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. 2024 Sep 5;16(17):1133–1148. doi: 10.1080/17501911.2024.2388018

LncRNA SSTR5-AS1 promotes esophageal carcinoma through regulating ITGB6/JAK1/STAT3 signaling

Zhaohui Tang a, Yongjun Jiang a, Yuyu Zong a, Sijuan Ding a, Chen Wu a, Zhangwen Tang a, Lin Liao a, Shaohui Jiang a, Ruoting Tang a, Fang Li a, Pengfei Luo a,*
PMCID: PMC11457597  PMID: 39234955

Abstract

Aim: To investigate function of somatostatin receptor 5 antisense RNA 1 (SSTR5-AS1) in esophageal carcinoma (ESCA).

Materials & methods: The cellular function was assessed using EdU staining and Transwell assay. The localization of SSTR5-AS1 was measured using fluorescence in situ hybridization staining.

Results: SSTR5-AS1 shRNA repressed invasion and migration and induced apoptosis in ESCA cells. SSTR5-AS1 was distributed in cytoplasm, and it regulated its subunit integrin beta 6 (ITGB6) via eukaryotic translation initiation factor 4A3 (EIF4A3). SSTR5-AS1 shRNA inactivated ITGB6 and JAK1/STAT3 signaling. SSTR5-AS1 silencing attenuated the malignant behavior of ESCA cells through the ITGB6-mediated JAK1/STAT3 axis.

Conclusion: SSTR5-AS1 promotes tumorigenesis of ESCA by interacting with EIF4A3 to regulate ITGB6/JAK1/STAT3 axis, which serves a basis for discovering strategies against ESCA.

Keywords: : EIF4A3, ESCA, ITGB6, JAK1/STAT3, SSTR5-AS1

Plain Language Summary

The development of esophageal carcinoma (ESCA) seriously affects the health of people. Although great efforts have been made for curing ESCA, the outcomes remain limited. In this research, we used large amounts of experiments about the molecular biology. As expected, we found knockdown of lncRNA SSTR5-AS1 could inhibit the tumorigenesis of ESCA through mediation of its subunit integrin beta 6 /JAK1/STAT3 axis. Thus, our research provided new molecular targets for ESCA treatment.

Plain language summary

Article highlights.

  • Somatostatin receptor 5 antisense RNA 1 (SSTR5-AS1) was upregulated in esophageal carcinoma (ESCA).

  • Knockdown of SSTR5-AS1 inhibited the malignant behavior of ESCA cells.

  • SSTR5-AS1 interacted with EIF4A3.

  • EIF4A3 interacted with Integrinβ6 (ITGB6).

  • SSTR5-AS1 enhanced the mRNA stability of ITGB6 through binding with EIF4A3.

  • Silencing of SSTR5-AS1 inhibited the tumor growth of ESCA in vivo.

  • ITGB6 positively regulated JAK1/STAT3 signaling.

  • SSTR5-AS1 promoted the progression of ESCA via ITGB6-mediated JAK1/STAT3 signaling.

  • SSTR5-AS1 might act as a diagnostic marker in ESCA.

1. Introduction

According to data published in the Global Cancer Statistics 2020, approximately 20,000,000 people are diagnosed with cancer annually, and over 10,000,000 people die from malignant tumors worldwide [1]. Esophageal carcinoma (ESCA) accounts for up to 3.1% of all new cancer cases, with its incidence ranking eighth among all tumors. More importantly, ESCA accounts for 5.5% of all cancer-related deaths and ranks sixth in terms of mortality [2]. Though nearly 90% of ESCA cases are esophageal squamous cell carcinoma (ESCC), the incidence andmortality rates of ESCA are gradually increasing to the point of even surpassing those of ESCC in some regions across Europe and North America [3,4]. The primary risk factors for ESCA mainly include obesity and gastroesophageal reflux disease, which is closely associated with chemical carcinogen exposure, cigarette smoking and alcohol consumption, a diet low on fruits or vegetables, high consumption of pickled vegetables or processed meat, hot drinks, etc. [5,6]. The 5-year survival rates of patients with ESCA usually range from 20 to 30%, depending mainly on the tumor stage at initial diagnosis and the therapeutic strategy, such as surgery combined with neoadjuvant therapy (radiotherapy and chemotherapy) [7,8]. Although considerable efforts have been made in the treatment of ESCA, conventional treatments have shown limited efficacy and potential adverse effects [9,10]. Hence, more effective therapeutic strategies are urgently needed to improve the prognosis of patients with ESCA.

Long noncoding RNAs (LncRNAs) are transcripts longer than 200 nucleotides that can act as not only tumor promoters or tumor suppressors in the progression of malignant cancer [11–13] but also crucial mediators in the development of ESCA [14,15]. In addition, dysregulation of lncRNAs could participate in the progression of ESCA. For example, Liang et al. found that the upregulation of lncRNA LINC01088 attenuated the progression of ESCA by regulating the nucleophosmin 1 (NPM1)-human double minute 2 (HDM2)-p53 axis [16]. Cheng et al. found that lncRNA tumor protein translationally controlled 1-antisense RNA 1 (TPT1-AS1) could significantly increase ESCA cell invasion via mediation of the miR-26a/high mobility group AT-hook 1 (HMGA1) axis [17]. Kong et al. showed that overexpression of small nucleolar RNA host gene 1 (SNHG1) significantly promoted the tumorigenesis of ESCA via regulation of the miR-216a-3p/transmembrane bax inhibitor motif-containing 6 (TMBIM6) axis [14], whereas Hu et al. revealed that LINC00963 promoted cisplatin resistance in ESCA by interacting with miR-10a to upregulate the expression of spindle and kinetochore-associated complex subunits 1 (SKA1) [18]. Meanwhile, the level of somatostatin receptor 5 antisense RNA 1 (SSTR5-AS1) was found to be upregulated in ESCA [19], revealing that SSTR5-AS1 can serve as a mediator of ESCA. However, the detailed mechanism underlying the function of SSTR5-AS1 remains unexplored.

Reports have shown that lncRNAs can regulate the progression of cancers by binding with RNA binding proteins (RBPs). For instance, Liu et al. demonstrated that integrin-α9 antisense RNA 1 (ITGA9-AS1) upregulated integrin-α9 (ITGA9) by targeting miR-4765 and recruiting heterogeneous nuclear ribonucleoprotein U (HNRNPU) to inhibit the proliferation and apoptosis of non-small cell lung cancer cells [20], whereas Wang et al. revealed that A1BG antisense RNA 1 (A1BG-AS1) could promote adriamycin resistance of breast cancer by recruiting insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) to upregulate ABCB1 in an m6A-dependent manner [21]. In addition, RBPs can bind with transcript or nontranscript RNAs to regulate disease progression [22,23]. Meanwhile, eukaryotic translation initiation factor 4A3 (EIF4A3), a member of the RBP family, is able to act as an oncogene in ESCA [24]. For example, lncRNA SNHG16 was able to promote the tumorigenesis of ESCA by interacting with EIF4A3 [25]. Similarly, Xue et al. revealed that lncRNA LUESCC promoted tumorigenesis of ESCA by targeting the miR-6785-5p/Neurensin-2 (NRSN2) axis [26]. In the mentioned study, predictions using starBase (https://rnasysu.com/encori/) indicated that SSTR5-AS1 might interact with EIF4A3, although the detailed relationship between SSTR5-AS1 and EIF4A3 remains largely unknown. Moreover, Integrin beta 6 (ITGB6) acts as a mediator of multiple biological functions during the progression of various cancers [27] and has been closely associated with tumor stage [28]. Previous studies have shown that ITGB6 could promote the malignant behavior of ESCA cells [29,30]. Furthermore, it evidence suggests that the inactivation of JAK1/STAT3 could inhibit the progression of ESCA and that silencing of ITGB6 could inhibit the phosphorylation of p-STAT3 and p-JAK1 in cervical carcinoma [31]. Nevertheless, the detailed relationships between SSTR5-AS1, EIF4A3 and ITGB6/JAK1/STAT3 signaling in ESCA needs further investigations.

Based on the aforementioned findings, we can hypothesize that SSTR5-AS1 may interact with EIF4A3 to modulate ITGB6/JAK1/STAT3 signaling, which further aggravates the tumorigenesis of ESCA. We believe that the current study provides evidence regarding a potentially new target for the treatment of ESCA.

2. Materials & methods

2.1. Gene expression profiling interactive analysis (GEPIA) database

The level of SSTR5-AS1 in ESCA tissues (n = 182) and normal tissues (n = 286) or the prognosis of patients with ESCA were explored using the GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=boxplot). GEPIA is a bioinformatics tool used to investigate the expression level of target genes in cancers, as well as explore the relationship between target genes and the prognosis of patients with malignant tumors. Data were analyzed using Log2FC as previously described [32].

2.2. Cell culture

ESCA cell lines (ECA-109, TE-1, TE-8 and KYSE410) and human endometrial epithelial cells were obtained from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM, Invitrogen, USA) containing 1% penicillin, streptomycin (Sigma, USA) and 10% fetal bovine serum (FBS, Gibco, USA), after which the cells were cultured in an incubator at 37°C and 5% CO2.

2.3. Cell transfection

shRNA negative control (sh-NC), SSTR5-AS1 shRNA (sh-SSTR5-AS1, GenePharma), EIF4A3 shRNA (sh-EIF4A3), ITGB6 shRNA (sh-ITGB6), pcDNA3.1, oe-EIF4A3 and oe-SSTR5-AS1 were purchased from GenePharma. In addition, cells were transfected with lentiviruses-expressed SSTR5-AS1 shRNA during knockdown of SSTR5-AS1. When ECA109 and KYSE410 cells reached 70%, cells (3 × 105) were transfected with sh-NC (10 μM), sh-SSTR5-AS1 (10 μM), sh-EIF4A3 (10 μM), sh-ITGB6 (10 μM), pcDNA3.1 (1 μg/μl), oe-EIF4A3 (1 μg/μl) or oe-SSTR5-AS1 (1 μg/μl) using Lipofectamine 3000 (Invitrogen) as per the manufacturer’s instruction.

2.4. RT-qPCR

Total RNA was isolated from cells and tumor tissues using TRIzol (Takara, Tokyo, Japan) according to the manufacturer’s instruction [33], and then the RNA was quantified using KingFisher Apex (Invitrogen). PrimeScript RT (cat. no. RR014A) was used for reversing RNA into cDNA. SYBR Premix Ex Taq II procured from ELK Bioscience (cat. no. EQ007) was applied for real-time PCR with Applied Biosystems 7900 Real-Time PCR System, which was procured from Applied Biosystems (Foster City, CA, USA). The RT-qPCR protocol was as follows: 60°C for 15 min and 90°C for 1 min, followed by 40 cycles at 90°C for 15 s and 55°C for 45 s. 2-ΔΔCt method was performed to quantify the relative expression levels of SSTR5-AS1 and ITGB6. The primers used were as follows: SSTR5-AS1: 5′-ACTACAGGTGCCATCAGACC-3′ (F) and 5′-AGCCTGCCATCCTAACACTT-3′ (R); ITGB6: 5′-ACTGGCCAGCTACTTACTGTG-3′ (F) and 5′-TTTTGGGGTTGTGACTTCACTG-3′ (R); and β-actin: 5′-GCCTCGCCTTTGCCGATCC-3′ (F) and 5′-GCGCGGCGATATCATCATCCA-3′ (R).

2.5. Western blot

Total protein was isolated from cells or tissues using RIPA buffer (Beyotime, Shanghai, China). Thereafter, the isolated proteins were quantified using BCA kit (cat. no P0009; Beyotime) and then separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the separated proteins were transferred onto PVDF membranes (Beyotime), which were then incubated with the primary antibodies overnight at 4°C after blocking with 5% skim milk. The primary antibodies used were as follows: anti-Bax (ab32503, 1:1,000), anticleaved caspase-3 (ab32042, 1:500), anti-Bcl-2 (ab182858, 1:1,000), anti-JAK1 (ab133666, 1:1,000), anti-STAT3 (ab68153, 1:1,000), anti-p-JAK1 (ab138005, 1:1,000), anti-p-STAT3 (ab76315, 1:2,000), anti-EIF4A3 (ab32485, 1:1,000), anti-ITGB6 (ab187155, 1:10,000) and anti-GAPDH (ab8245, 1:1,000). Subsequently, the membranes were incubated with the secondary antibody (HRP-conjugated; ab288151, 1:5000) at room temperature for 1 h. The protein bands were then visualized using the ECL kit (cat. no. P00185; Beyotime). All antibodies were purchased from Abcam (Cambridge, MA, USA).

2.6. CCK8 assay

Transfected ESCA cells were resuspended and subsequently seeded into 96-well plates, with a total of five wells allocated to each group. Each well was filled with 5 × 103 cells and 100 μl of DMEM, which was then incubated at 37°C with 5% CO2 for 48 h. The 10-μl CCK-8 solution (Beyotime) was pipetted into each well, followed by a 2 h incubation period. The absorbance (450 nm) of the cells was measured using a light microreader (SMZ745, Nikon, Tokyo, Japan).

2.7. 5-Ethynyl-2′-deoxyuridine (EdU) staining

ESCA cells were inoculated into 96-well plates (2 × 104 cells/well) 24 h after transfection. After culturing the cells for 18–24 h in DMEM, they were treated with 50 μM EdU (cat. no. C00715; BeyoClick™, Beyotime) for 2 h at 37°C, fixed with 4% paraformaldehyde, incubated with 2 mg/ml glycine for 5 min, incubated with PBS containing 0.5% Triton X-100 for 10 min, dipped in 1× Apollo staining solution, and finally incubated with 100 ml of 1× Hoechst 33342 for 30 min. EdU-positive cells were quantified via fluorescence microscopy (Nikon, Tokyo, Japan).

2.8. Fluorescence in situ hybridization (FISH) detection

ESCA cells were inoculated in 24-well plates. After growing to a suitable density, the medium was discarded, and the cells were washed with PBS. In addition, fluorescence-conjugated SSTR5-AS1 probe was synthesized using RiboBio (Guangzhou, China). Subsequently, the cells were fixed with 4% PFA paraformaldehyde and then washed, after which prehybridization solution was added at 65°C for 1 h. Thereafter, the hybridization solution containing the probe was added and reacted in the dark at 65°C in a hybridizer for 48 h. Afterward, the cells were washed with 4× and 2× SSC and then subjected to DAPI staining. The slices were observed via fluorescence microscopy after being sealed. RNA FISH probes were designed and synthesized using RiboBio.

2.9. Cell apoptosis detection

Transfected ESCA cells (2 × 104 cells/well) were cultured in DMEM overnight. Thereafter, the cells were trypsinized, washed and resuspended in Annexin V Binding Buffer (cat. no. 556454; Becton, Dickinson and Company, Franklin Lake, NJ, USA) after centrifugation (955 × g) for 5 min. The cells were then stained with 5 μl fluorescein isothiocyanate (FITC) and 5 μl propidium in the dark for 15 min. Afterward, the cells were analyzed using flow cytometry (BD) to test the cell apoptosis rate. In addition, the apoptotic cells were calculated using FlowJo (version 2.0; BD).

2.10. Transwell assay

For cell invasion and migration analysis, Transwell assays were performed. Cell invasion assays were performed using a 24-well Mill cell chamber (Corning 3422). The upper chamber was pretreated with 100 μl of Matrigel. The ESCA cells (2 × 105) were seeded into the upper chamber in media with 1% FBS, with the density being adjusted to approximately 1.0 × 106 cells per chamber. RPMI1640 medium with 10% FBS was added to the lower chamber. After 24 h of incubation at 37°C, the Transwell chamber was rinsed twice with PBS (5 min per time), fixed using 5% glutaraldehyde at 4°C, and stained with 0.1% crystal violet for 30 min. The Transwell chamber was washed twice with PBS and then observed under a microscope (original magnification, ×200). The number of invaded cells was regarded as a reflection of the invasion ability.

2.11. RNA immunoprecipitation (RIP)

Anti-Argonaute 2 (AGO2), anti-EIF4A3, and anti-IgG antibody (EMD Millipore) were applied for the immunoprecipitation of the lyzed cells. Finally, RT-qPCR analysis was performed to detect the purified RNA.

2.12. RNA pull-down

Cells (1 × 107) were lyzed using RNA lysis buffer (Beyotime), which consisted of 25 mM Tris-HCl (pH 8.0), 10 mM EDTA and 50 mM glucose, for 3 min after collection. Magnetic beads were applied to treat the SSTR5-AS1 probe for 2 h, after which the cell lysate was applied to incubate the SSTR5-AS1 probe at 4°C overnight. After the complex bound to the magnetic beads was eluted, protein extraction was performed. Thereafter, western blot analysis was performed to determine the level of EIF4A3.

2.13. RNA stability assay

Cells were treated with actinomycin D (5 μg/ml). RNA extraction was performed from 0 to 9 h. Actinomycin D (5 μg/ml) was performed to handle sh-SSTR5-AS1, sh-EIF4A3, or sh-NC and total RNA was isolated. After reverse transcribing the total RNA into cDNA, RT-qPCR was performed.

2.14. In vivo study

BALB/c nude mice (n = 12; 6–8 weeks old) were purchased from Vital River (Beijing, China) and housed within a dedicated SPF facility. ESCA cells stably expressing SSTR-AS1 shRNA were transplanted subcutaneously into each mouse using a method described previously [34]. The tumor volume was measured weekly as described previously [35]. At the end of the experiments, the mice were sacrificed, and the tumors were collected and weighted. The expression of cleaved caspase-3 was determined using immunohistochemical staining as previously reported [36]. All in vivo experiments were performed in accordance with National Institutes of Health guide for the care and use of laboratory animals, following a protocol approved by the Ethics Committees of the Central Hospital of Yongzhou (No.2023020301).

2.15. Immunohistochemical staining

Tissues were fixed and cut into 5 μm thick sections. Next, sections were heated in a microwave (with sodium citrate buffer) for the purpose of the antigen retrieval after being deparaffinized and rehydrated. The samples were then incubated with H2O2 (3%) for 30 min and then blocked in serum for another 30 min. Thereafter, the samples were incubated with anti-cleaved caspase-3 (cat. no. #9661; cell signaling, 1:400) at 4°C and then with HRP at 37°C for 30 min. Diaminobenzidine (DAB, Beyotime) was used for color development. Finally, the tissues were observed under a light microscope.

2.16. Statistical analysis

All statistical analyses were performed using GraphPad Prism (version 8.0). Data were presented as mean ± standard deviation. Differences between two groups were determined using Student’s t-test. Comparison between more than two groups were performed using one-way analysis of variance. In all analyses, a p-value of <0.05 indicated statistical significance.

3. Results

3.1. SSTR5-AS1 levels were upregulated in ESCA

To explore the role of SSTR5-AS1 in ESCA, the GEPIA database was used. As indicated in Figure 1A, SSTR5-AS1 levels were higher in ESCA tissues than in normal control tissues. In addition, high SSTR5-AS1 levels promoted poor prognosis from ESCA (Figure 1B). Meanwhile, the expression of SSTR5-AS1 was much higher in ESCA cells than in human endometrial epithelial cells (Figure 1C). Moreover, SSTR5-AS1 levels were significantly more upregulated in ECA-109 and KYSE410 cells than in other ESCA cells (Figure 1C). Hence, ECA-109 and KYSE410 cells were selected for subsequent experiments. Taken together, the expression of SSTR5-AS1 was upregulated in ESCA.

Figure 1.

Figure 1.

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SSTR5-AS1 levels were upregulated in ESCA. (A) SSTR5-AS1 levels in ESCA and adjacent tissues were explored using the GEPIA database. (B) The relationship between the expression of SSTR5-AS1 and survival of patients with ESCA was investigated using the GEPIA database. (C) The relative mRNA concentrations of SSTR5-AS1 in HEECs, ECA-199, TE-1, TE-8 and KYSE410 cells were examined using RT-qPCR. N = 3, *p < 0.05; **p < 0.01; ***p < 0.001.

ESCA: Esophageal carcinoma; GEPIA: Gene expression profiling interactive analysis; HEECs: Human endometrial epithelial cells; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1.

3.2. Knockdown of SSTR5-AS1 inhibited ESCA cell invasion & migration

To determine the function of SSTR5-AS1 in ESCA, ESCA cells were transfected with SSTR5-AS1 shRNA, after which RT-qPCR was performed to evaluate the transfection efficiency. As shown in Figure 2A, SSTR5-AS1 shRNA decreased the level of SSTR5-AS1 in ESCA cells. Silencing of SSTR5-AS1 notably reduced the proliferation and viability of ESCA cells (Figure 2B & C). Moreover, SSTR5-AS1 shRNA significantly attenuated ESCA cell invasion and migration (Figure 2D & E). Downregulation of SSTR5-AS1 induced considerable apoptosis of ESCA cells (Figure 2F). Meanwhile, SSTR5-AS1 shRNA markedly inhibited the protein level of Bcl-2 and upregulated the expressions of cleaved caspase-3 and Bax in ESCA cells (Figure 2G). Knockdown of SSTR5-AS1 downregulated the expression of p-JAK1 and p-STAT3 proteins in ESCA cells (Figure 2H). In summary, silencing of SSTR5-AS1 significantly reduced the invasion, proliferation and migration of ESCA cells.

Figure 2.

Silencing of SSTR5-AS1 inhibited the proliferation, invasion, and migration of ESCA cells. ECA-109 and KYSE410 cells were transfected with sh-NC or sh-SSTR5-AS1. (A) The relative mRNA concentration of SSTR5-AS1 in ESCA cells was determined using RT-qPCR. (B) The viability of ESCA cells was assessed using the CCK8 assay. (C) ESCA cell proliferation was examined via EdU staining. (D & E) The invasion and migration of ESCA cells were determine using the Transwell assay. (F) The apoptosis of ESCA cells was examined using flow cytometry. (G) The relative protein concentrations of Bax, cleaved caspase-3, and Bcl-2 in ESCA cells were examined via western blot analysis. (H) The relative protein concentrations of JAK1, p-JAK1, p-STAT3 and STAT3 were investigated via western blot analysis. N = 3, *p < 0.05; **p < 0.01; ***p < 0.001.

Bax: Bcl-2-associated Xprotein; Bcl-2: B-cell lymphoma protein-2; CCK8: Cell Counting Kit-8; EdU: 5-Ethynyl-2-deoxyuridine; ESCA: Esophageal carcinoma; JAK1: Janus kinase-1; RT-qPCR: Real-time reverse transcriptase-polymerase chain reaction; sh-NC: shRNA negative control; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1; SSTR5-AS1 shRNA: sh-SSTR5-AS1; STAT3: Signal transducer and activator of transcription 3.

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3.3. SSTR5-AS1 regulated the mRNA stability of ITGB6 through recruiting EIF4A3

For the purpose of investigating the localization of SSTR5-AS1, RT-qPCR and FISH assays were performed. We revealed that SSTR5-AS1 was distributed mainly in the ESCA cell cytoplasm (Figure 3A & B). In addition, SSTR5-AS1 was found to interact with EIF4A3 (Figure 3C & D). Consistently, anti-EIF4A3 increased the enrichment of ITGB6 in ESCA cells (Figure 3E). Moreover, knockdown of SSTR5-AS1 was able to inhibit EIF4A3 levels (Figure 3F). Furthermore, knockdown of EIF4A3 decreased the expression of ITGB6 (Figure 3G & H). EIF4A3/SSTR5-AS1 shRNA reduced the half-life of ITGB6 mRNA (Figure 3I). Furthermore, downregulation of SSTR5-AS1 decreased the levels of ITGB6, p-JAK1 and p-STAT3 in ESCA cells, which were partially reversed by the overexpression of EIF4A3 (Figure 3J). Taken together, SSTR5-AS1 regulated the mRNA stability of ITGB6 by recruiting EIF4A3.

Figure 3.

SSTR5-AS1 regulated the mRNA stability of ITGB6 by recruiting EIF4A3. (A) Relative mRNA concentration of SSTR5-AS1 in the cytoplasm or nucleus of ESCA cells was determined using RT-qPCR. (B) The localization of SSTR5-AS1 in ESCA cells was observed using the FISH assay. (C & D) The interaction between SSTR5-AS1 and EIF4A3 was determined using RIP and RNA pull-down assays. (E) The interaction between EIF4A3 and ITGB6 was explored using the RIP assay. (F) ESCA cells were transfected with sh-NC or sh-SSTR5-AS1. The relative protein concentration of EIF4A3 in ESCA cells was examined using western blot analysis. (G & H) ESCA cells were transfected with sh-EIF4A3 or sh-NC. The relative mRNA and protein concentrations of ITGB6 in ESCA cells were assessed using RT-qPCR and western blot analysis, respectively (I) ESCA cells were treated with actinomycin D and transfected with sh-NC, sh-EIF4A3, or sh-SSTR5-AS1. The half-life of ITGB6 mRNA was determined using RNA stability assay. (J) ESCA cells were treated with sh-NC, sh-SSTR5-AS1 or sh-SSTR5-AS1 + oe-EIF4A3. The relative protein concentrations of ITGB6, JAK1, p-JAK1, STAT3, and p-STAT3 in ESCA cells were examined using western blot analysis. N = 3, *p < 0.05; **p < 0.01; ***p < 0.001.

ESCA: Esophageal carcinoma; EIF4A3: Eukaryotic translation initiation factor 4A3; FISH: Fluorescence in situ hybridization; ITGB6: Integrin β6; JAK1: Janus kinase-1; RIP: RNA immunoprecipitation; RT-qPCR: Real-time reverse transcriptase-polymerase chain reaction; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1; STAT3: Signal transducer and activator of transcription 3.

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3.4. SSTR5-AS1 promoted the malignant behavior of ESCA cells by modulating ITGB6-mediated JAK1/STAT3 signaling

To explore the role of ITGB6 in SSTR5-AS1-mediated ESCA cell growth, ESCA cells were transfected with ITGB6 shRNA. As illustrated in Figure 4A, downregulation of ITGB6 decreased the levels of ITGB6 in ESCA cells. Silencing of ITGB6 markedly reduced the proliferation and viability of ESCA cells, a phenomenon that was reversed by the overexpression of SSTR5-AS1 (Figure 4B & C). Consistently, silencing of ITGB6 attenuated the invasion and migration of ESCA cells, which were greatly abolished in the presence of oe-SSTR5-AS1 (Figure 4D & E). Downregulation of ITGB6 markedly induced the apoptosis of ESCA cells, whereas upregulation of SSTR5-AS1 reversed the apoptotic effects of ITGB6 shRNA (Figure 5A). Furthermore, silencing of ITGB6 greatly inhibited the levels of Bcl-2, p-JAK1, and p-STAT3 and upregulated the expression of Bax and cleaved caspase-3, whereas overexpression of SSTR5-AS1 showed the opposite effects (Figure 5B & C). Taken together, SSTR5-AS1 promoted the malignant behavior of ESCA cells by mediating ITGB6-mediated JAK1/STAT3 signaling.

Figure 4.

Figure 4.

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SSTR5-AS1 promoted the malignant behavior of ESCA cells by mediating ITGB6/JAK1/STAT3 signaling. (A) ECA-109 and KYSE410 cells were treated with sh-NC or sh-ITGB6. The relative mRNA concentration of ITGB6 in ESCA cells was determine using RT-qPCR. (B) ESCA cells were transfected with sh-NC, sh-ITGB6, sh-ITGB6 + pcDNA3.1, and sh-ITGB6 + oe-SSTR5-AS1. The viability of ESCA cells was determined using the CCK8 assay. (C) The proliferation of ESCA cells was assessed using EdU staining. (D & E) The invasion and migration of ESCA cells were determined using the Transwell assay. N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. 

CCK8: Cell Counting Kit-8; EdU: 5-Ethynyl-2-deoxyuridine; ESCA: Esophageal carcinoma; ITGB6: Integrinβ6; RT-qPCR: Real-time reverse transcriptase-polymerase chain reaction; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1.

Figure 5.

Overexpression of SSTR5-AS1 inhibited the apoptosis of ESCA cells by regulating ITGB6/JAK1/STAT3 signaling. ESCA cells were transfected with sh-NC, sh-ITGB6, sh-ITGB6 + pcDNA3.1 and sh-ITGB6 + oe-SSTR5-AS1. (A) ESCA cell apoptosis was evaluated using flow cytometry. (B) The relative protein concentrations of Bax, cleaved caspase-3, and Bcl-2 in ESCA cells were investigated using western blot analysis. (C) The relative protein concentrations of JAK1, p-JAK1, p-STAT3, and STAT3 in ESCA cells were investigated using western blot analysis. N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. 

Bax: Bcl-2-associated Xprotein; Bcl-2: B-cell lymphoma protein-2; ESCA: Esophageal carcinoma; ITGB6: Integrinβ6; JAK1: Janus kinase-1; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1; STAT3: Signal transducer and activator of transcription 3.

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3.5. Silencing of SSTR-AS1 significantly attenuated the growth of ESCA tumors in vivo

To further confirm the function of SSTR-AS1 in ESCA, a xenograft mice model was established. Our data revealed that silencing of SSTR-AS1 significantly inhibited the tumor size in mice (Figure 6A), whereas SSTR-AS1 shRNA markedly inhibited the relative mRNA concentrations of SSTR-AS1 and ITGB6 and increased the protein levels of cleaved caspase-3 in tumor tissues of mice (Figure 6B–D). In addition, knockdown of SSTR-AS1 obviously decreased the relative protein concentrations of ITGB6, p-STAT3 and p-JAK1 in mice (Figure 6D). All these results indicated that silencing of SSTR-AS1 could inhibit the tumorigenesis of ESCA in vivo.

Figure 6.

Figure 6.

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Knockdown of SSTR5-AS1 significantly attenuated the growth of ESCA tumors in vivo by mediating ITGB6/JAK1/STAT3 signaling. (A) The tumor tissues of mice were captured, and the tumor volume was measured weekly. (B) The relative mRNA concentrations of SSTR5-AS1 and ITGB6 in mouse tissues were measured by RT-qPCR. (C) The protein level of cleaved caspase-3 in mice was determined using IHC staining. (D) The relative protein concentrations of ITGB6, STAT3, p-STAT3, JAK1, and p-JAK1 in mouse tissues were examined using western blot analysis. N = 6, *p < 0.05; **p < 0.01; ***p < 0.001. 

ESCA: Esophageal carcinoma; ITGB6: Integrinβ6; IHC: Immunohistochemistry; JAK1: Januskinase-1; RT-qPCR: Real-time reverse transcriptase-polymerase chain reaction; STAT3: Signal transducer and activator of transcription 3; SSTR5-AS1: Somatostatin receptor 5 antisense RNA 1.

4. Discussion

Studies have shown that among patients with ESCA, Chinese populations have a poorer prognosis than do other populations worldwide [37]. In addition, ESCA is often diagnosed at the advanced stages, during which the prognosis is unsatisfactory [38]. Hence, developing a new therapeutic strategy for the treatment of ESCA is necessary. The current study found that the level of SSTR5-AS1 was upregulated in ESCA and that silencing of SSTR5-AS1 attenuated the malignant behaviors of ESCA cells by recruiting EIF4A3 to regulate ITGB6/JAK1/STAT3 signaling. Given that the abnormal regulation of mRNAs could serve as a theoretical basis for the early diagnosis of ESCA, SSTR-AS1 can be regarded as marker for the early diagnosis of ESCA. More importantly, this paper has been the first to suggest the role of SSTR5-AS1 as an oncogene in ESCA. In addition, SSTR-AS1 could be considered one of detection markers for the early diagnosis of ESCA.

LncRNAs were closely associated with the progression of ESCA. For example, Yu et al. found that lncRNA CTC-490G23.2 was able to control the metastasis of ESCA tumors by binding PTBP1 to enhance alternative splicing of CD44 [39]. Knockdown of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) could increase the radiosensitivity of ESCA by negatively modulating the Warburg effect [40]. SSTR5-AS1 was found to be upregulated in patients with ESCA [41]. In addition, recent studies have found that SSTR5-AS1 played an important role in cancer. Indeed, one study found that SSTR5-AS1 promoted cell proliferation and epithelial–to–mesenchymal transition in prostate cancer [42]. Another study revealed that the expression of SSTR5-AS1 was upregulated in patients with gastric cancer and that those with high expression of SSTR5-AS1 had poorer overall and disease-free survival rates [43]. Consistent with previous research, the current study suggests that the level of SSTR5-AS1 was upregulated in ESCA cells and that knockdown of SSTR5-AS1 suppressed the migration and invasion of ESCA cells and induced their apoptosis. Therefore, our study has been the first to explore the mechanism by which SSTR5-AS1 affects ESCA.

On the other hand, ITGB6 and JAK/STAT were involved in the progression of malignant tumors. For example, Zhang et al. found that ITGB6 could aggravate the malignant process of pancreatic cancer via the JAK2/STAT3 signaling pathway [44]. In addition, JAK/STAT signaling participated in tumor progression [45,46]. More importantly, ITGB6 and JAK/STAT acted as crucial modulators of ESCA progression [47,48]. However, the detailed relationship between SSTR5-AS1 and the ITGB6/JAK1/STAT3 axis in ESCA remains unexplored. Thus, the current study has been the first to suggest that SSTR5-AS1 could be involved in the progression of ESCA by regulating the ITGB6/JAK1/STAT3 axis.

Reports have found that SSTR5-AS1 could regulate the progression of tumors through multiple pathways. For example, Xue et al. found that SSTR5-AS1 could facilitate gemcitabine resistance by stabilizing NONO in gallbladder carcinoma [49]. The same study also revealed that SSTR5-AS1 could function as a ceRNA to regulate carbonic anhydrase 2 (CA2) by sponging miR-15b-5p for the development and prognosis of hepatitis B virus-related hepatocellular carcinoma [50]. Another study by Yuan et al. suggested that SSTR5-AS1 could act as a prognostic marker to promote the proliferation and epithelial–to–mesenchymal transition of prostate cancer cells [51]. RBPs can play important roles across various types of cancers, and lncRNAs are able to interact with RBPs. Yang et al. revealed that HOXC cluster antisense RNA 1 (HOXC-AS1) showed oncogenic effects in ESCA by binding with IGF2BP2 to enhance the mRNA stability of SIRT1 [24]. Chen et al. also showed that lncRNA LINC00200 obviously promoted the malignant progression of MYCN-amplified neuroblastoma by binding with IGF2BP3 to increase the mRNA stability of Zic family member 2 (ZIC2) [52]. In the current study, we found that SSTR5-AS1 could interact with EIF4A3. One previous study found that ITGB6 could serve as an oncogene that was able to activate the JAK1/STAT3 pathway [31]. ITGB6 promoted the malignant behavior of ovarian cancer through the activation of transforming growth factor-beta1 [28]. Transforming growth factor-beta1 has been considered as a vital factor in fibrosis, which promotes the invasive ability of cancer cells [53,54]. Similarly, JAK1/STAT3 signaling has been proven to promote tumor progression [55]. Moreover, our findings also suggest that ITGB6 might be the promoter in JAK1/STAT3 signaling. Furthermore, our results indicated that the upregulation of SSTR5-AS1 decreased the effects of sh-ITGB6 on the cellular function of ESCA, which suggests that SSTR5-AS1 could activate ITGB6/JAK1/STAT3 signaling by interacting with EIF4A3.

Our findings revealed that SSTR5-AS1 could aggravate the tumorigenesis of ESCA by interacting with EIF4A3 to regulate ITGB6/JAK1/STAT3 signaling, potentially shedding new light on therapeutic strategies for the treatment of ESCA. Nonetheless, the current study has some shortcomings worth noting. First, several mechanisms by which SSTR5-AS1 regulates the progression of ESCA remain unexplored. Second, the mechanism underlying the function of ITGB6 in JAK1/STAT3 signaling needs to be further investigated. As such, more investigations are needed in the coming future.

5. Conclusion

Our findings revealed the level of SSTR5-AS1 was upregulated in ESCA and that silencing of SSTR5-AS1 significantly inhibited the malignant behavior of ESCA cells. In addition, our results showed that SSTR5-AS1 could bind with EIF4A3 to enhance the mRNA stability of ITGB6. Furthermore, we found that SSTR5-AS1 promoted the tumorigenesis of ESCA through ITGB6-mediated JAK1/STAT3 signaling. Overall, we believe that the findings provided herein could serve as a theoretical basis for exploring new targets against ESCA.

Funding Statement

This work was supported by Scientific research project of Hunan Provincial Health and Family Planning Commission (No. 202202085648).

Author contributions

Z Tang, Y Jiang, Y Zong designed the study; S Ding, C Wu, Z Tang supervised the study; L Liao, S Jiang performed the experiments. R Tang, F Li, P Luo edited the manuscript. All authors agreed with the submission.

Financial disclosure

This work was supported by Scientific research project of Hunan Provincial Health and Family Planning Commission (No. 202202085648). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

Medical writing and editorial support were provided by Enago, the editing brand of Crimson Interactive Consulting Co., Ltd. The service is funded by the research grant.

Ethical conduct of research

All in vivo experiments were performed in accordance with National Institutes of Health guide for the care and use of laboratory animals, following a protocol approved by the Ethics Committees of the Central Hospital of Yongzhou (No. 2023020301).

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