. 128:861–875. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Zuo X, Chen Z, Gao W, Zhang Y, Wang J, Wang J, Cao M, Cai J, Wu J and Wang X: M6A-mediated upregulation of LINC00958 increases lipogenesis and acts as a nanotherapeutic target in hepatocellular carcinoma. J Hematol Oncol. 13:52020. View Article : Google Scholar : PubMed/NCBI | |
|
Zhao H, Zheng GH, Li GC, Xin L, Wang YS, Chen Y and Zheng XM: Long noncoding RNA LINC00958 regulates cell sensitivity to radiotherapy through RRM2 by binding to microRNA-5095 in cervical cancer. J Cell Physiol. 234:23349–23359. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Chen M, Xu Z, Zhang Y and Zhang X: LINC00958 promotes the malignancy of nasopharyngeal carcinoma by sponging microRNA-625 and thus upregulating NUAK1. Onco Targets Ther. 12:9277–9290. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Chen S, Chen JZ, Zhang JQ, Chen HX, Qiu FN, Yan ML, Tian YF, Peng CH, Shen BY, Chen YL and Wang YD: Silencing of long noncoding RNA LINC00958 prevents tumor initiation of pancreatic cancer by acting as a sponge of microRNA-330-5p to down-regulate PAX8. Cancer Lett. 446:49–61. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Luo Z, Han Z, Shou F, Li Y and Chen Y: LINC00958 accelerates cell proliferation and migration in non-small cell lung cancer through JNK/c-JUN signaling. Hum Gene Ther Methods. 30:226–234. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Hu H, Kong Q, Huang XX, Zhang HR, Hu KF, Jing Y, Jiang YF, Peng Y, Wu LC, Fu QS, et al: Longnon-coding RNA BLACAT2 promotes gastric cancer progression via the miR-193b-5p/METTL3 pathway. J Cancer. 12:3209–3221. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Ren Y, Zhao C, He Y, Xu H and Min X: Long non-coding RNA bladder cancer-associated transcript 2 contributes to disease progression, chemoresistance and poor survival of patients with colorectal cancer. Oncol Lett. 18:2050–2058. 2019.PubMed/NCBI | |
|
Zhu J, Zhu Z, Cai P, Gu Z and Wang J: Bladder cancer-associated transcript 2 contributes to nephroblastoma progression. J Gene Med. 24:e32922022. View Article : Google Scholar : PubMed/NCBI | |
|
Binang HB, Wang YS, Tewara MA, Du L, Shi S, Li N, Nsenga AGA and Wang C: Expression levels and associations of five long non-coding RNAs in gastric cancer and their clinical significance. Oncol Lett. 19:2431–2445. 2020.PubMed/NCBI | |
|
Chen BJ, Byrne FL, Takenaka K, Modesitt SC, Olzomer EM, Mills JD, Farrell R, Hoehn KL and Janitz M: Transcriptome landscape of long intergenic non-coding RNAs in endometrial cancer. Gynecol Oncol. 147:654–662. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Jiang Y, Qiao Z, Jiang J and Zhang J: LINC00958 promotes endometrial cancer cell proliferation and metastasis by regulating the miR-145-3p/TCF4 axis. J Gene Med. 23:e33452021. View Article : Google Scholar : PubMed/NCBI | |
|
Khachigian LM: The Yin and Yang of YY1 in tumor growth and suppression. Int J Cancer. 143:460–465. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Sarvagalla S, Kolapalli SP and Vallabhapurapu S: The two sides of YY1 in cancer: A friend and a foe. Front Oncol. 9:12302019. View Article : Google Scholar : PubMed/NCBI | |
|
Thomas MJ and Seto E: Unlocking the mechanisms of transcription factor YY1: Are chromatin modifying enzymes the key? Gene. 236:197–208. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Qiao K, Ning S, Wan L, Wu H, Wang Q, Zhang X, Xu S and Pang D: LINC00673 is activated by YY1 and promotes the proliferation of breast cancer cells via the miR-515-5p/MARK4/Hippo signaling pathway. J Exp Clin Cancer Res. 38:4182019. View Article : Google Scholar : PubMed/NCBI | |
|
Berchuck A, Iversen ES, Lancaster JM, Pittman J, Luo J, Lee P, Murphy S, Dressman HK, Febbo PG, West M, et al: Patterns of gene expression that characterize long-term survival in advanced stage serous ovarian cancers. Clin Cancer Res. 11:3686–3696. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Seligson D, Horvath S, Huerta-Yepez S, Hanna S, Garban H, Roberts A, Shi T, Liu X, Chia D, Goodglick L and Bonavida B: Expression of transcription factor Yin Yang 1 in prostate cancer. Int J Oncol. 27:131–141. 2005.PubMed/NCBI | |
|
Luo J, Zhou X, Ge X, Liu P, Cao J, Lu X, Ling Y and Zhang S: Upregulation of Ying Yang 1 (YY1) suppresses esophageal squamous cell carcinoma development through heme oxygenase-1. Cancer Sci. 104:1544–1551. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Zhao G, Li Q, Wang A and Jiao J: YY1 regulates melanoma tumorigenesis through a miR-9~RYBP axis. J Exp Clin Cancer Res. 34:662015. View Article : Google Scholar : PubMed/NCBI | |
|
Yang Y, Zhou L, Lu L, Wang L, Li X, Jiang P, Chan LK, Zhang T, Yu J, Kwong J, et al: A novel miR-193a-5p-YY1-APC regulatory axis in human endometrioid endometrial adenocarcinoma. Oncogene. 32:3432–3442. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Masood N, Basharat Z, Khan T and Yasmin A: Entangling relation of micro RNA-let7, miRNA-200 and miRNA-125 with various cancers. Pathol Oncol Res. 23:707–715. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Xiao M, Iglinski-Benjamin KC, Sharpe O, Robinson WH and Abrams GD: Exogenous micro-RNA and antagomir modulate osteogenic gene expression in tenocytes. Exp Cell Res. 378:119–123. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Jiang T, Sui D, You D, Yao S, Zhang L, Wang Y, Zhao J and Zhang Y: MiR-29a-5p inhibits proliferation and invasion and induces apoptosis in endometrial carcinoma via targeting TPX2. Cell Cycle. 17:1268–1278. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Wang P, Zeng Z, Shen X, Tian X and Ye Q: Identification of a multi-RNA-type-based signature for recurrence-free survival prediction in patients with uterine corpus endometrial carcinoma. DNA Cell Biol. 39:615–630. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Cui Y, Xie M and Zhang Z: LINC00958 involves in bladder cancer through sponging miR-378a-3p to elevate IGF1R. Cancer Biother Radiopharm. 35:776–788. 2020.PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Thomas GJ, Lewis MP, Hart IR, Marshall JF and Speight PM: AlphaVbeta6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9. Int J Cancer. 92:641–650. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Wang L, Zhong Y, Yang B, Zhu Y, Zhu X, Xia Z, Xu J and Xu L: LINC00958 facilitates cervical cancer cell proliferation and metastasis by sponging miR-625-5p to upregulate LRRC8E expression. J Cell Biochem. 121:2500–2509. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Xie M, Fu Q, Wang PP and Cui YL: STAT1-induced upregulation lncRNA LINC00958 accelerates the epithelial ovarian cancer tumorigenesis by regulating Wnt/β-catenin signaling. Dis Markers. 2021:14050452021. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang D, Ma Q, Wang Z, Zhang M, Guo K, Wang F and Wu E: β2-adrenoceptor blockage induces G1/S phase arrest and apoptosis in pancreatic cancer cells via Ras/Akt/NFκB pathway. Mol Cancer. 10:1462011. View Article : Google Scholar : PubMed/NCBI | |
|
Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E and Seiki M: A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 370:61–65. 1994. View Article : Google Scholar : PubMed/NCBI | |
|
Roberts PJ and Der CJ: Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 26:3291–3310. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Fatima R, Akhade VS, Pal D and Rao SM: Long noncoding RNAs in development and cancer: Potential biomarkers and therapeutic targets. Mol Cell Ther. 3:52015. View Article : Google Scholar : PubMed/NCBI | |
|
Cheetham SW, Gruhl F, Mattick JS and Dinger ME: Long noncoding RNAs and the genetics of cancer. Br J Cancer. 108:2419–2425. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Ouyang D, Li R, Li Y and Zhu X: A 7-lncRNA signature predict prognosis of uterine corpus endometrial carcinoma. J Cell Biochem. 120:18465–18477. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Yuan C, Zhu X, Han Y, Song C, Liu C, Lu S, Zhang M, Yu F, Peng Z and Zhou C: Elevated HOXA1 expression correlates with accelerated tumor cell proliferation and poor prognosis in gastric cancer partly via cyclin D1. J Exp Clin Cancer Res. 35:152016. View Article : Google Scholar : PubMed/NCBI | |
|
Park GH, Song HM and Jeong JB: The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells. Food Chem Toxicol. 95:142–148. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Liu H, Zeng Z, Wang S, Li T, Mastriani E, Li QH, Bao HX, Zhou YJ, Wang X, Liu Y, et al: Main components of pomegranate, ellagic acid and luteolin, inhibit metastasis of ovarian cancer by down-regulating MMP2 and MMP9. Cancer Biol Ther. 18:990–999. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Wang X, Yang B, She Y and Ye Y: The lncRNA TP73-AS1 promotes ovarian cancer cell proliferation and metastasis via modulation of MMP2 and MMP9. J Cell Biochem. 119:7790–7799. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Wang Y, Zhu Y, Zhang L, Tian W, Hua S, Zhao J, Zhang H and Xue F: Insulin promotes proliferation, survival, and invasion in endometrial carcinoma by activating the MEK/ERK pathway. Cancer Lett. 322:223–231. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Wang F, Chen Q, Huang G, Guo X, Li N, Li Y and Li B: BKCa participates in E2 inducing endometrial adenocarcinoma by activating MEK/ERK pathway. BMC Cancer. 18:11282018. View Article : Google Scholar : PubMed/NCBI | |
|
Zhao Z, Sun W, Guo Z, Zhang J, Yu H and Liu B: Mechanisms of lncRNA/microRNA interactions in angiogenesis. Life Sci. 254:1169002020. View Article : Google Scholar : PubMed/NCBI | |
|
Paraskevopoulou MD and Hatzigeorgiou AG: Analyzing MiRNA-LncRNA interactions. Methods Mol Biol. 1402:271–286. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Liu C, Zhang YH, Deng Q, Li Y, Huang T, Zhou S and Cai YD: Cancer-related triplets of mRNA-lncRNA-miRNA revealed by integrative network in uterine corpus endometrial carcinoma. Biomed Res Int. 2017:38595822017.PubMed/NCBI | |
|
Kong D, Hou Y, Li W, Ma X and Jiang J: LncRNA-ZXF1 regulates P21 expression in endometrioid endometrial carcinoma by managing ubiquitination-mediated degradation and miR-378a-3p/PCDHA3 axis. Mol Oncol. 16:813–829. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Rong D, Dong Q, Qu H, Deng X, Gao F, Li Q and Sun P: m6A-induced LINC00958 promotes breast cancer tumorigenesis via the miR-378a-3p/YY1 axis. Cell Death Discov. 7:272021. View Article : Google Scholar : PubMed/NCBI | |
|
Painter JN, Kaufmann S, O'Mara TA, Hillman KM, Sivakumaran H, Darabi H, Cheng THT, Pearson J, Kazakoff S, Waddell N, et al: A common variant at the 14q32 endometrial cancer risk locus activates AKT1 through YY1 binding. Am J Hum Genet. 98:1159–1169. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Bhaskar Rao D, Panneerpandian P, Balakrishnan K and Ganesan K: YY1 regulated transcription-based stratification of gastric tumors and identification of potential therapeutic candidates. J Cell Commun Signal. 15:251–267. 2021. View Article : Google Scholar : PubMed/NCBI |
