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Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

ABSTRACT Endometrial carcinoma (EC) originates from the endometrium and is one of the most common tumors in female patients, and its incidence has continued to increase in recent decades. LncRNAs are involved in the pathogenesis and metastasis of a variety of malignant tumors, which indicates that lncRNAs can be used as tumor diagnostic markers and potential therapeutic targets. In this study, we analyzed the RNA transcripts of EC cells from The Cancer Genome Atlas (TCGA) and first reported a novel lncRNA, BMPR1B-AS1, that was more highly expressed in endometrial cancer tissues than in adjacent tissues, and BMPR1B-AS1 could promote endometrial cancer cell proliferation and metastasis. Bioinformatics prediction and experimental results both suggested that BMPR1B-AS1 could modulate the malignant behaviors of endometrial cancer cell lines by sponging miR-7-2-3p to modulate DCLK1, and a DCLK1 inhibitor blocked the activation of the PI3K/Akt/NF-κB signaling pathway. Collectively, this study suggests that the BMPR1B-AS1/miR-7-2-3p/DCLK1 axis contributes to the proliferation and metastasis of endometrial cancer cells via the PI3K/Akt/NF-κB pathway.


Introduction
Endometrial carcinoma (EC) originates from the endometrium and is one of the most common tumors in female patients, and its incidence has continued to increase in recent decades [1]. Surgery combined with chemotherapy and radiotherapy can notably prolong the survival of patients with early EC. However, due to the lack of effective treatments for recurrent and metastatic tumors, the relative 5-year survival rate of EC patients has not significantly improved [1]. Therefore, a novel nonsurgical therapeutic option is required [2].
Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs with lengths of greater than 200 nucleotides [3]. LncRNAs are important regulators of gene expression and chromatid modification and are involved in a wide variety of physiological and pathological processes throughout the body [3,4]. Notably, lncRNAs are involved in the pathogenesis and metastasis of a variety of malignant tumors, which indicates that lncRNAs can be used as tumor diagnostic markers and potential therapeutic targets [3,5]. However, the number of disease-related lncRNAs is far lower than the total number of known lncRNAs [6].
MicroRNAs (miRNAs) are endogenous, singlechain, noncoding RNAs that are approximately 22 nucleotides in length; miRNAs play important roles in cell proliferation, differentiation and apoptosis [7]. miRNAs can inhibit and degrade specific genes through cleavage or transcriptional repression [7]. Compared with that in normal cells, lncRNA and miRNA expression in cancer cells may be upregulated or downregulated, and lncRNAs and miRNAs regulate the development and metastasis of cancers [8][9][10].
LncRNAs can function via various mechanisms; in one such mechanism, lncRNAs may exert their biological effects through their ability to act as competitive endogenous RNAs (ceRNAs) for miRNAs [11]. Typically, miRNAs function as negative regulators of gene expression, and in the presence of ceRNAs, the negative regulatory effects of miRNAs on their targets is impaired [7,12,13]. Various lncRNAs were reported to function as ceRNAs and play crucial roles in the pathological mechanisms of EC. For instance, lncRNA NEAT1 promotes EC cell proliferation and invasion by regulating the miR-144-3p/EZH2 axis [10]. LINC00261 expression is downregulated in endometrial carcinoma cells and increases FOXO1 protein levels by reducing miR-182, miR-183, miR-153, miR-27a, and miR-96 expression to inhibit endometrial carcinoma cell proliferation, migration, and invasion [9]. Despite this knowledge, the key lncRNAs associated with EC remain to be identified and could be potential targets for EC treatment.
In this study, we analyzed the RNA transcripts of EC cells from The Cancer Genome Atlas (TCGA) and first reported a novel lncRNA, BMPR1B-AS1, that was more highly expressed in endometrial cancer tissues than in adjacent tissues. We explored the potential function of BMPR1B-AS1 in endometrial cell lines, and the results indicated that BMPR1B-AS1 could modulate the malignant behaviors of endometrial cell lines via the miR-7-2-3p/DCLK1 axis, and a DCLK1 inhibitor could block the activation of the PI3K/Akt/ NF-κB signaling pathway. Collectively, our findings suggested that the BMPR1B-AS1/miR-7-2-3p/ DCLK1 axis contributes to the proliferation and metastasis of endometrial cancer cells via the PI3K/Akt/NF-κB pathway, providing new targets for EC diagnosis and treatment.

Bioinformatics analysis
The RNA transcription data from 405 EC samples were obtained from TCGA. The para-cancerous tissues and carcinoma tissues were distinguished according to the rules for naming TCGA samples, and the differential expression patterns were analyzed with restriction [the mean values of FPKM in the two groups were both greater than 1, |log2 fold change(FC)| >1.0, False Discovery Rate(FDR) <0.05]. MiRanda was used to predict potential target genes, and combined with literature review and test screening, miR-7-2-3p and DCLK1 were selected for further studies.

Patients and specimens
Ten cancer tissues and paired adjacent nontumor tissues (obtained from sites at least 2 cm from the tumor edge) were obtained from patients who were pathologically diagnosed with EC during surgery at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) from October 2018 to April 2020. All the fresh specimens were rapidly frozen in liquid nitrogen and then stored in a − 80°C freezer. The protocol of this study was approved by the ethics committee of The First Affiliated Hospital of Zhengzhou University, and all the patients provided informed consent.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissue samples or cultured cells with TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer's instructions.
The RNA concentration and quality were confirmed using a NanoDrop One C Microvolume UV-Vis Spectrophotometer (Thermo, Waltham, USA). cDNA was generated using PrimeScript RT Master Mix (Takara). Then, RT-qPCR was conducted with SYBR Green qPCR Mix (BioTeke, Beijing, China). Each sample was standardized to the expression of U6 or β-actin as an internal control gene. Relative gene expression was calculated using the 2 -ΔΔCT method. All the primer sequences are listed in Table 1.

Fluorescence in situ hybridization
Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China). Fluorescent in situ hybridization experiments were conducted using a FISH Kit (GenePharma) according to the manufacturer's instructions. Briefly, Ishikawa cells were fixed in 4% paraformaldehyde (PFA, Biosharp, Hefei, China) at room temperature for 15 min and then washed with PBS. The cells were then permeabilized with Triton X-100 (Beyotime, Shanghai, China) at 4°C for 15 min. Subsequently, the cells were incubated with DIG-labeled BMPR1B-AS1 probes or Control-FISH probes at 55°C for 4 h and washed with 2× PBS for 10 min. The signals were detected with SABC (streptavidin-biotin complex)-FITC (BOSTER). DAPI was used to counterstain the nuclei. Images were obtained by confocal microscopy. The probe sequences are presented in Table 1.

Cell proliferation
Cell proliferation was monitored with a cell counting kit-8 (CCK8, Dojin, Kumamoto, Japan). Briefly, a total of 3 × 10 3 cells were seeded in each well of a 96-well plate and cultured at 37°C in 5% CO 2 . At specific time points, 10 μl CCK-8 solution was added to each well, and the plate was incubated at 37°C for 1 h. Then, cell viability was measured by a microplate reader (Thermo) at 450 nm.

Transwell assay
Cells (5 × 10 4 cells/well for migration assays or 2 × 10 5 cells/well for invasion assays) were seeded in 200 μl serum-free media into the upper chambers, which were covered with (invasion assays) or without (migration assays) 40 μl Matrigel (BD, New Jersey, USA). The lower chambers contained 600 μl medium supplemented with antibiotics and 10% FBS. After 24 h, the cells that had invaded or migrated were fixed with 4% PFA (Biosharp) for 20 minutes, and the cells in the lower chambers were stained with 0.4% crystal violet for 20 minutes. Finally, the cells were washed with PBS and counted using an inverted microscope (Olympus Corporation; magnification, 200x) with the image processing program ImageJ software version 1.8 (National Institute of Health). Images from 3 random fields of view were captured for each group for analysis.

Wound healing assay
Cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well and cultured for 12 h. When the cell monolayer reached 80% confluence, a 200-μl pipette tip was used to create scratch wounds, and the cells were washed twice with serum-free medium to remove the cell debris. The cells were cultured in serum-free medium, and scratchwound healing was observed by microscopy (Olympus Corporation) and recorded at 0 h, 24 h, and 48 h after scratching.

Cell cycle and apoptosis analysis
Cell cycle assays were performed using a Cell Cycle Detection kit (Beyotime). After digestion and collection, the cells were washed twice with precooled PBS and fixed with 70% ethanol (Aladdin, Shanghai, China) at 4°C overnight. Then, the ethanol was removed, and the cells were washed with PBS. After adding 100 μl RNaseA to the samples, the cells were incubated in a 37°C water bath for 30 minutes. Finally, 500 μl PBS containing 50 μg/ml propidium iodide (PI) was added to the samples and incubated at 4°C in the dark for 30 minutes. A NovoCyte TM Flow Cytometer (ACEA Biosciences, San Diego, USA) was used to analyze the results. An Annexin V-FITC kit (Beyotime) was used to assess cell apoptosis. After collection, the cells were washed twice with PBS, and 500 μl binding buffer was added to resuspend the cells. The samples were mixed well with 5 μl Annexin V-FITC and 10 μl PI and incubated at room temperature in the dark for 15 minutes. A NovoCyte TM Flow Cytometer (ACEA Biosciences) was used to analyze the results.

Tumor xenografts in nude mice
5-week-old female BALB/c nude mice used for this study were purchased from Beijing Huafukang Bioscience Co., ltd., China and maintained in pathogen-free conditions. The animals were divided into 5 groups: Lv-BMPR1B-AS1 group, Lv-BMPR1B-AS1+ agomir-7-2-3p group, Lv-BMPR1B-AS1+ agomir-7-2-3p NC group, Lv-NC group, and blank Hec-1b group. Each mouse was injected subcutaneously in the right flank area with 5 × 10 6 cells. The agomir-7-2-3p(1 OD, GenePharma) or agomir NC(1 OD) was administrated by intra-tumoral injection once every 3 days after 10 days of inoculation(to allow for tumor growth). The tumor volume was calculated and recorded every 3 days according to the formula: volume(mm 3 ) = length(mm) × width(mm) 2 /2. After 25 days, all the mice were sacrificed, and the tumors were removed and weighed. The tumor tissues were subjected to RT-qPCR, and immunohistochemistry.

Immunohistochemistry (IHC)
The tissues were deparaffinized and rehydrated, and then were placed in a retrieval box filled with citric acid antigen retrieval buffer (PH6.0) for antigen retrieval in a microwave oven. DCLK1 antibody (JA11-03, HUABIO) and corresponding secondary antibody were sequentially incubated. Afterward, the freshly prepared DAB (Servicebio, Wuhan, China) solution was applied onto each section, and the nuclei were counterstained with hematoxylin.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, USA). Student's t-test was used to analyze the differences between two groups. Spearman's correlation analysis was employed to determine the correlation between two parameters. All the experiments were repeated at least three independent times. The data are presented as the mean ± SD. Statistical significance was defined as p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The expression of BMPR1B-AS1 was upregulated in endometrial cancer tissues
Analysis of the differential expression of lncRNAs between the paracancerous tissues and carcinoma tissues showed that the expression of 35 lncRNAs was decreased, while the expression of 39 lncRNAs was increased (the mean values of FPKM in the two groups were both greater than 1, |log2 fold change(FC)| >1.0, FDR <.005) (Figure 1a, Sup 1 and 2). Among these lncRNAs with altered expression, the expression of lncRNA BMPR1B-AS1 was most significantly increased. Then, we collected 28 tumor tissues and corresponding paracancerous tissues from EC patients who were pathologically diagnosed, and RT-qPCR was used to detect the expression of BMPR1B-AS1 in these samples. The results showed that the expression of BMPR1B-AS1 in the tumor tissues was significantly higher than that in the adjacent tissues ( Figure 1b).

Overexpression of BMPR1B-AS1 promotes malignant behaviors of Hec-1b cells
Because of the higher expression of BMPR1B-AS1 in EC tissues, we further explored the oncogenic role of BMPR1B-AS1 in EC cells. First, different expression levels of BMPR1B-AS1 were detected in EC cell lines (Ishikawa, Hec-1a and Hec-1b cells) by RT-qPCR, and the results demonstrated that Ishikawa cells had the highest expression, Hec-1a had intermediate expression, and Hec-1b had the lowest expression (Figure 1c). Then, we used Lv-BMPR1B-AS1 to overexpress BMPR1B-AS1 in Hec-1b cells. The BMPR1B-AS1 overexpression efficiency was confirmed by RT-qPCR ( Figure 2a). According to the CCK8 assay, BMPR1B-AS1 overexpression significantly promoted cell growth compared with Lv-NC ( Figure 2b). Both wound healing assays and Transwell migration assays revealed that overexpression of BMPR1B-AS1 facilitated the migration of Hec-1b cells (Figure 2c, 2e, 2g, and 2h). Transwell invasion assays showed that the invasion capability of Hec-1b cells was enhanced after BMPR1B-AS1 overexpression (Figure 2d and 2f). Subsequently, apoptosis was inhibited in the Lv-BMPR1B-AS1 group (Figure 2i and 2k). With BMPR1B-AS1 overexpression, we observed increased accumulation of cells in the S-phase and decreased accumulation of cells in the G0/G1 phase, which was consistent with the CCK8 results. (Figure 2j and 2l). In addition, the expression of E-cadherin, N-cadherin, Cyclin D1 and CDK4, which act as key players in tumor cell dissemination and proliferation, was examined by western blotting (Figure 2m-q). The results indicated that overexpression of BMPR1B-AS1 led to decreased expression of E-cadherin but increased expression of N-cadherin, Cyclin D1 and CDK4. These results collectively suggested that

Knockdown of BMPR1B-AS1 expression inhibits the malignant behaviors of Ishikawa cells
Next, Ishikawa cells were transfected with Lv-sh-BMPR1B-AS1 to knockdown the expression of BMPR1B-AS1. The knockdown efficiency was determined by RT-qPCR (Figure 3a). According to the CCK8 assay, transfection with Lv-sh-BMPR1B-AS1 significantly inhibited cell growth compared with transfection with Lv-sh-NC (Figure 3b). Both wound healing assays and Transwell migration assays revealed that knockdown of BMPR1B-AS1 expression inhibited the migration of Ishikawa cells (Figure 3c, 3e, 3g, and 3h). Transwell invasion assays showed that the invasion of Ishikawa cells was inhibited after BMPR1B-AS1 knockdown (Figure 3d and 3f). Subsequently, the apoptosis rate was significantly increased in the Lv-sh-BMPR1B-AS1 group (Figure 3i and 3k). With BMPR1B-AS1 knockdown, we observed increased accumulation of cells in the G0/G1 phase and decreased accumulation of cells in the S-phase, which was consistent with the CCK8 results (Figure 3j and 3l). Western blotting assays indicated that knockdown of BMPR1B-AS1 expression led to increased expression of E-cadherin but decreased expression of N-cadherin, Cyclin D1 and CDK4 (Figure 3m-q).
These results collectively suggested that knockdown of BMPR1B-AS1 expression inhibited the proliferation, migration and invasion of Ishikawa cells while promoting apoptosis.

Downregulation of miR-7-2-3p expression reverses the inhibition of Ishikawa cells mediated by the knockdown of BMPR1B-AS1 expression.
Cotransfection with the miR-7-2-3p inhibitor promoted the proliferation of the BMPR1B-AS1 -knockdown cell lines (Figure 6a). Transwell assays and wound healing assays both showed that BMPR1B-AS1 knockdown cell lines cotransfected with the miR-7-3p inhibitor had higher migration and invasion capabilities than those cotransfected with the miR-7-2-3p inhibitor NC (Figure 6b-g). Cotransfection with the miR-7-2-3p inhibitor inhibited apoptosis and cell cycle arrest, and these effects were reversed by knockdown of BMPR1B-AS1 expression in Ishikawa cells (Figure 6h-k).

DCLK1 is a target gene of miR-7-2-3p
DCLK1 was predicted to be a target gene of miR-7-2-3p (Figure 7a). A dual-luciferase assay was conducted to elucidate whether miR-7-2-3p can interact with DCLK1. The results showed that cotransfection of DCLK1-WT and the miR-7-2-3p mimic decreased the luciferase activity of the cells, whereas cotransfection of DCLK1-MUT and the miR-7-2-3p mimic and cotransfection of DCLK1-WT and the miR-7-2-3p mimic NC did not change the luciferase activity of the cells (Figure 7b and 7c). Also, we found a negative relationship between miR-7-2-3p expression and DCLK1 mRNA expression in EC tissues (Figure 7d).

Effects of DCLK1 on the PI3K, Akt, NF-κB expression levels in Ishikawa and Hec-1b cells
We further explored the mechanism by which DCLK1 regulates endometrial cancer cells. It has been reported that DCLK1 promotes cancer progression via the PI3K/Akt/NF-κB axis in other cancers [14]. Therefore, we hypothesized that DCLK1 may regulate the biological behaviors of endometrial cancer cells via the PI3K/Akt/NF-κB pathway. DCLK1-IN-1 is an effective inhibitor of DCLK1 15 . Ishikawa cells and Hec-1b cells were treated with 1 μM DCLK1-IN-1 for 1, 4 and BMPR1B-AS1 knockdown increased the accumulation of cells in the G0/G1 phase and decreased the accumulation of cells in the S-phase. M, N, O, P, Q, Western blot and analysis showed that the protein level of E-cadherin was increased, but the protein levels of N-cadherin, Cyclin D1 and CDK4 were decreased in the BMPR1B-AS1 knockdown group. Sh, short hairpin. The data are representative of three independent experiments. Bars, ±SD. **P < .01, ***P < .001, ****P < 0.0001.  24 hours. DMSO treatment for the same time points was used as a control. As shown in Figure 8, the levels of p-PI3K p85, p-Akt and p-p65 were decreased in the DCLK1-IN-1 group, but we did not observe any significant changes in the total PI3K p85, Akt or p65 levels. Collectively, our results suggested that the PI3K/Akt/NF-κB pathway may be involved in DCLK1-mediated endometrial cancer progression.

Discussion
EC-related lncRNAs have been studied in recent years, and when dysregulated, these lncRNAs act as oncogenes or tumor suppressors. Although an increasing number of studies about lncRNAs have been published in recent years, the study of lncRNAs in EC is still in its infancy [8]. lncRNAs can be divided into different categories, including intronic lncRNAs, bidirectional lncRNAs, intergenic lncRNAs, enhancer RNAs, circular RNAs (circRNAs), pseudogenes, sense lncRNAs, and natural antisense transcripts (NATs), based on their genomic location of origin or their relationship with protein-coding genes [15,16]. NATs are transcribed from the DNA strand opposite to that of the sense transcript and can exert their effects locally, distally, in cis or in trans, and they can also function in multiple subcellular compartments [16][17][18][19]. Aberrant expression of NATs is closely related to cancer development [16]. Different mechanisms underlying the biological functions involved in tumorigenesis depend on the subcellular localization of NATs; among these subcellular localizations, cytoplasmic NATs mainly regulate RNA stability and/or mRNA translation [17,20]. IFN-α1 AS is transcribed from the positive strand of chromosome 9, which is the strand opposite the IFNA1 locus (9p22) and overlaps with the conserved secondary structure (CSS) region of IFN-α1 mRNA. This NAT can interact with IFN-α1 mRNA in the cytoplasm by competing for the miR-1270 binding site, preventing the miRNA-induced destabilization of IFN-a1 mRNA [21]. Similarly, BACE1-AS prevents miR-485-5pinduced BACE1 mRNA repression by competing for the same site [22]. LOXL1-AS1 can upregulate the expression of RAP1B, a member of the oncogene family, and then play oncogenic roles in the growth and metastasis of EC cells by sponging miR-28-5p [23]. ZEB1-AS1 expression is significantly upregulated in colon adenocarcinoma (COAD) and can promote PAK2 expression by sponging miR-455-3p, thus facilitating COAD cell growth and metastasis [24]. In the present study, we first defined BMPR1B-AS1 as a cytoplasmic tumor promoter that functions via the miR-7-2-3p/DCLK1 axis through bioinformatics analysis and experiments. miR-7 is a gene that has attracted substantial attention in the field of tumor research, and its expression is downregulated in a variety of tumor cells. As a tumor suppressor gene, miR-7 participates in tumor growth, development, invasion and metastasis through a variety of signaling pathways [ [25][26][27]. miR-7 has three subtypes, namely, miR-7-1, miR-7-2, and miR-7-3, which originate from three different genes. However, these subtypes can be processed into the same mature miR-7 sequence, which comprises 23 nucleotides [28]. miR-7-2-3p is an antisense miRNA star product of miRNA-7-229. Additionally, miR-7-2-3p is generally considered to be a cancer suppressor. miR-7-2-3p expression is downregulated in gallbladder carcinoma (GBC), and upregulation of miR-7-2-3p expression can reverse epithelial-mesenchymal transition (EMT) and decrease gallbladder carcinoma cell metastasis in vitro and in vivo [29]. miR-7-2-3p plays a role in thyroid cancer tumorigenesis by influencing critical pathways, and deficiency in miR-7-2-3p is closely associated with lymph node metastases (LNMs) [30,31]. The correlation of decreased miR-7-2-3p expression with decreased overall survival and progression-free survival in patients with locally advanced esophageal adenocarcinoma (EAC) demonstrated that miR-7-2-3p may serve as a promising prognostic marker in EAC [32]. However, miR-7-2-3p may promote the tumorigenesis process of pancreatic neuroendocrine tumors [33]. This study demonstrated that BMPR1B-AS1 functions as an oncogenic factor by directly sponging miR-7-2-3p and inhibiting its expression, suggesting that miR-7-2-3p is a suppressor of EC. DCLK1 (doublecortin-like kinase 1) is a member of the microtubule-associated protein (MAP) family and has been identified as a tumor stem cell marker in gastrointestinal tumors [34,35]. DCLK1 is overexpressed in gastric cancer, pancreatic cancer and many other malignant tumors, and DCLK1 overexpression induces the development, metastasis and EMT of tumor cells [14,[36][37][38]. DCLK1 is the direct target gene of miR-7-2-3p in GBC [29]. In this study, the regulatory effect of miR-7-2-3p on DCLK1 was also verified, and DCLK1 expression was positively correlated with the progression of endometrial cancer. Additionally, the DCLK1-positive cell ratio and mean density were both increased in EC tissues (77.5066 ± 15.1461% and 0.3947 ± 0.0577, respectively) compared with paracarcinoma tissues (75.4211 ± 14.0169% and 0.3724 ± 0.0473, respectively), even though there was no significant difference, while the DCLK1 mean density in advanced EC (0.4299 ± 0.0713) was significantly higher than that in early-stage EC (0.3781 ± 0.0431) (p < 0.05) (data not shown). Nevertheless, few studies have examined the role of DCLK1 in the endometrium. DCLK1 expression is upregulated in the cells of the uterine cavity of endometriosis patients before dissemination into the peritoneal cavity, indicating that the eutopic upregulation of DCLK1 expression is crucial for endometriotic cell survival before the establishment of endometriotic lesions [39]. This finding may indicate that DCLK1 is associated with endometrial cell migration capability, which is partially consistent with our findings. It has been reported that DCLK1 plays an oncogenic role via the PI3K/Akt/NF-κB signaling pathway in colorectal cancer, and activation of the PI3K/Akt/NF-κB pathway improves cell proliferation and metastasis, which are key factors in cancer progression [14,[40][41][42]. To elucidate whether the PI3K/Akt/NF-κB pathway is also involved in the DCLK1-mediated progression of endometrial cancer, we treated Hec-1b and Ishikawa cells with a DCLK1 inhibitor and then assessed the levels of activated PI3K, Akt and NF-κB. We observed that the DCLK1 inhibitor continuously blocked the levels of phosphorylated PI3K, phosphorylated Akt and NF-κB subunit p65 in Hec-1b cells and Ishikawa cells.
This is the first study to define BMPR1B-AS1 as an oncogenic factor. We studied the molecular functions that BMPR1B-AS1 performs and the corresponding mechanisms from the perspective of the popular ceRNA theory both in vitro and in vivo. It has been widely thought that the regulation of gene expression by antisense RNAs is a posttranscriptional phenomenon and that antisense RNAs function only as repressors of the translation of specific mRNAs. However, several reports have demonstrated that antisense RNAs may silence their sense genes at the transcriptional level, affecting DNA methylation and histone modifications [17,18,43]. Because BMPR1B expression is significantly decreased in EC samples compared to benign samples [44], it is necessary to further investigate whether BMPR1B-AS1 plays a role in the regulation of BMPR1B transcription.
In conclusion, we first showed that lncRNA BMPR1B-AS1 plays a critical role in the progression of EC by competitively binding to miR-7-2-3p in order to mediate DCLK1-induced PI3K/Akt/NF-κB pathway activation. BMPR1B-AS1 may be a potential therapeutic target for the treatment of EC; however, further investigation is still required to determine the mechanism underlying the role of BMPR1B-AS1 in EC.