{'fraction_non_alphanumeric': 0.03973008331611926, 'fraction_numerical': 0.060662397576258346, 'mean_word_length': 5.089727463312369, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Knockdown of hsa_circ_0043691 restrains the progression of gastric cancer by decoying miR‐1294 to target pre‐leukemia transcription factor 3

Abstract Background Circular RNAs (circRNAs) have been found to render pivotal effects in gastric cancer (GC). However, the effect of hsa_circ_0043691 in GC still needs to be further unveiled. Methods The contents of hsa_circ_0043691, miR‐1294, and pre‐leukemia transcription factor 3 (PBX3) were examined using qRT‐PCR or Western blot assay. In vitro colony formation, transwell, wound healing, flow cytometry, tube formation assays, glutaminolysis corresponding kit and in vivo Xenograft mice model were utilized to evaluate cell functions. The relationship between miR‐1294 and hsa_circ_0043691 or PBX3 was further verified. Results The levels of hsa_circ_0043691 and PBX3 were upregulated, whereas miR‐1294 expression was diminished in GC tissues and cells. Hsa_circ_0043691 deficiency significantly inhibited GC cell progression and glutaminolysis metabolism. Mechanistically, hsa_circ_0043691 was directly bound to miR‐1294 to modulate PBX3 expression. Besides, silencing of hsa_circ_0043691 impeded tumor growth in vivo. Conclusion Hsa_circ_0043691 knockdown repressed GC malignant phenotypes by miR‐1294/PBX3 axis, which exhibited a novel therapeutic target for GC treatment.

For instance, the high expression of circGSK3B in hepatocellular carcinoma (HCC) was verified and upregulated circGSK3B enhanced HCC cell malignant phenotype. 10 The high expression of circ_0006220 in non-small-cell lung carcinoma (NSCLC) was closely involved in promoting cell growth, migration, and invasion. 11 In addition, vast data have shown that circRNA regulates the function of drug resistance in human tumors. For example, circ_0014235 could mediate biological progress and gefitinibresistance in NSCLC. 12 Bai et al. 13 disclosed that hsa_circ_0004674 reinforced the doxorubicin (DXR) resistance in osteosarcoma (OS) via Wnt/β-catenin signal pathway. Hsa_circ_0043691 is originated from 5′-nucleotidase, cytosolic IIIB (NT5C3L), located in chr17: 39983677-39983878 and contains 201 nucleotides. Hsa_ circ_0043691 was observably overexpressed in GC, 14 indicating that hsa_circ_0043691 may have a vital function in GC progression. Therefore, it was essential to further exploit the possible molecular mechanisms of hsa_circ_0043691 in GC.
Accordingly, the potential role and functional mechanism of hsa_circ_0043691 in GC progression were explored, which may help to reveal a novel rationale and therapeutic candidate for GC.

| Clinical human specimens
Thirty-nine pairs of fresh GC samples and paired non-cancerous tissue samples were removed from GC patients at Fuyong People's Hospital. No donors in this paper had received preoperative treatment and signed written informed consent prior to surgery.
All acquired tissue samples were quickly transferred to a liquid nitrogen container. Our work was supported by the Research Ethics Committee of Fuyong People's Hospital.

| Cell transfections
Several GC cell lines, including N87, HGC27, and AGS, were bought from ATCC. Human normal gastric cell (GES-1) and GC cell line (MKN-45) were commercially gained from Chinese Academy of Sciences (Shanghai, China). The RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) was used to culture cells under a moist environment with 5% CO 2 at 37°C.

| qRT-PCR
Total RNA was isolated through trizol reagents (TaKaRa) in line with the guidebook. The cDNA was obtained by using the PrimeScriptVR RT kit (Promega). SYBR Green PCR Master Mix (BioTNT Biotechnologies) was employed to measure the expression levels of mRNAs and genes, and all primer sequences were presented in Table 1. The relative expression level of hsa_circ_0043691, miR-1294 and PBX3 was determined by the 2 −ΔΔC t quantification method and normalized to β-actin and U6.

| CircRNA stability analysis and subcellular localization analysis
To analyze hsa_circ_0043691 stability, total RNA from GC cells were

| Tube formation assay
Human umbilical vein endothelial cells (HUVECs) were employed to assess the angiogenetic ability. The 96-well plates were smeared with 80 μl/well Matrigel (BD Bioscience). Then, HUVEC cells and GC cells of AGS and MKN-45 were co-cultured for 24 h.
The capillary-like branches were observed, imaged, and counted using a microscope.

| Glutamine, glutamate, and α-KG level assay
The cultured GC cells were suspended in assay Buffer for 24 h, and then the glutamine, glutamate determination kit or α-KG colorimetric assay kit (BioVision) were used to detect the concentrations of glutamine, glutamate or α-KG base on the manufacturer's project.

| Dual-luciferase reporter assay
The online websites of circinteractome and targetscan were applied to measure the relationship between miR-1294 and hsa_circ_0043691 or PBX3. The wild-type (WT) and mutant (MUT) binding sequences of miR-1294 on hsa_circ_0043691 or PBX3 were designed, amplified and assembled into the pmirGLO vectors (Promega) to establish luciferase reporter vectors (hsa_circ_0043691 WT, PBX3 3′UTR WT, hsa_circ_0043691 MUT, or PBX3 3′UTR MUT). Subsequently, the above vectors were co-transfected with miRNA mimics or miR-1294 mimic into AGS and MKN-45 cells. Finally, luciferase activity was investigated using the dual-luciferase reporter assay kit (Promega).

| RIP and pull-down assays
RIP assay was implemented using the Magna RIP kit (Millipore).
In short, the experimental cells were lysed using RIP lysis buffer (Millipore) for 48 h, followed by cultivating with AGO2 or IgG antibody overnight at 4°C to prepare co-precipitated RNAs. Ultimately, the enrichment of miR-1294 and hsa_circ_0043691 was assayed by qRT-PCR.

| Animal experiments
All animal tests were authorized by the Animal Ethics Committee of Fuyong People's Hospital. Twelve 6-week-old BALB/c mice from Vital River (Beijing, China) were randomly divided into two groups (six mice/group). 6 × 10 6 MKN-45 cells transfected with sh-hsa_ circ_0043691 or its control (sh-NC) were hypodermically implanted into the nude mice. The xenograft volume was monitored every seven days. The tumors were harvested after 28 days, then weighed, and further used for additional assays.

| Statistical analysis
With three independent experiments, the data in this work were presented as the mean ± standard deviation (SD). Student's t test, or ANOVA, was employed to assess the difference between two groups or among multiple groups. The survival outcomes of GC patients were analyzed by Kaplan-Meier method, and the relationships among hsa_circ_0043691, miR-1294 and PBX3 in GC tissues were measured using the Pearson correlation coefficient. p < 0.05 was deemed as statistically different.

| Hsa_circ_0043691 expression was heightened in GC
The qRT-PCR assay exhibited that hsa_circ_0043691 expression was prominently higher in GC tissues (tumor) compared to matched non-tumor tissues (normal) ( Figure 1A). The expression of hsa_circ_0043691 was higher in stage III (n = 20) than that in stage I + II (n = 19) ( Figure 1B) and higher in the lymph node metastasis (n = 18) than that in non-metastasis tissues (n = 21) ( Figure 1C).
Meanwhile, hsa_circ_0043691 high expression was closely linked to larger tumor size (>3 cm) (n = 17) in comparison to smaller tumor size (≤3 cm) (n = 22) ( Figure 1D). Furthermore, GC patients with high hsa_circ_0043691 expression level showed shorter survival rates ( Figure 1E). The expression level of hsa_circ_0043691 was observably higher in four GC cell lines (N87, HGC27, AGS, and MKN-45) than that in normal GES-1 cells ( Figure 1F). Hsa_circ_0043691 was looped and comprised exons 8 of its parental gene NT5C3L ( Figure S1A). Then the stability of hsa_circ_0043691 was con- that hsa_circ_0043691 could be detected only in cDNA, as no products were detected in the extracted gDNA ( Figure S1B). In addition, hsa_circ_0043691 was located in GC cells' cytoplasm by qRT-PCR assay ( Figure 1K,L). Therefore, these findings indicated that cicrN-T5C3L, with a stable structure in cytoplasmic, might play an oncogenic role in GC patients.

| Hsa_circ_0043691 knockdown curbed GC cell growth, metastasis and angiogenesis but promoted apoptosis
To research the effect of hsa_circ_0043691 on GC cells, the lossof-function analysis was implemented. In relation to the control  Figure 2I).

| Knockdown of hsa_circ_0043691 decreased glutaminolysis in GC cells
The effect of hsa_circ_0043691 on energy metabolism was analyzed in this work. Down-regulation of hsa_circ_0043691 markedly inhibited glutaminolysis metabolites, including glutamine, glutamate, and α-KG levels ( Figure 3A-C). Meanwhile, glutaminolysis-related proteins (ASCT2 and GLS1) were quantified by Western blot assay.
The outcomes disclosed that hsa_circ_0043691 absence predominantly suppressed the protein levels of ASCT2 and GLS1 in AGS and MKN-45 cells ( Figure 3D,E). The results disclosed that knockdown of hsa_circ_0043691 regulated glutaminolysis in GC.

| Hsa_circ_0043691 targeted miR-1294 in GC cells
To evaluate the potential mechanism of hsa_circ_0043691, the downstream miRNAs of hsa_circ_0043691 were analyzed. As shown in Figure 4A, hsa_circ_0043691 and miR-1294 had complementary binding sites. MiR-1294 abundance was increased by miR-1294 mimic and reduced by miR-1294 inhibitor transfection in AGS and MKN-45 ( Figure 4B). Enrichment of miR-1294 restricted the luciferase reporter activity of hsa_circ_0043691 WT group, but not the hsa_circ_0043691 MUT group (Figure 4C,D). Additionally, in RIP assay, miR-1294 and hsa_circ_0043691 had a higher enrichment in Anti-Ago2 group compared with Anti-IgG group ( Figure 4E,F).
Simultaneously, high abundance of hsa_circ_0043691 was enriched by Bio-miR-1294 WT, but not Bio-miR-1294 MUT or Bio-NC ( Figure 4G). The abundance of miR-1294 was particularly reduced in GC tissue samples ( Figure 4H), and miR-1294 expression was inversely associated with hsa_circ_0043691 level in GC tissues ( Figure 4I). Similarly, a declined miR-1294 level was observed in GC cells ( Figure 4J). Furthermore, deletion of hsa_circ_0043691 remarkably increased miR-1294 expression, while this effect was partly overturned by co-transfection of miR-1294 inhibitor in GC cells ( Figure 4K).

| MiR-1294 inhibitor abrogated the effects of hsa_circ_0043691 silencing in GC cells
To additionally realize the regulatory mechanism of hsa_circ_0043691 and miR-1294, rescue assays were conducted in GC cells with the transfections of si-NC, si-cicrNT5C3L, si-cicrNT5C3L + in-miR-NC and si-cicrNT5C3L + in-miR-1294. The results exposed that miR-1294 partially overturned the effects of hsa_circ_0043691 deficiency on cell proliferation ( Figure 5A), migration and invasion ( Figure 5B-D). The changes in cell apoptosis and apoptosis-related protein levels (Bcl-2 and Bax) that were induced by si-hsa_circ_0043691 were largely weakened by miR-1294 inhibitor ( Figure 5E-G). The reduction in angiogenesis ( Figure 5H) and glutaminolysis metabolites including glutamine ( Figure 5I), glutamate ( Figure 5J), α-KG level ( Figure 5K), as well as the decline in expressions of ASCT2 and GLS1 ( Figure 5L,M) in GC cells with hsa_circ_0043691 deletion were rescued by miR-1296 inhibitor.

| PBX3 was targeted by miR-1294 in GC cells
Next, the target genes of miR-1294 were predicted. The results presented that miR-1294 and PBX3 3′UTR had complementary binding sites ( Figure 6A). The results disclosed that miR-1294 overexpression  Figure 6B,C). Moreover, PBX mRNA and protein levels were overtly upregulated in GC tissues ( Figure 6D,E). PBX3 was inversely related to miR-1294 expression ( Figure 6F) and positively linked to hsa_ circ_0043691 expression in GC tissues ( Figure 6G). Also, an elevation of PBX3 protein level was showed in GC cells ( Figure 6H). In addition, PBX3 protein level was reduced by miR-1294 mimic, while this suppressive effect was abrogated by PBX3 overexpression ( Figure 6I).
si-cicrNT5C3L transfection abated the level of PBX3, while the effect was counteracted by co-transfection of miR-1294 inhibitor ( Figure 6J).

| MiR-1294 hampered GC cell malignant phenotypes by targeting PBX3
The rescue experiments were conducted in GC cells to further clarify the interplay between miR-1294 and PBX3 in GC cells.

| DISCUSS ION
As one of the digestive system malignancies, GC is a serious threat to human health because of its difficult diagnosis and poor prognosis. 15,16 Currently, the treatment of GC in the clinic mainly includes radiotherapy and chemotherapy. 17 well as PI3K-Akt pathway to promote glutaminolysis. 28 Therefore, we speculated that hsa_circ_0043691 may also regulate glutaminolysis via these pathways, which needs to be further investigated in future.
In this work, hsa_circ_0043691 and miR-1294 were predicted to have binding sites by using circinteractome bioinformatics software, and this relationship was testified by multiple assays. Previous studies have found that miR-1294 plays an antitumor effect in the malignant phenotype of many of cancer, including esophageal cancer, 29 osteosarcoma, 30 and laryngeal squamous cell carcinoma cell (LSCC). 31 In GC tissues, downregulated miR-1294 was uncovered in GC tissue samples and cells. [32][33][34] In our paper, miR-1294 expression was also decreased in GC tissue sample, and overexpression of miR-1294 significantly hindered GC cell progression. Furthermore, hsa_circ_0043691 mediated the suppressive role in GC cells by sponging miR-1294.
PBX3 and miR-1294 3′UTR had complementary sites. Recent evidence has shown that PBX acted as an oncogene and was involved in regulating human malignant tumors, such as nasopharyngeal carcinoma (NPC), 35 ovarian cancer (OC) 36 and GC. 37 PBX3 content was prominently upregulated in GC tissues and cells, which was consistent with previous research. 38 Upregulation of PBX3 largely eliminated the antitumor action of miR-1294 on cell progression.
In conclusion, our study indicated that hsa_circ_0043691 was markedly upregulated in GC, and downregulation of hsa_circ_0043691 repressed GC cell malignant phenotype and glutaminolysis by regulating PBX3 via targeting miR-1294. Hence, hsa_circ_0043691 may serve as a potential target for GC management.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.