[Retracted] lncRNA NBAT1 Inhibits Cell Metastasis and Promotes Apoptosis in Endometrial Cancer by Sponging miR-21-5p to Regulate PTEN

Tian, Chunhua; Su, Jing; Ma, Zhao; Wu, Yang; Ma, Hongyun

doi:https://doi.org/10.1155/2022/9304392

Computational and Mathematical Methods in Medicine

On this page

Abstract Introduction Methods Results Discussion Conclusion Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article Retraction

!

This article has been Retracted. To view the article details, please click the ‘Retraction’ tab above.

Special Issue

Biomedical Computational Analysis Models based on Multi-Omics Data

View this Special Issue

Research Article | Open Access

Volume 2022 | Article ID 9304392 | https://doi.org/10.1155/2022/9304392

[Retracted] lncRNA NBAT1 Inhibits Cell Metastasis and Promotes Apoptosis in Endometrial Cancer by Sponging miR-21-5p to Regulate PTEN

Chunhua Tian,¹Jing Su,¹Zhao Ma,¹Yang Wu,¹and Hongyun Ma¹

Academic Editor: Gang Chen

Received14 Jun 2022

Accepted09 Jul 2022

Published20 Jul 2022

Abstract

Objective. Long noncoding RNA neuroblastoma-associated transcript 1 (NBAT1) is implicated in the progression of various cancers. Nevertheless, its biological function in endometrial cancer (EC) remains unknown. Methods. The levels of NBAT1, miR-21-5p, and PTEN in EC cells and EC tissues were examined by RT-qPCR. Western blot was carried out to assess the protein expression of PTEN. The dual-luciferase reporter assay was conducted to explore the interactions among NBAT1, miR-21-5p, and PTEN. The effect of NBAT1 on EC proliferation, metastasis, and apoptosis was evaluated by CCK-8, transwell assays, wound healing, and flow cytometry. miR-21-5p mimics or NBAT1+miR-21-5p were transfected into HEC-1A and Ishikawa cells to investigate whether NBAT1 regulated EC tumorigenesis via sponging miR-21-5p. Results. NBAT1 is downregulated, and miR-21-5p is upregulated in EC cells and tumor tissues. Overexpression of NBAT1 inhibits the proliferation, migration, and invasion abilities of EC cells and facilitated apoptosis. NBAT1 directly binds and negatively regulates miR-21-5p in EC. miR-21-5p mimics reverses the effect of lncRNA NBAT1 overexpression on the proliferation and migration of EC cells. PTEN is a downstream gene of miR-21-5p. lncRNA NBTA1 elevates PTEN expression via sponging miR-21-5p. Conclusions. lncRNA NBAT1 acts as a tumor suppressor in EC via regulating PTEN through sponging miR-21-5p.

1. Introduction

Endometrial cancer (EC) is currently the most frequent female reproductive tract cancer and remains the most lethal gynecologic malignant tumor [1, 2]. The morbidity of EC has boosted worldwide results from the growth of elderly individuals and rising rates of obesity [3]. The mortality of EC is on the rise, which seriously threatens women’s health in China [4]. Remarkably, it has been shown that recurrence and metastasis are critical stages in the formation and development of EC [5, 6]. The innovative diagnosis and therapy of EC have developed over recent years. However, various confines occurred which obstruct the effectiveness of EC therapy in practice [7]. Furthermore, the treatment of EC has a whole host of side effects, including infertility, lymphedema of the lower extremities, and distress [8]. Evidence has indicated that the expeditious proliferation of tumor cells and angiogenesis usually resulted in endometrial cancer recurrence [9]. Cancer metastasis is a critical obstruction to the effective cure for EC. However, the mechanisms underlying EC metastasis remain largely elusive. Therefore, it is crucial to perform experimental studies to explore the molecular mechanisms of EC metastasis to expose therapeutic targets to improve effective treatment for EC.

Long noncoding is a set of nucleotides in length, which modulates gene expression at the posttranscriptional levels [10]. lncRNAs play crucial roles in numerous biological functions and are intensely involved in tumorigenesis. Wei et al. [11] exposed that lncRNA-u50535 facilitated lung cancer development via controlling CCL20/ERK pathway. Pei et al. [12] revealed that lnc-SNHG1 operated as a ceRNA of miR-216b-5p, which was crucial in moderating the paclitaxel sensitivity of ovarian cancer cells. Likewise, lnc-NEAT1 has been observed to serve as a protumorigenic feature in colorectal cancer [13]. Ku et al. [14] revealed that lnc-LINC00240 inhibited non-small-cell lung cancer development by sponging miR-7-5p. Recently, increasing evidence indicated that lnc-RNAs were implicated in the formation and development of EC. For example, Li et al. [15] demonstrated that lnc-MONC restrains the grade malignancy of endometrial cancer stem cells (ECSCs) and endometrial cancer cells (ECCs) by ordering the miR-636/GLCE axis. Similarly, lncRNA HOXB-AS3 was upregulated in endometrial cancer tissues and cell lines, enhanced cell proliferation, and inhibited apoptosis in EC cells [16]. Therefore, lncRNAs exhibit a novel potential therapeutic aim for EC.

lncRNA NBAT1 is a functional lncRNA, which was first found in neuroblastoma [17, 18]. NBAT1 could facilitate the restraining of neuroblastoma by inhibiting proliferation and invasion of tumor cells, which might be recognized as a predictor of neuroblastoma prognosis [19]. Hu et al. [20] revealed that NBAT1 constrained breast cancer metastasis through interrelating with EZH2. So far, only one study has shown the high expression of NEAT1 in endometrial cancer tissues and cell lines, and NEAT1 overexpression promotes HEC-59 cell growth and invasive and migratory ability [21]. However, the effect of NBAT1 on EC apoptosis is still unclear, and its molecular mechanism of regulating EC biological behavior still needs to be further explored.

MicroRNAs (miRNAs) are endogenous noncoding RNAs with a length of about 18–24 nt and control gene expression through posttranscriptional repression [22]. lncRNAs might serve as miRNA sponges that contribute to the modulation of miRNAs on their targets [23]. For example, Dai et al. suggested that lnc-STYK1-2 repressed bladder cancer development by binding to miR-146b-5p to modulate ITGA2. Yang et al. [17] indicated that NBAT1 repressed osteosarcoma development and metastasis via cooperating with miR-21-5p. However, whether NBAT1 functions as a ceRNA to regulate the progression of EC has not yet been explored. However, whether miR-21-5p is targeted for regulation by NBAT1 in ECs remains unclear. Whether NBAT1 can regulate the biological behavior of EC cells based on ceRNA mechanism deserves further study.

This study measures the levels of NBAT1 in endometrial cancer cells and tumor tissues. Furthermore, the regulatory role of NBAT1 in the proliferation and metastasis of endometrial cancer cell lines and the potential molecular mechanism is also explored. The mechanism by which NBAT1 inhibits the biological behavior of EC cells by targeting the miR-21-5p/PTEN axis was analyzed by cellular experiments. This research might offer a novel diagnostic and therapeutic target for endometrial cancer.

2. Methods

2.1. Tissues Samples

EC samples and adjacent endometrial tissues were obtained from 20 EC patients undergoing surgical resection during 2016 to 2020 at hospital, . The paired adjacent normal tissues were elected >4 cm away from the tumor sample located. Three pathologists assessed the tumor tissues and adjacent normal samples. All the specimens were then gathered in liquid nitrogen and saved at -80 ̊C for further use. All patients signed consent, and the study was approved by the Ethics Committee of People’s Hospital of Ningxia Hui Autonomous Region (No.: 2020-KY-049).

2.2. Cell Culture

Human endometrial cancer cell lines (HEC-1A and Ishikawa) were obtained from ATCC (Rockville, MD, USA), and the hESC was obtained from Wicell (Madison, WI). HEC-1A and Ishikawa cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin-streptomycin solutions (Invitrogen, Carlsbad, CA, USA) at 37°C with 5% CO₂ in a humidified incubator.

2.3. Cell Transfection

miR-21 mimic, lncRNA-NBAT1 (OE-lncRNA-NBAT1), siRNA targeting lnc-NBAT1, and NC were purchased from GenePharma (Shanghai, China). Cell transfection was conducted using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfection efficiency was validated using RT-qPCR.

2.4. Cell Proliferation Assay

Endometrial cell lines, HEC-1A, and Ishikawa were seeded at 96-well plates ( cells per well) and cultured at 37°C with 5% CO₂. The next day, cells were infected with the vectors as follows: control, OE-lncRNA-NBAT1, OE-NC, miR-21 mimic, and OE-lncRNA-NBAT1+miR-21 mimic for 48 h. Next, CCK-8 reagent (APExBio, Houston, TX, USA) was added to the corresponding wells and maintained for 4 h. The absorbance (450 nm) was assessed by a microplate reader (Bio-Tek, Winooski, U.S.A.).

2.5. Matrigel Assay

Cell migration was measured using 24-well transwell filters (BD Biosciences). HEC-1A and Ishikawa cells were added to the upper chamber of a transwell chamber, and 20% FBS was added to the lower chamber. Subsequently, the lower chamber cells were fixed with methanol, then stained with 0.1% crystal violet, and counted using a light microscope (Leica, Germany). Cell invasion assays were performed with Matrigel (Corning) at 37°C for 30 min and were constant with the migration assay.

2.6. Dual-Luciferase Reporter Assay

The full length of NBAT1 was ligated into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Ribobio, China) to construct wild-type (WT) pmirGLO-lncRNA. Furthermore, the pmirGLO-lncRNA mutant (MUT) was developed in which the binding sites of miR-21-5p were mutated. The 3UTR of PTEN mRNA was amplified from cDNA derived from the total RNA of HEC1A and Ishikawa cells, and cells were transfected with pmirGLO vector (Ribobio, China), pmirGLO-PTEN-3UTR-wild type (WT). Mutation reporter vector, with a mutation in the 3UTR complementary to the seed sequence of miR-21-5p, was created by PCR. HEC1A cells were cotransfected with the reporter vectors combined with miR-21-5p or negative controls mimics. After 48 h of transfection, the cells were gathered, and luciferase assays were carried out with the Dual-Luciferase reporter Gene Assay Kit (Beyotime, China). Fluorescence intensity was detected by an F-4500 Fluorescence Spectrophotometer (Hitachi, Japan).

2.7. Flow Cytometry

After transfection, HEC-1A and Ishikawa cells were splashed with phosphate-buffered saline (PBS) and gathered into the centrifuge pipe. After centrifugation, cells were stained using Apoptosis Analysis Kit (Beyotime Biotechnology, Shanghai, China). Flow cytometry was performed using a Flow cytometer (BD Biosciences, Detroit, MI, U.S.A.), and the apoptosis was estimated.

2.8. Wound Healing Assay

Cell migration was identified by the scratch assay. HEC-1A and Ishikawa cells were seeded in 6-well microplates and grown to 95% confluence. The adherent cells were scraped by a 10 μL tip to generate wounds and then were splashed with PBS three times, and the medium was substituted with a serum-free culture medium. Images were photographed instantly after the wounding, and the sizes of scratches were assessed.

2.9. Quantitative Reverse Transcription PCR

Total RNA was isolated from the cells by TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized using M-MLV reverse transcriptase (GeneCopoeia, Inc.). The RT-qPCR was conducted using SYBR Green (Bio-Rad Laboratories, Inc.) on a real-time PCR system (Thermo Fisher Scientific, Inc.). GAPDH and U6 were applied as the endogenous controls for NBAT1, PTEN, and miR-21-5p expression, respectively. The following primers were used: PTEN forward, 5-TGGATTCGACTTAGACTTGACCT-3; PTEN reverse, 5-GGTGGGTTATGGTCTTCAAAAGG-3; NBAT1 forward, 5-GCAGCTCAGATGAAGAAACTG-3, NBAT1 reverse, 5-GCAATATCCAAATCCTGCCTC-3; miR-21-5p forward, 5-GCACCTAGCTTATCAGACTGA-3, miR-21-5p reverse, 5-GTGCAGGGTCCGAGGT-3; and GAPDH forward, 5-CGCTCTCTGCTCCTCCTGTTC-3, GAPDH reverse, 5-ATCCGTTGACTC CGACCTTCAC-3. The relative expression was evaluated via the delta-delta CT method.

2.10. Western Blot

Western blot was conducted as described previously [24] with the following antibodies: anti-PTEN (138G6, CST 9559 1 : 1000) and anti-GAPDH (1 : 10000, Sigma, St. Louis, MO). The total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with a protease inhibitors cocktail (Roche). The proteins were measured using a BCA assay kit (Pierce, Rockford, IL, USA) according to the standard protocol.

2.11. Statistical Analysis

Results were evaluated using SPSS 20 software (SPSS Inc., Chicago, IL, USA) and are stated as . Paired Student’s -test and ANOVA were applied for statistical analysis. was considered to demonstrate significance.

3. Results

3.1. lncRNA NBAT1 Is Downregulated in Endometrial Cancer Cells and Tumor Tissues

To assess the association of lncRNA NBAT1 expression with EC progression, we first examined the expression of NBAT1 in endometrial cancer cells and tumor tissues. NBAT1 expression in 20 paired EC and adjacent endometrial tissues was measured by qRT-PCR. The result indicated that NBAT1 expression was markedly reduced in EC tissues in reference to that in the paired adjacent endometrial tissues (Figure 1(a), ). To further confirm the dysregulation of NBAT1 in EC, we also determined the expression of NBAT1 in human Ishikawa and HEC-1A cells. We found a much lower expression of lnc-NBAT1 in human Ishikawa and HEC-1A cells in compared to hESCs (Figure 1(b), ). These findings implied that NBAT1 was downregulated in endometrial cancer cells and tumor tissues.

(a)

(b)

3.2. Overexpression of lnc-NBAT1 Inhibits EC Cell Viability, Migration, and Invasion and Promotes Apoptosis

Subsequently, we elucidated whether NBAT1 is implicated in the modulation of EC development. First, we constructed Ishikawa and HEC-1A cell lines solidly expressing the NBAT1 overexpression. The transfection efficiency of NBAT1 was validated via RT-qPCR. As expected, overexpression constructs efficiently upregulated NBAT1 expression in Ishikawa and HEC-1A cells (Figure 2(a), ). CCK-8 assay exposed that overexpression of NBAT1 remarkably decreased the viability of Ishikawa and HEC-1A cells (Figure 2(b), ). Furthermore, wound healing analysis validated that the migratory ability of human Ishikawa and HEC-1A cells was markedly reduced when NBAT1 was overexpressed (Figure 2(c), and ). Transwell assay revealed that overexpression of NBAT1 significantly attenuated Ishikawa and HEC-1A cell invasion and migration (Figure 2(d), ). In addition, overexpression of NBAT1 increased apoptosis in both Ishikawa and HEC-1A cells (Figure 2(e), ). These findings implied that overexpression of lncRNA NBAT1 hindered cell viability, migration, and invasion and facilitated cell apoptosis in EC.

(a)

(b)

(c)

(d)

(e)

Figure 2

Overexpression of lncRNA NBAT1 inhibits EC cell viability, migration, invasion, and promotes cell apoptosis. (a) The transfection efficiency of NBAT1 was confirmed by RT-qPCR. (b) The cell viability of EC cells was measured using CCK-8. (c) The migration of EC cells was measured using a wound healing assay and the corresponding quantitative results. (d) The invasion of EC cells was measured using transwell assays and the corresponding quantitative results. (e) The apoptosis of EC cells was measured using flow cytometry assay and the corresponding quantitative results. Flow cytometry analysis, respectively. Bar: 100 μm. vs. the control group.

3.3. lncRNA NBAT1 Directly Binds and Negatively Regulates the Expression of miR-21-5p in EC

Then, we explored the underlying molecular mechanisms of lncRNA NBAT1 in regulating EC metastasis and apoptosis. Precious study[25] has found putative binding sites between miR-21-5p and NBAT1, as shown in Figure 3(a). To further validate the association between NBAT1 and miR-21 in EC cells, WT-NBAT1 and MUT-NBAT1 were constructed into the dual-luciferase vectors. As exhibited in Figure 3(b), the relative luciferase activity was markedly reduced in the HEC-1A cells cotransfected with miR-21-5p mimics and the luciferase vector containing WT-NBAT1 when in compared with the cells transfected with miR-NC (). In contrast, no noticeable difference in the luciferase activity was found in the MUT-NBAT1-transfected cells between miR-NC and miR-21-5p mimics groups. Furthermore, the relative miR-21-5p expression was much higher in EC tissues than that in adjacent normal tissues (Figure 3(c), ), which was significantly increased in human Ishikawa and HEC-1A cells compared with that in the hESCs (Figure 3(d), and ). Also, we detected the miR-21-5p and NBAT1 expression in HEC-1A and Ishikawa cells with knockdown of lnc-NBAT1. The results demonstrated that knockdown of NBAT1 markedly upregulated miR-21-5p levels () and downregulated NBAT1 in HEC-1A and Ishikawa cells (Figure 3(e), and ). In brief, these findings suggested that NBAT1 directly bound and negatively regulated the expression of miR-21-5p in EC cell lines.

(a)

(b)

(c)

(d)

(e)

Figure 3

lncRNA NBAT1 directly binds and negatively regulates the expression of miR-21 in EC. (a) The putative binding sites of miR-21-5p on NBAT1 transcripts. (b) The binding ability between NBAT1 and miR-21-5p in HEC-1A cells was assessed using the luciferase reporter assay in HEC-1A cells. Overexpression of miR-21-5p significantly decreased the luciferase activity of WT-NBAT1, whereas no significant changes were observed in MUT-NBAT1. (c) The relative expression of miR-21-5p in EC and the adjacent tissues was measured by RT-qPCR. (d) The relative expression of miR-21-5p in hESCs, HEC-1A, and Ishikawa cells and was detected by RT-qPCR. (e) The relative expressions of lncRNA NBAT1 and miR-21-5p in EC cells were detected by RT-qPCR. vs. adjacent tissues or hESC or sh-NC; vs. miR-NC or hESC or sh-NC.

3.4. miR-21-5p Reverses the Effect of lncRNA NBAT1 on Proliferation and Migration of EC Cell Lines

To investigate whether NBAT1 regulated EC tumorigenesis through miR-21-5p, miR-21-5p mimics or NBAT1+miR-21-5p was transfected into HEC-1A and Ishikawa cells. RT-qPCR exposed that the decreased expression of NBAT1 was restored by miR-21-5p mimics in both HEC-1A and Ishikawa cells (Figure 4(a), ). The CCK-8 assay exposed that NBAT1 restricted EC cell viability (), while miR-21-5p mimics inverted the inhibitory effect of NBAT1 overexpression on HEC-1A and Ishikawa cells viability (, Figure 4(b)). Flow cytometry analysis demonstrated that NBAT1 overexpression was associated with a marked increase in EC cell apoptosis (, ), but miR-21-5p mimics significantly recovered the increase of apoptotic rate induced by NBAT1 overexpression in HEC-1A and Ishikawa cells ( and , Figure 4(c)). Also, we found that an obvious decrease in migratory ability of Ishikawa and HEC1A cells induced by NBAT1 overexpression was restored by miR-21-5p mimics ( and , Figure 4(d)). Transwell assay revealed that the decreased invasion and migration induced by NBAT1 overexpression was recovered by miR-21-5p mimics, as well ( and , Figure 4(e)). Overall, the above findings implied that NBAT1 regulated the proliferation, invasion, and migration of EC cell lines by sponging miR-21-5p.

(a)

(b)

(c)

(d)

(e)

Figure 4

miR-21-5p reverses the effect of lncRNA NBAT1 on proliferation and migration of EC cells. (a) The transfection efficiency of NBAT1 and miR-21-5p in EC cells was confirmed via RT-qPCR analysis. (b) The proliferation of EC cells was measured using CCK-8. (c) The apoptosis of EC cells was measured using flow cytometry assay and the corresponding quantitative results. (d) The migration of EC cells was measured using a wound healing assay and the corresponding quantitative results. (e) The invasion of EC cells was measured using transwell assays and the corresponding quantitative results. Bar: 100 μm. vs. the control group; ^#, ^##, and ^### vs. the NBAT1 group; ^& and ^&&& vs. the miR-21-5p mimics group.

3.5. NBTA1 Elevates PTEN Expression via Sponging miR-21 in EC

Prior research has exposed that PTEN was a potential target of miR-21-5p [26]. The present study proved the binding site between miR-21-5p and PTEN (Figure 5(a)). Dual-luciferase reporter assay showed that miR-21-5p mimics markedly repressed the luciferase activity of the WT-PTEN (), while there are no differences in the MUT-PTEN (Figure 5(b)), suggesting that PTEN might sponge miR-21-5p in EC cells. RT-qPCR was carried out to assess the PTEN expression in endometrial cancer tissues and the adjacent endometrial tissues. A noticeable decrease in PTEN expression was identified in endometrial cancer tissues (Figure 5(c), ). Additionally, the result of Western blot exhibited that the protein expression of PTEN was much lower in HEC-1A and Ishikawa cells than in hESC (Figure 5(d)). Furthermore, NBAT1 enhanced the protein expression of PTEN in HEC-1A and Ishikawa cell lines (Figures 5(e) and 5(f)). On the contrary, miR-21-5p mimics markedly suppressed the protein expression of PTEN in HEC-1A and Ishikawa cells (Figures 5(e) and 5(f)), suggesting that PTEN is a critical downstream target of miR-21-5p. Consequently, these findings suggested that NBTA1 elevated PTEN expression via sponging miR-21 in EC.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 5

lncRNA NBTA1 elevates PTEN expression via sponging miR-21 in EC cells. (a) The complementary sequence of miR-21-5p in PTEN transcripts. (b) The binding ability between PTEN and miR-21-5p in HEC-1A cells was assessed using a luciferase reporter assay. (c) The relative mRNA expression of PTEN in EC and adjacent normal tissues was assessed using RT-qPCR. (d) The protein expression of PTEN in normal human ESC and EC cell lines was detected using Western blot. (e) The protein expression of PTEN in HEC-1A cells transfected with NBAT1 or/and miR-21-5p mimics was detected by Western blot. (f) The protein expression level of PTEN in Ishikawa cells transfected with NBAT1 or/and miR-21-5p mimics was detected by Western blot. vs. adjacent tissues; vs. miR-NC.

4. Discussion

Endometrial cancer (EC) is one of the most frequent gynecological malignancies worldwide [27]. The morbidity of EC has boosted worldwide results from the growth of elderly individuals and rising rates of obesity [28]. EC was sorted into two types based on the histologic features, estrogen-dependent (type I) endometrial cancer, and estrogen-independent type II nonestrogen-dependent with an inclination of recurrence [29]. The prognosis of EC is impacted by many elements, including tumor stage, histologic sort, differentiation level, and degree of myometrial invasion [30]. Although EC is often diagnosed with incipient stage owing to anomalous vaginal hemorrhage, the prognostic factors and survival rate of metastatic EC remain the focus of the current research. As a result, the underlying molecular mechanisms that induce EC require to be exposed to comprehend the underlying mechanisms of EC metastasis.

Recently, numerous studies have proposed that lncRNAs function a critical role in modulating gene expression at both transcriptional and posttranscriptional levels [31]. Increasing evidence supported that lncRNAs were implicated in various cancers involving EC development [32]. For example, lnc-PICSAR affected REV3L expression and improved DDP resistance in cutaneous squamous cell carcinoma [24]. Moreover, lnc-HSD17B11-1:1 was found to enhance the expression of MACC1, thus promoting CRC progression [33]. Evidence has supported the tumor suppressor role of lncRNA NBTA1 in various human tumors, including renal carcinoma and gastric cancer. Gao and Chen [34] suggested that low expression of NBAT-1 could promote GC development by downregulating PTEN expression. Xue et al. [35] implied that NBAT-1 could inhibit RCC cell proliferation and invasion. To further clarify the expression characteristics of lncRNA NBTA1 in EC, 20 clinical EC samples and normal tissues were also collected, and it was showed that the level of NBTA1 in EC tissues was significantly lower than that in normal tissues. And lncRNA NBTA1 levels in human EC cell lines (HEC-1A and Ishikawa) are also significantly lower than that in normal hESC. In this study, HEC-1A and Ishikawa cell models overexpressing NBTA1 were constructed. The results showed that overexpression of NBTA1 significantly inhibited the ability of migration and invasion and promoted apoptosis of HEC-1A and Ishikawa cells. These results are consistent with previous findings that lncRNA NBTA1 might function as a tumor suppressor and valuable prognostic indicator for EC.

Generally, lncRNAs show function depends on ceRNA to constrain miRNA and lncRNAs bind miRNA[36]. Numerous studies confirm a crucial role of lncRNAs in sponging-specific miRNAs, a type of small, noncoding RNAs comprising 18–25 nucleotides that could suppress gene expression at the posttranscriptional level [37]. In addition, an increasing number of findings have implied the effect of ceRNAs on endometrial cancer pathology. Zhou et al. [38] demonstrated that long noncoding RNA H19 promoted the tumorigenesis of endometrial cancer by regulating miR-20b-5p/AXL/HIF-1α axis. Pan et al.[39] suggested that LINC01016 accelerated endometrial cancer development through mediating miR-302a-3p/SATB1 pathway. Based on above, it is essential to perform studies on the comprehension of the ceRNAs formed by lncRNAs and miRNAs implicated in EC metastasis. Therefore, it is speculated that lncRNA NBTA1 might play the role of ceRNA and is involved in the development of EC.

In this study, bioinformatics analysis predicted that miR-21-5p was a potential target of NBTA1 and validated by the luciferase activity reporter assay. As reported previously, miR-21-5p inhibits non-small-cell lung cancer progression, colon adenocarcinoma, and thyroid papillary carcinoma [26, 40, 41]. In this study, it was confirmed through cell experiments that NBTA1 was targeted to bind to miR-21-5p and overexpression of miR-21-5p not only promoted the proliferation and invasion of EC and inhibited apoptosis but also significantly blocked the effect of NBTA1 on EC cells. These observations indicated that NBAT1 could play a tumor-suppressing role in EC cells by connecting with miR-21-5p to regulate the target genes negatively.

Additionally, miRNA targets are critical portions of the ceRNA network [42]. PTEN is a well-known secondary messenger of PI3K and is a tumor-suppressing gene implicated in inhibiting tumor cells’ growth and differentiation [43, 44]. Recent evidences demonstrate that PTEN serves as a tumor suppressor in EC [45, 46]. In addition, emerging evidence shown that PTEN was a target mRNA of miR-21-5p and miR-21-5p accelerated cancer development by restraining PTEN [47, 48]. To determine whether the miR-21-5p/PTEN targeting relationship exists in ECs and whether this regulatory axis is targeted by NBAT1, further experiments are performed. We found that miR-21-5p bound to the 3-UTR of PTEN mRNA. Furthermore, overexpression of NBAT1 promoted PTEN protein expression in EC cells, while overexpression of miR-21-5p suppressed PTEN protein levels and overexpression of miR-21-5p blocked the promotion of PTEN by NBAT1. This suggested that the mechanism by which NBAT1 promoted PTEN was inseparable from targeting miR-21-5p.

5. Conclusion

In conclusion, this research reveals that lncRNA NBAT1 elevated PTEN expression via sponging miR-21-5p to promote EC proliferation and metastasis. NBAT1 functions as a miR-21-5p sponge to enhance PTEN level, thereby repressing cell proliferation and promoting apoptosis in EC cell lines. Our study indicated that inhibition of NBAT1 might serve as a novel molecular target of gene therapy for EC.

Data Availability

The simulation experiment data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval

This study was approved by the Ethics Committee of People’s Hospital of Ningxia Hui Autonomous Region (No. 2020-KY-049).

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

Chunhua Tian, Hongyun Ma, and Jing Su performed the experiment; Hongyun Ma, Chunhua Tian, and Zhao Ma contributed significantly to analysis and manuscript preparation; Chunhua Tian, Hongyun Ma, and Yang Wu performed the data analyses and wrote the manuscript; Chunhua Tian, Yang Wu, and Jing Su helped perform the analysis with constructive discussions. The authors declare that all data were generated in-house and that no paper mill was used. Chunhua Tian and Jing Su contributed equally to this work and are the co-first authors.

Acknowledgments

This study supported by the Natural Science Foundation of Ningxia Province (No. 2020AAC03336).

References

Y. Zhang, F. Z. Jiang, W. Bao et al., “SOX17 increases the cisplatin sensitivity of an endometrial cancer cell line,” Cancer Cell International, vol. 16, no. 1, pp. 1–9, 2016.
View at: Publisher Site | Google Scholar
A. Torres, J. Kozak, A. Korolczuk et al., “In vitro and in vivo activity of miR-92a–locked nucleic acid (LNA)–inhibitor against endometrial cancer,” BMC Cancer, vol. 16, no. 1, p. 822, 2016.
View at: Publisher Site | Google Scholar
W. A. Khan, A. S. Alsamghan, and M. Khan, “Endometrial cancer patients have high affinity antibodies for estrogen metabolite–receptor aggregate: a potential biomarker for EC,” Journal of Obstetrics and Gynaecology Research, vol. 46, no. 10, pp. 2115–2125, 2020.
View at: Publisher Site | Google Scholar
X. Yu, B. Zhou, Z. Zhang et al., “Significant association between IL-32 gene polymorphisms and susceptibility to endometrial cancer in Chinese Han women,” Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine, vol. 36, no. 7, pp. 5265–5272, 2015.
View at: Publisher Site | Google Scholar
G. Emons, G. Fleckenstein, B. Hinney, A. Huschmand, and W. Heyl, “Hormonal interactions in endometrial cancer,” Endocrine-Related Cancer, pp. 227–242, 2000.
View at: Publisher Site | Google Scholar
J. Zhu, T. A. O’Mara, D. Liu et al., “Associations between genetically predicted circulating protein concentrations and endometrial cancer risk,” Cancers, vol. 13, no. 9, p. 2088, 2021.
View at: Publisher Site | Google Scholar
G. Zhang, X. Yu, Z. Sun, L. Zhu, and J. Lang, “Value of endometrial thickness in diagnosis of endometrial hyperplasia during selective estrogen receptor modulator therapy in premenopausal breast cancer patients,” Reproduction, vol. 50, no. 8, p. 101929, 2021.
View at: Publisher Site | Google Scholar
M. Avila, B. Fellman, and R. R. Broaddus, “Mismatch repair deficiency predicts worse survival despite adjuvant treatment in stage I endometrial cancer: opportunities for novel adjuvant therapy,” Gynecologic Oncology, vol. 159, p. 201, 2020.
View at: Publisher Site | Google Scholar
J. Guo, X. Cui, X. Zhang, H. Qian, H. Duan, and Y. Zhang, “The clinical characteristics of endometrial cancer with extraperitoneal metastasis and the value of surgery in treatment,” Technology in Cancer Research & Treatment, vol. 19, p. 153303382094578, 2020.
View at: Publisher Site | Google Scholar
Z. Zhao, W. Sun, Z. Guo, B. Liu, H. Yu, and J. Zhang, “Long noncoding RNAs in myocardial ischemia-reperfusion injury,” Oxidative Medicine and Cellular Longevity, vol. 2021, 15 pages, 2021.
View at: Publisher Site | Google Scholar
W. Wei, X. Zhao, J. Zhu et al., “lncRNA-u50535 promotes the progression of lung cancer by activating CCL20/ERK signaling,” Oncology Reports, vol. 42, no. 5, 2019.
View at: Publisher Site | Google Scholar
M. L. Pei, Z. X. Zhao, and T. Shuang, “Dysregulation of lnc-SNHG1 and miR-216b-5p correlate with chemoresistance and indicate poor prognosis of serous epithelial ovarian cancer,” Journal of Ovarian Research, vol. 13, no. 1, p. 144, 2020.
View at: Publisher Site | Google Scholar
X. Wang, G. Jiang, W. Ren, B. Wang, C. Yang, and M. Li, “lncRNA NEAT1 regulates 5-Fu sensitivity, apoptosis and invasion in colorectal cancer through the MiR-150-5p/CPSF4 axis,” Oncotargets and Therapy, vol. Volume 13, pp. 6373–6383, 2020.
View at: Publisher Site | Google Scholar
G. W. Ku, Y. Kang, S. L. Yu et al., “lncRNA LINC00240 suppresses invasion and migration in non-small cell lung cancer by sponging miR-7-5p,” BMC Cancer, vol. 21, no. 1, p. 44, 2021.
View at: Publisher Site | Google Scholar
Y. Li, J. Huo, J. He, and X. Ma, “lncRNA MONC suppresses the malignant phenotype of endometrial cancer stem cells and endometrial carcinoma cells by regulating the MiR-636/GLCE axis,” Cancer Cell International, vol. 21, no. 1, p. 331, 2021.
View at: Publisher Site | Google Scholar
Y. Xing, X. Sun, F. Li et al., “Long non-coding RNA (lncRNA) HOXB-AS3 promotes cell proliferation and inhibits apoptosis by regulating ADAM9 expression through targeting miR-498-5p in endometrial carcinoma,” The Journal of International Medical Research, vol. 49, no. 6, p. 030006052110135, 2021.
View at: Publisher Site | Google Scholar
C. Yang, G. Wang, J. Yang, and L. Wang, “Long noncoding RNA NBAT1 negatively modulates growth and metastasis of osteosarcoma cells through suppression of miR-21,” American Journal of Cancer Research, vol. 7, no. 10, pp. 2009–2019, 2017.
View at: Google Scholar
L. Wei, M. Ling, S. Yang, Y. Xie, C. Liu, and W. Yi, “Long noncoding RNA NBAT1 suppresses hepatocellular carcinoma progression via competitively associating with IGF2BP1 and decreasing c-Myc expression,” Human Cell, vol. 34, no. 2, pp. 539–549, 2021.
View at: Publisher Site | Google Scholar
F. Shao and Z. Wang, “Effect of the downregulation of long non-coding RNA NBAT1 expression on biological behavior of bladder cancer 5637 cells,” Tumori, vol. 37, no. 1, pp. 19–26, 2017.
View at: Google Scholar
P. Hu, J. Chu, Y. Wu et al., “NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2,” Oncotarget, vol. 6, no. 32, pp. 32410–32425, 2015.
View at: Publisher Site | Google Scholar
Z. Li, D. Wei, C. Yang, H. Sun, T. Lu, and D. Kuang, “Overexpression of long noncoding RNA, NEAT1 promotes cell proliferation, invasion and migration in endometrial endometrioid adenocarcinoma,” Biomedicine & Pharmacotherapy, vol. 84, pp. 244–251, 2016.
View at: Publisher Site | Google Scholar
S. Morales, F. Gulppi, P. Gonzalez-Hormazabal et al., “Association of single nucleotide polymorphisms in pre-miR-27a, pre-miR-196a2, pre-miR-423, miR-608 and pre-miR-618 with breast cancer susceptibility in a South American population,” BMC Genetics, vol. 17, no. 1, p. 109, 2016.
View at: Publisher Site | Google Scholar
S. Ghafouri-Fard, A. Abak, S. Tavakkoli Avval et al., “Contribution of miRNAs and lncRNAs in osteogenesis and related disorders,” Biomedicine & Pharmacotherapy, vol. 142, no. 2, article 111942, 2021.
View at: Publisher Site | Google Scholar
D. Wang, X. Zhou, J. Yin, and Y. Zhou, “Lnc-PICSAR contributes to cisplatin resistance by miR-485-5p/REV3L axis in cutaneous squamous cell carcinoma,” Open Life Sciences, vol. 15, no. 1, pp. 488–500.
View at: Publisher Site | Google Scholar
Z. Liu, D. Xie, and H. Zhang, “Long noncoding RNA neuroblastoma-associated transcript 1 gene inhibits malignant cellular phenotypes of bladder cancer through miR-21/SOCS6 axis,” Cell Death & Disease, vol. 9, no. 10, pp. 1–3, 2018.
View at: Publisher Site | Google Scholar
D. Lv, Q. Bi, Y. Li et al., “Long non‑coding RNA MEG3 inhibits cell migration and invasion of non‑small cell lung cancer cells by regulating the miR‑21‑5p/PTEN axis,” Molecular Medicine Reports, vol. 23, no. 3, 2021.
View at: Publisher Site | Google Scholar
M. Sinreih, N. Hevir, and T. L. Rizner, “Altered expression of genes involved in progesterone biosynthesis, metabolism and action in endometrial cancer,” Chemico-Biological Interactions, vol. 202, no. 1-3, pp. 210–217, 2013.
View at: Publisher Site | Google Scholar
S. Capriglione, F. Plotti, A. Miranda et al., “Further insight into prognostic factors in endometrial cancer: the new serum biomarker HE4,” Expert Review of Anticancer Therapy, vol. 17, no. 1, pp. 9–18, 2017.
View at: Publisher Site | Google Scholar
F. Xue, H. Li, and S. Jiao, “A study on the clinicopathological characteristics of estrogen-dependent and estrogen-independent endometrial carcinoma,” Chinese Journal of Clinical Oncology, 2000.
View at: Google Scholar
G. Corrado, V. Laquintana, R. Loria et al., “Endometrial cancer prognosis correlates with the expression of L1CAM and miR34a biomarkers,” Journal of Experimental & Clinical Cancer Research, vol. 37, no. 1, p. 139, 2018.
View at: Publisher Site | Google Scholar
Y. Han, M. K. Ali, K. Dua, E. Spiekerkoetter, and Y. Mao, “Role of long non-coding RNAs in pulmonary arterial hypertension,” Cell, vol. 10, no. 8, p. 1892, 2021.
View at: Publisher Site | Google Scholar
W. Xin, X. Liu, J. Ding et al., “Long non-coding RNA derived miR-205-5p modulates human endometrial cancer by targeting PTEN,” American Journal of Translational Research, vol. 7, no. 11, pp. 2433–2441, 2015.
View at: Google Scholar
W. Zhang, B. Wang, Q. Wang et al., “Lnc-HSD17B11-1:1 functions as a competing endogenous RNA to promote colorectal cancer progression by sponging miR-338-3p to upregulate MACC1,” Frontiers in Genetics, vol. 11, p. 628, 2020.
View at: Publisher Site | Google Scholar
Y. Gao and J. Chen, “Inhibition of cancer cell growth by oleanolic acid in multidrug resistant liver carcinoma is mediated via suppression of cancer cell migration and invasion, mitochondrial apoptosis, G2/M cell cycle arrest and deactivation of JNK/p38 signalling pathway,” Journal of B.U.ON.: official journal of the Balkan Union of Oncology, vol. 24, no. 5, pp. 1964–1969, 2019.
View at: Google Scholar
S. Xue, S. Wang, J. Li et al., “lncRNA NBAT1 suppresses cell proliferation and migration via miR-346/GSK-3β axis in renal carcinoma,” IUBMB Life, vol. 71, no. 11, pp. 1720–1728, 2019.
View at: Publisher Site | Google Scholar
Q. H. Zhu and M. B. Wang, “Molecular functions of long non-coding RNAs in plants,” Genes, vol. 3, no. 1, pp. 176–190, 2012.
View at: Publisher Site | Google Scholar
M. Wang, S. Zheng, X. Li et al., “Integrated analysis of lncRNA-miRNA-mRNA ceRNA network identified lncRNA EPB41L4A-AS1 as a potential biomarker in non-small cell lung cancer,” Frontiers in Genetics, vol. 11, 2020.
View at: Publisher Site | Google Scholar
H. Zhu, Y. M. Jin, X. M. Lyu, L. M. Fan, and F. Wu, “Long noncoding RNA H19 regulates HIF-1α/AXL signaling through inhibiting miR-20b-5p in endometrial cancer,” Cell Cycle (Georgetown, Texas), vol. 18, no. 6, pp. 1–11, 2019.
View at: Google Scholar
X. Pan, D. Li, J. Huo, F. Kong, H. Yang, and X. Ma, “LINC01016 promotes the malignant phenotype of endometrial cancer cells by regulating the miR-302a-3p/miR-3130-3p/NFYA/SATB1 axis,” Cell Death & Disease, vol. 9, no. 3, p. 303, 2018.
View at: Publisher Site | Google Scholar
H. Zhang, Y. Cai, L. Zheng, Z. Zhang, X. Lin, and N. Jiang, “lncRNA BISPR promotes the progression of thyroid papillary carcinoma by regulating miR-21-5p,” International Journal of Immunopathology and Pharmacology, vol. 32, p. 205873841877265, 2018.
View at: Google Scholar
W. Yu, K. Zhu, Y. Wang, H. Yu, and J. Guo, “Overexpression of miR-21-5p promotes proliferation and invasion of colon adenocarcinoma cells through targeting CHL1,” Molecular Medicine, vol. 24, no. 1, p. 36, 2018.
View at: Publisher Site | Google Scholar
Y. X. Song, J. X. Sun, J. H. Zhao et al., “Non-coding RNAs participate in the regulatory network of CLDN4 via ceRNA mediated miRNA evasion,” Communications, vol. 8, no. 1, 2017.
View at: Publisher Site | Google Scholar
R. Parsons, Identification of Extracellular Form of PTEN That Can Be Used to Treat Tumors, U.S. Patent and Trademark Office, Washington, DC, 2012.
G. Xun, W. Hu, and B. Li, “PTEN loss promotes oncogenic function of STMN1 via PI3K/AKT pathway in lung cancer,” Scientific Reports, vol. 11, no. 1, p. 14318, 2021.
View at: Publisher Site | Google Scholar
B. Djordjevic, B. A. Barkoh, R. Luthra, and R. R. Broaddus, “Relationship between PTEN, DNA mismatch repair, and tumor histotype in endometrial carcinoma: retained positive expression of PTEN preferentially identifies sporadic non-endometrioid carcinomas,” Modern Pathology, vol. 26, no. 10, pp. 1401–1412, 2013.
View at: Publisher Site | Google Scholar
Q. Wang, Y. Yang, and X. Liang, “Retracted article: lncRNA CTBP1-AS2 sponges miR-216a to upregulate PTEN and suppress endometrial cancer cell invasion and migration,” Journal of Ovarian Research, vol. 13, no. 1, 2020.
View at: Publisher Site | Google Scholar
O. Peralta-Zaragoza, J. Deas, A. Meneses-Acosta et al., “Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells,” BMC Cancer, vol. 16, no. 1, p. 215, 2016.
View at: Publisher Site | Google Scholar
H. Shen, F. Zhu, J. Liu et al., “Alteration in Mir-21/PTEN expression modulates gefitinib resistance in non-small cell lung cancer,” PLoS One, vol. 9, no. 7, article e103305, 2014.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2022 Chunhua Tian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies