Glutamine transporter SLC1A5 inhibits autophagy-mediated CD276 degradation to promote esophageal cancer progression

The aim of this study was to explore the expression of long non-coding ribonucleic acid (lncRNA) nuclear receptor subfamily 2 group F member 1-antisense RNA 1 (NR2F1-AS1) in esophageal squamous cell carcinoma (ESCC) tissues and cells and to investigate its effects on ESCC proliferation and metastasis. The expression level of NR2F1-AS1 in 51 pairs of ESCC tissues and corresponding adjacent tissues was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Meanwhile, NR2F1-AS1 expression in ESCC cells was measured via qRT-PCR as well. Subsequently, specific interference sequences of NR2F1-AS1 were designed, synthesized, and transiently transfected into ESCC cells. 48 h later, qRT-PCR assay was performed to detect the interference efficiency. The effects of small interfering (si)-NR2F1-AS1 on the proliferation of ESCC cells were determined through cell counting kit-8 (CCK-8) and colony formation assay. Wound healing and transwell assays were conducted to investigate the influences of si-NR2F1-AS1 on the migration and invasion of ESCC cells. Additionally, the changes in the expressions of epithelial-mesenchymal transition (EMT) molecular markers were detected by Western blotting. QRT-PCR assay revealed that the expression level of NR2F1-AS1 was significantly up-regulated in 42 of 51 cases of ESCC tissues (42/51, 82.4%). Compared with esophageal mucosal epithelial HET-1A cells, NR2FA-AS1 was highly expressed in ESCC cells. CCK-8 and colony formation assay indicated that the proliferation of ESCC cells decreased remarkably after interference in NR2F1-AS1 expression. The results of wound healing and transwell assays showed that the migration and metastasis of cells were significantly lower in si-NR2F1-AS1 group than those in si-NC group. Western blotting demonstrated that the expressions of EMT molecular markers were changed after interfering with NR2F1-AS1 expression. NR2F1-AS1 was highly expressed in ESCC tissues and cells. Furthermore, high expression of NR2F1-AS1 promoted the proliferation and metastasis of ESCC cells by modulating EMT.

E2F1-activated NRSN2 promotes esophageal squamous cell carcinoma progression through AKT/mTOR pathway

Objective: To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. Methods: RAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells. Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. Results: The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, P=0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, P=0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues (P<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes (P=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes (n=10) exhibited high expression of RAB23 (P<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells in vitro and in vivo, but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, P<0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, P<0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Conclusions: Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells.

Increasing studies indicate that circular RNAs (circRNAs) play critical roles in tumor metabolism of multiple cancers. However, the contribution of circRNAs in glutamine metabolism of esophageal squamous cell carcinoma (ESCC) remains elusive. The objective of this research was to investigate the role and mechanism of circRNA hsa_circ_0001093 (circ_0001093) in the glutamine metabolism and tumorigenesis of ESCC. Circ_0001093, microRNA-579-3p (miR-579-3p) and glutaminase (GLS) expressions in ESCC tissues and cell lines were measured by qRT-PCR, tissue array or Western blot. Cell proliferation, invasion and migration were assessed by CCK-8 or transwell assays. Glutamine consumption, glutamate and ATP production were detected by indicated assay kits. The relationships between circ_0001093 and miR-579-3p or GLS mRNA were investigated by bioinformatics analysis, RNA pull-down, luciferase reporter and RNA immunoprecipitation (RIP) assays. Here, we found that circ_0001093 expression was up-regulated in ESCC tissues and cell lines. Increased circ_0001093 expression predicted an unfavourable prognosis, and was associated with the lymph node metastasis, TNM staging and tumor size in ESCC tissues. Circ_0001093 knockdown suppressed cell proliferation, invasion, migration and glutamine metabolism of ESCC cells, while circ_0001093 over-expression showed the opposite effects. Mechanistically, circ_0001093 acted as a competing endogenous RNA (ceRNA) by sponging miR-579-3p, thereby increasing GLS expression. Furthermore, the inhibitory effects of circ_0001093 knockdown on the invasion, migration and glutamine metabolism were partly rescued by miR-579-3p inhibition or GLS over-expression in ESCC cells. Additionally, miR-579-3p expression was down-regulated in ESCC tissues, while GLS expression was up-regulated. In conclusion, this study first provides evidence that the circ_0001093/miR-579-3p/GLS regulatory network can affect glutamine metabolism and malignant phenotype of ESCC, which can further impact ESCC progression.

BackgroundFibulin‐4, namely, EFEMP2, is an essential matricellular protein associated with a variety of malignancies. The aim of this study was to explore the role of fibulin‐4 in the progression of esophageal squamous cell carcinoma (ESCC), as well as its effect on ESCC sensitivity to apatinib treatment.MethodsThe expression of fibulin‐4 in ESCC tissues and cell lines was detected. Stably transfected ESCC cells were established by transducing lentiviral vectors for silencing or overexpressing the fibulin‐4 gene into ESCC cells, and a subcutaneous xenograft tumor model of ESCC in mice was successfully established. IHC, RT–qPCR and western blotting were used to detect the expression of related genes and proteins. The CCK8 assay, EdU cell proliferation assay, wound healing assay, transwell assay and flow cytometry were used to evaluate the proliferation, invasion, migration and apoptosis of ESCC cells. After mice were sacrificed, the transplanted tumors were resected, and their volumes were measured.ResultsThe expression of fibulin‐4 was significantly increased in both ESCC tissues and cell lines, and the high expression was closely related to the poor clinicopathological features. Downregulation of fibulin‐4 inhibited the proliferation, invasion and migration of ESCC cells in vitro and in vivo. Meanwhile, fibulin‐4 knockdown inhibited autophagy of tumor cells by activating the Akt–mTOR signaling pathway and significantly promoted apatinib‐induced apoptosis of ESCC cells.ConclusionOur study showed that fibulin‐4 is an oncogene that can promote ESCC progression and inhibit apoptosis. Downregulation of fibulin‐4 enhances the sensitivity of ESCC cells to apatinib by inhibiting cellular protective autophagy through activating the Akt–mTOR signaling pathway.

Esophageal squamous cell carcinoma (ESCC) ranks as the fourth leading cause of tumor-related deaths in China. Circ_0050444 has been revealed to be downregulated in ESCC tissues, however, its function and molecular mechanism underlying ESCC progression is unknown. Therefore, we attempted to clarify the functional role and molecular mechanism of circ_0050444 underlying ESCC progression. RT-qPCR and RNase R digestion assays were used to evaluate circ_0050444 expression and stability characteristics in ESCC cells. Gain-of-function assays were conducted to clarify circ_0050444 role in ESCC cell malignant behaviors. Bioinformatics and mechanism experiments were performed to assess the relationship between circ_0050444 or C10orf91 and miR-486-3p in ESCC cells. Rescue assays were conducted to evaluate the regulatory function of the circ_0050444-miR-486-3p-C10orf91 axis in ESCC cellular processes. Circ_0050444 expression was found to be downregulated both in ESCC patient tissues and cell lines. Functionally, circ_0050444 overexpression repressed ESCC cell proliferative, migratory, and invasive capabilities in cultured cells. Mechanistically, circ_0050444 was found to be competitively bound with miR-486-3p to upregulate C10orf91 in ESCC cells. Moreover, the impact of circ_0050444 elevation on ESCC cell proliferation, migration, and invasion was countervailed by C10orf91 silencing. Circ_0050444 presents downregulation and functions as a tumor suppressor in ESCC progression. Circ_0050444 suppresses ESCC proliferation, migration, and invasion through sponging miR-486-3p to upregulate C10orf91, providing a potential new direction for seeking therapeutic plans for ESCC.

Background: Long noncoding RNAs (lncRNAs), a class of noncoding RNA nucleotides >200 bp, has been demonstrated to play vital role in the development of cancer. FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) has been reported as an lncRNA which acts as a tumor-promoting effect in some cancers. However, the role of it in esophageal squamous cell carcinoma (ESCC) and its potential regulatory mechanism was unclear now.Methods: qRT-PCR was used to detect the levels of FEZF1-AS1 and mRNA CTNNB1 (β-catenin) in ESCC tissues and cells. Cell transfection experiments were used to knock down or overexpress the level of FEZF1-AS1 in EC1 and EC9706 cell lines. WST-1 assays, cell cycle assays, scratch wound assays, migration, and invasion assays were used to evaluate the function of FEZF1-AS1 in ESCC progression.Results: FEZF1-AS1 was remarkably upregulated in ESCC tissues and cell lines. Silencing of FEZF1-AS1 significantly inhibited the migration and invasion of ESCC cells, while overexpression of FEZF1-AS1 notably accelerated ESCC migration and invasion. Meanwhile, the levels of FEZF1-AS1 had no effect on ESCC cell proliferation and cell cycle. We also found that β-catenin was upregulated in ESCC tissues, and the level of it was positively correlated with the expression of FEZF1-AS1. Silencing of FEZF1-AS1 could decrease the mRNA and protein level of β-catenin, while overexpression FEZF1-AS1 could lead to the contrary.Conclusion: Our results suggested that the expression of lncRNA FEZF1-AS1 played an important role in ESCC progression, especially the motility of the tumor. FEZF1-AS1 may provide us with a new sight for ESCC treatment.

Long intergenic non-coding RNA 01232 (LINC01232) was identified as a critical regulator of the development of pancreatic adenocarcinoma. The present study investigated the expression and regulatory roles of LINC01232 in esophageal squamous cell carcinoma (ESCC). The main aim of the present study was to elucidate the underlying mechanisms through which LINC01232 affects the malignancy of ESCC. Initially, LINC01232 expression in ESCC was analyzed using the TCGA and GTEx databases and was confirmed using reverse transcription-quantitative polymerase chain reaction. ESCC cell proliferation, apoptosis and migration and invasion were assessed using the Cell Counting kit-8 assay, flow cytometric analysis, and migration and invasion assays, respectively. ESCC tumor growth in vivo was examined using a xenograft mouse model. As shown by the results, a high LINC01232 expression was detected in ESCC tissues and cell lines. LINC01232 downregulation suppressed the proliferation, migration and invasion of ESCC cells, and promoted cell apoptosis in vitro. In addition, LINC01232 depletion restricted tumor growth in vivo. Mechanistically, LINC01232 was shown to function as an microRNA-654-3p (miR-654-3p) sponge in ESCC cells, and hepatoma-derived growth factor (HDGF) was identified as a direct target of miR-654-3p. LINC01232 could bind competitively to miR-654-3p and decrease its expression in ESCC cells, thereby promoting HDGF expression. Rescue experiments reconfirmed that the effects of LINC01232 deficiency in ESCC cells were restored by increasing the output of the miR-654-3p/HDGF axis. On the whole, the present study demonstrates that LINC01232 plays a tumor-promoting role during the progression of ESCC by regulating the miR-654-3p/HDGF axis. The LINC01232/miR-654-3p/HDGF pathway may thus provide a novel theoretical basis for the management of ESCC.

Objective To investigate the expression of ATPase family AAA domain-containing protein 2 (ATAD2) and kinesin family member 4A (KIF4A) in esophageal squamous cell carcinoma (ESCC) tissues and their association with clinicopathological features and to explore the role of ATAD2 in regulating KIF4A expression and biological functions in ESCC cells and the effect of aspirin on their expression. Methods The mRNA and protein expression of ATAD2 and KIF4A in the tissues of patients with ESCC were measured by RT-qPCR and immunohistochemistry, and the correlation between the expression of mRNA and clinicopathological characteristics was analyzed. Western blot and RT-qPCR were used to detect the interference efficiency and KIF4A expression after si-ATAD2 transfection in EC109 and KYSE30 cells. CCK-8 and Transwell assay were performed to investigate the effects of ATAD2 and aspirin on proliferation, migration, and invasion of ESCC cells. The effect of aspirin on the expression of ATAD2 and KIF4A in ESCC cells was measured by RT-qPCR and Western blot. Results The expression of ATAD2 and KIF4A was upregulated in ESCC tissues, and both were correlated with the differentiation grades and lymph node metastasis. Knockdown of ATAD2 in ESCC cells significantly inhibited cell proliferation, migration, and invasion. Compared to the negative control group, the proliferation, migration, and invasion ability of ESCC cells in the aspirin-treated groups were decreased, and the expression of ATAD2 and KIF4A in ESCC cells was decreased after treating with aspirin for 48 h. Conclusion The expression levels of ATAD2 and KIF4A are elevated in ESCC. ATAD2 promotes proliferation, migration, and invasion of ESCC cells by regulating KIF4A. Aspirin can inhibit the malignant behavior of ESCC cells by downregulating ATAD2 and KIF4A.

Background: Exosome plays an important role in cell communication and cell environment. But the mechanism of how exosome derived from cancer-associated fibroblast (CAF) influent esophageal squamous cell carcinoma (ESCC) progression is not clear. In this work, compared with normal fibroblast-derived exosomes (NDEs), we found that CAF derived exosomes (CDEs) can enhance the growth, migration, and invasion of ESCC cells obviously. Therefore, we would like to clarify the role of CDEs in ESCC progression. Methods and Results: Using microRNA array to analyze the different miRNAs in the CDEs and NDEs, we got a series of up-regulation miRNAs and two down-regulation miRNAs in CDEs. Among them, miR-3656 shows a significantly highest level. Then, we transfected miR-3656 mimics to the ESCC cells and found the mimics increased the growth, migration, and invasion of ESCC cells in cell proliferation assay. cell scratch tests and chamber invasion experiments. We established recombinant ESCC cells with miR-3656 high expression by the lentiviral system. The cells were injected into mice to form the xenograft tumor. The results showed the miR-3656 can enhance the tumor formation. After that, aided by TargetScan software, we found ACAP2, one of miR3656 targets, was significantly down-regulating in ESCC cells at the high level of miR-3656. When ACAP2 expression was knocked-down by siRNA, the proliferation and migration of ESCC cells was increased, which indicated ACAP2 a potential anti-tumor factor. Furthermore, we detected the different proteins between ESCC cells with high expression miR-3656 and normal ESCC cells through LC_MS; the results were analyzed by Integrate Pathway Analysis (IPA) system and many differential proteins were found to be involved of PI3K/AKT and Wnt pathways. Conclusions: In this study, we found the CDEs promote the progression of their sounding ESCC cells in vitro and in vivo. The main reason is that CDEs carry some tumor-promoting miRNAs that influent the target ESCC cells. Especially, miR-3656 expresses the highest level of miRNAs in CDEs. It not only decreases the expression of ACAP2 but also impacts the expression of proteins involved PI3K/AKT and Wnt pathways in ESCC cells. The results indicate the CDEs playing a very important role in ESCC progression. Citation Format: Xiaojia Chen, Yuan Jin, Bihui Zhang, Xiaocen LI, Baoqing Tian, Qiang Wang, An Hong. Exosome from CAF-ESCC aggravates tumor progression by its containing molecular miR-3656 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1987.

To investigate the expression, role and the underlying mechanism of miR-124 in esophageal squamous cell carcinoma (ESCC). A total of 25 pairs of ESCC and adjacent tissues were collected for analysis. The human normal esophageal epithelial cell line (Het-1A) and human esophageal cancer cell lines (EC-1) was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were transfected with miR-124 mimics and inhibitors. A series of assays, including Cell-Counting-Kit-8 (CCK-8), Transwell migration and invasion assays, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and luciferase reporter gene assay, were performed to detect the effects of miR-124 on ESCC cells. Spearman's correlation analysis was used to evaluate the relationship of miR-124 expression and signal transducer and activator of transcription-3 (STAT3) expression. miR-124 was significantly downregulated in ESCC tissues and cells. Overexpression of miR-124 inhibited the proliferation, migration and invasion of ESCC cells in vitro, while inhibition of miR-124 promoted these processes (all P<0.05). miR-124 was found to specifically bind to the 3'-untranslated region (3'UTR) of STAT3. The expression level of STAT3 was obviously higher in tumor tissues and cells compared to normal adjacent tissues and cells (all P<0.05). Moreover, miR-124 expression was negatively associated with STAT3 levels. Restoring STAT3 expression in ESCC cells transfected with miR-124 mimics partially reversed the inhibitory effects of miR-124 mimics on cell proliferation, migration, and invasion. Conversely, inhibiting STAT3 expression in ESCC cells transfected with miR-124 inhibitors partially abolished the promoting effects of miR-124 inhibitors on the proliferation, migration and invasion of ESCC cells. miR-124 inhibits the proliferation, migration and invasion of ESCC cells by downregulating STATS, indicating that miR-124 may serve as a molecular candidate for treating ESCC.

Esophageal squamous cell carcinoma (ESCC) is a type of digestive tract malignant tumor that severely threatens human health. The long non-coding RNA BRAF activated non-coding RNA (BANCR) and insulin-like growth factor 1 receptor (IGF1R) are associated with various types of cancer; however, it remains unclear whether BANCR can regulate IGF1R expression in ESCC. In the present study, the expression levels of BANCR, IGF1R mRNA and microRNA-338-3p (miRNA/miR-338-3p) in ESCC tissues or cells were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The levels of IGF1R, E-cadherin, N-cadherin, Vimentin, p-Raf-1, p-MEK1/2 and p-ERK1/2 were measured by western blot analysis. The proliferation, migration and invasion of ESCC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) or Transwell assays. The relationship between miR-338-3p and BANCR or IGF1R was predicted using starBase2.0 and confirmed by dual-luciferase reporter assay. The role of BANCR in ESCC in vivo was confirmed through a tumor xenograft assay. It was found that BANCR and IGF1R were upregulated, while miR-338-3p was down-regulated in ESCC tissues and cells. Both BANCR and IGF1R knockdown suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells. IGF1R enhancement reversed BANCR knockdown-mediated effects on the proliferation, migration, invasion and EMT of ESCC cells. BANCR regulated the Raf/MEK/ERK pathway by regulating IGF1R expression. Notably, BANCR regulated IGF1R expression by sponging miR-338-3p. Moreover, BANCR silencing inhibited tumor growth in vivo. On the whole, the findings of the present study demonstrate that BANCR inhibition blocks ESCC progression by inactivating the IGF1R/Raf/MEK/ERK pathway by sponging miR-338-3p.

Objective: To investigate the role of microRNA-139-5p (miR-139-5p) in the occurrence and development of esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion of ESCC cells and its molecular mechanisms. Methods: Seventy-five cases of ESCC tissues and paired normal tissues were obtained from thoracic surgery of the First Affiliated Hospital of Zhengzhou University from February 2017 to March 2018. Experiment was divided into two group: ESCC (n=75) and normal esophageal tissues (n=75).GEO datasets and real-time quantitative PCR (qRT-PCR) were used to detect the expression of miR-139-5p in ESCC tissues and cells. miR-139-5p inhibitor, miR-139-5p mimic, negative control, control siRNA, T-box transcliption factor 1(TBX1) siRNA, pcDNA3.1 and pcDNA3.1-TBX1 were transfected into ESCC Eca109 and TE1 cells. qRT-PCR was used to detect the expressions of miR-139-5p and TBX1 in transfected ESCC cells. Cell counting kit 8 (CCK-8) and Transwell chamber were employed to detect cell proliferation and invasion of ESCC cells, respectively. Dual-Luciferase Reporter assay was used to analyze the interaction between miR-139-5p with TBX1. qRT-PCR, Western blot and immunohistochemistry were utilized to detect the expression of TBX1 in ESCC tissues. Western blot was used to detect the expressions of E-cadherin, N-cadherin and Vimentin after transfection. Results: The level of miR-139-5p in ESCC tissues was significantly lower than that in normal tissues (1.17±0.43 vs 5.16±3.62,P<0.001). Log-rank test showed that the survival rate of ESCC patients with high miR-139-5p level (n = 43) was significantly higher than that with low miR-139-5p level (n=32) (67.44% vs 25.00%, P = 0.005). The expression level of miR-139-5p in ESCC cells was significantly lower than that of normal esophageal epithelial cell Het-1A (all P<0.001). The proliferation and invasion ability of ECA109 and TE1 cells with high expression of miR-139-5p were significantly lower than those transfected with negative control (NC) (all P<0.05). Dual-Luciferase Reporter assay showed that miR-139-5p could bind to the 3'-untranslated region of TBX1. miR-139-5p mimic or inhibitor suppressed or promoted the expression of TBX1 protein in Eca109 and TE1 cells, respectively (all P<0.05). Downregulation of TBX1 significantly suppressed proliferation and invasion of ECA109 and TE1 cells, while overexpression of TBX1 significantly promoted proliferation and invasion of ECA109 and TE1 cells (all P<0.05). In addition, pcDNA3.1-TBX1 partially reversed the inhibition of miR-139-5p-mediated invasion ability (all P<0.05), while TBX1 siRNA partially reversed the enhancement of miR-139-5p inhibitor-mediated invasion ability (all P<0.05). Conclusion: miR-139-5p suppressed ESCC cell proliferation and invasion by targeting TBX1.

Long noncoding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6) has been shown to be upregulated in esophageal squamous cell carcinoma (ESCC). However, its detailed function in ESCC remains unknown. We investigated its specific roles in ESCC cell proliferation, invasion, and migration. Gene expression was evaluated by quantitative reverse transcriptase polymerase chain reaction and western blot. The subcellular localization of lncRNA SNHG6 was determined using subcellular location assay. Luciferase reporter assay and RNA pull-down assay were applied to determine the interaction between lncRNA SNHG6, miR-186-5p, and hypoxia-inducible factor 1α (HIF1α). The cell proliferation, migration, and invasion abilities were evaluated by using cell count kit-8, colony formation assay, and transwell migration and invasion assays. LncRNA SNHG6 and HIF1α were upregulated, while miR-186-5p was downregulated in ESCC tissues and cell lines. Knockdown of lncRNA SNHG6 inhibits ESCC cell proliferation, migration, and invasion. A negative correlation between lncRNA SNHG6 and miR-186-5p expression was found in ESCC tissues. Similarly, there is a positive correlation between lncRNA SNHG6 and HIF1α expression in ESCC tissues. Conversely, miR-186-5p expression was negatively correlated with HIF1α expression in ESCC tissues. Furthermore, lncRNA SNHG6 was identified as a decoy for miR-186-5p, thereby promoting the expression of miR-186-5p target HIF1α. More significantly, restoration of SNHG6 or HIF1α could reverse the inhibitory effect of miR-186-5p on ESCC cell proliferation, migration, and invasion. Downregulation of SNHG6 inhibited the proliferation, migration, and invasion of ESCC cells through regulating miR-186-5p/HIF1α axis, providing a novel therapeutic target for ESCC.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in human cancers, including esophageal squamous cell carcinoma (ESCC). In the current study, we identified CCAT2 as a relevant lncRNA and investigated its role in the progression of ESCC. RT-qPCR was adopted to detect CCAT2 expression in collected clinical samples, ESCC cell lines, and a normal cell line. We tested the correlation between CCAT2 expression and the prognosis of ESCC. RT-qPCR or immunoblotting was adopted to detect the expression of relevant factors in ESCC tissues or cells. Cell proliferation, apoptosis, migration, and invasion were examined by colony formation assay, flow cytometry, scratch assay, and Transwell assay, respectively, while subcutaneous tumorigenesis in nude mice was adopted to examine the role of CCAT2 in tumorigenesis of ESCC cells in vivo. Bioinformatics analysis, dual luciferase reporter assay, and RIP were conducted for the target relationship profiling. Me-RIP was adopted to detect m6A modification level of TK1 in ESCC tissues or cells. Upregulated CCAT2, IGF2BP2, and TK1 expression and inhibited miR-200b expression were observed in ESCC cells and tissues. CCAT2 bound to miR-200b and reduced its expression, leading to upregulated IGF2BP2 expression. IGF2BP2 improved TK1 mRNA stability to enhance its expression by recognizing its m6A modification. CCAT2 promoted the migration and invasion of ESCC cells in vitro, and tumorigenesis in vivo by upregulating TK1 expression, while overexpression of miR-200b reversed these effects of CCAT2. Overall, this study suggests that CCAT2 competitively binds to miR-200b to alleviate its inhibitory effects on IGF2BP2 expression, resulting in elevated TK1 expression, and an ensuing promotion of the development of ESCC.