CircRNA RNA hsa_circ_0008234 Promotes Colon Cancer Progression by Regulating the miR-338-3p/ETS1 Axis and PI3K/AKT/mTOR Signaling

Simple Summary Circular RNAs (circRNAs) exert an important role in cancer progression. Meanwhile, considering its widespread regulatory effect and the potential of noninvasive testing, understanding the role of circRNA in cancer is particularly meaningful. Our study identified the circRNA hsa_circ_0008234 in colon cancer based on an open-accessed circRNAs expression profile. Moreover, we found that hsa_circ_0008234 could promote proliferation, invasion, and migration abilities of colon cancers, which was partly dependent on the competitive endogenous RNA mechanism (miR-338-3p/ETS1 axis). Meanwhile, we discovered that PI3K/AKT/mTOR signaling is the downstream pathway of the has_circ_0008234/miR-338-3p/ETS1 axis, which improves the effect network of circRNAs in colon cancer. Abstract Circular RNAs (circRNAs) have been shown to play a crucial role in cancer occurrence and progression. This present work investigated the link between hsa_circ_0008234 and colon cancer. Data retrieved from GSE172229 was used to compare the circRNA profiles of colon cancer and surrounding non-tumorous tissues. The amount of RNA and protein in the molecules was determined using quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. The cell proliferation ability was assessed using CCK8, EdU, colon formation, and nude mice tumorigenesis tests. Cell invasion and migration abilities were evaluated using transwell wound healing and mice lung metastasis model. Hsa_circ_0008234 piqued our interest because bioinformatics and qRT-PCR analyses revealed that it is upregulated in colon cancer tissue. Cell phenotypic studies suggest that hsa_circ_0008234 may significantly increase colon cancer cell aggressiveness. Mice experiments revealed that inhibiting hsa_circ_0008234 significantly reduced tumor growth and metastasis. Moreover, the fluorescence in situ hybridization experiment demonstrated that hsa_circ_0008234 is primarily found in the cytoplasm, implying that it potentially functions via a competitive endogenous RNA pathway. These findings indicated that hsa_circ_0008234 may act as a “molecular sponge” for miR-338-3p, increasing the expression of miR-338-target 3p’s ETS1. In addition, the traditional oncogenic pathway PI3K/AKT/mTOR signaling was found to be the potential downstream pathway of the hsa_circ_0008234/miR-338-3p/ETS1 axis. In conclusion, hsa_circ_0008234 increases colon cancer proliferation, infiltration, and migration via the miR-338-3p/ETS1/PI3K/AKT axis; therefore, it could serve as a target and a focus for colon cancer therapy.


Introduction
Colon cancer is one of the most prevalent malignant tumors and one of the leading causes of cancer mortality deaths across the globe [1]. Although the mechanism is not fully understood, relevant evidence indicates that the incidence of colon cancer is often affected by multiple factors, such as heavy drinking, poor diet, genetic susceptibility, and so on [2]. Surgery remains the frontline therapy choice for resectable colon cancer, with a 5-year survival rate of nearly 60% in early-stage patients [3]. However, the efficacy of the treatment in advanced stages remains unsatisfactory [4]. In this view, it is critical to examine the unknown mechanisms of colon cancer to provide a theoretical basis for the underlying therapeutic target.
Circular RNA is an endogenous RNA with a special single-stranded closed structure, including exonic circRNAs, exon-intron circRNA, and circularized intron RNA [5]. Exonic circRNAs function primarily in the cytoplasm, whereas intron circRNA function primarily in the nuclei [6]. CircRNA functions in multiple ways, including competitive endogenous RNA (ceRNA), protein interaction, and encoding polypeptides [7]. The abnormal expressions of many circRNA have been observed in cancers, indicating their crucial role in tumor development and genesis [8]. CircRNAs have recently been linked to cancers [9]. Huang et al. discovered that the circRNA hsa_circ_104348 could promote the progression of liver cancer via the miR-187-3p/RTKN2 axis, which relies on the Wnt/β-catenin pathway [10]. Furthermore, Ma et al. discovered that the circRNA hsa_circ_0004872 potentially inhibits cancer progression via the miR-224/Smad4/ADAR1 successive regulatory circuit [11]. Meanwhile, Zheng et al. demonstrated that the circRNA circPPP1R12A encoded a novel protein known as circPPP1R12A-73aa, which could promote colon cancer pathogenesis and metastasis via the Hippo-YAP signaling pathway [12]. Furthermore, Li et al. discovered that circPRRC2A could promote angiogenesis and metastasis in renal cell cancer via the epithelial-mesenchymal transition and activation of TRPM3 [13]. These broad effect patterns of circRNAs make them promising as clinical targets of cancer [14]. While previous research looked into the effect of hsa_circ_0008234 on colon cancer (Table S1) [12,15], the role of the hsa_circ_0008234 function in colon cancer remains unclear.
In this present investigation, we assessed the expression sequence and biological activity of hsa_circ_0008234 in colon cancer. Hsa_circ_0008234 was first identified using the circRNA expression profile of GSE172229, revealing a high expression value in colon cancer tissue and cells. The FISH test revealed predominant levels of hsa_circ_0008234 within the cell cytoplasm, indicating its potential link to the ceRNA pathway. In vitro and in vivo studies revealed that hsa_circ_0008234 might promote the growth, infiltration, and migration of colon cancer. In addition, hsa_circ_0008234 was discovered to potentially increase ETS1 expression via miR-338-3p, which may explain its cancer-promoting activity. The PI3K/AKT/mTOR signaling pathway was revealed as the downstream pathway of the hsa_circ_0008234/miR-338-3p/ETS1 axis.

Methods and Materials
The circRNA expression profile of colon cancer and adjacent non-tumor was retrieved from the GEO database, GSE172229 (Platform: GPL28148; Agilent-084217 CapitalBio Technology Human CircRNA Array v2 (Beijing, China). Open-accessed transcriptional profiles and clinical information of colon cancer patients were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed circRNAs analysis was performed in the R program (v. 4.0.0) (limma package) with the threshold of |logFC > 1| and p value < 0.05. Heatmap was plotted using the heatmap package. Gene set enrichment analysis (GSEA) was performed to explore the biological effect based on Hallmark gene set [16].

Cell Lines Culture and Transfections
The normal colon epithelial cell NCM460 and the four colon cancer cell lines HCT116, SW480, HCT8, and DLD-1, were all laboratory stocks. Cells were cultivated in RPM1640 culture media and replaced every four days. Jima Com (Shanghai, China) provided the short hairpin RNAs (shRNAs) of hsa_circ_0008234 and the control vector, as well as the miR-338-3p, mimics, inhibitor, and ETS1 overexpressed plasmids. Lipofectamine 2000 was used to transfect cells following the standard protocol. The process includes: 1. 5 × 10 5 cells were inoculated on a 6-well plate, and 2 mL of complete medium was added. Cells converged to 70-90% before transfection; 2. Add 3 µg plasmid into 100 µL serum-free medium and shake it gently; 3. Add 4 µL Lipofectamine 2000 to 100 µL serum-free medium, mix slowly, and leave it at room temperature for 5 min; 4. Mix the diluted Lipofectamine reagent with the plasmid, shake it slowly, and place it at room temperature for 20 min to form the plasmid-Lipofectamine mixture; 5. According to the instructions, add 200 µL plasmid-Lipofectamine mixture to the cell hole containing 800 µL serum-free medium and slowly shake the cell plate; 6. Remove the old medium and add the complete medium after 6 h of culture under conventional conditions (5% CO 2 and at 37 • C).

Cell Proliferation Assay
The cell proliferation ability was assessed using CCK8, colony formation, and 5-ethyl-2 -deoxyuridine (EdU) assays. A CCK8 assay was performed using a CCK8 instrument (Dojindo, Shanghai, China). Colony formation and EdU test were performed as previously described [17]. For colony formation assay, inoculations of 500 cells per well were performed into 6-plate wells; for CCK8 assay, 2000 cells per well were inoculated into 96 well plates; for EdU assay, a total of 4 × 10 5 cells were inoculated into each 6-plate well.

Transwell Assay
Cell infiltration and migration potential were assessed using the transwell assay, as previously described [17]. A medium without serum was added to the upper chamber, while a medium with 20% serum was used for the lower chamber. A total of 4 × 10 4 cells were added into the upper compartment for 12 h. Finally, cells were fixed with 4% formaldehyde and stained with crystal violet.

Wound Healing Assay
The wound-healing assay process was performed as previously described [18]. Inoculated cells were cultured to 85-95% confluency in 6-well plates. The scratch was made using a 200 µL tip. Using a microscope camera, photos of the cell scratches were taken at 0 and 24 h.

Fluorescence In Situ Hybridization (FISH)
The FISH technique was employed to establish where circRNAs are located subcellularly. Briefly, the FISH assay was performed in accordance with the protocol of the FISH Kit (RiboBio). The cells were then repaired, made permeable, and placed in a prehybridization buffer environment for 30 min. The hsa_circ_0008234 FISH probe was incorporated into the cells overnight at room temperature, along with a previously heated hybridization buffer to a higher temperature. DAPI dye was used for nuclear staining.

RNA Immunoprecipitation (RIP)
The RIP assay was performed using an RIP kit provided by Millipore, Burlington, MA, USA. Briefly, 100 µL of RIP wash buffer was prepared for washing, then added and mixed into a 50 µL suspension of magnetic beads. After that, AGO2 and IgG antibodies were applied and incubated for 30 min. The lysates were placed in a beads-antibody complex, and all the tubes were rotated and incubated at 4 • C for 1 night. The immunoprecipitated RNA was investigated using qRT-PCR.

Luciferase Reporter Assay
The hsa_circ_0008234 and ETS1 sequences, in both their wild-type and mutant forms, were cloned into pGL3-control luciferase reporter vectors. The luciferase reporter vectors co-transfection into control and miR-mimics cells employing lipofectamine 2000. The luciferase activities of the luciferase reporter vectors were assessed using a dual-luciferase reporter assay kit (Promega, Beijing, China).

Immunohistochemistry (IHC)
IHC was used to detect Ki67 in the tumor tissues from nude mice. Briefly, tissues of cell xenografts were fixed with 4% formaldehyde for 24 hours and cut into 4 µm paraffin sections. Anti-Ki67 antibody (1:200 dilution) was incubated overnight at 4 • C after sections were treated with 10 mmol/L sodium citrate buffer. Cells with homogeneous Ki67 staining were considered positive.

Animal Models
The xenograft model and tumor metastasis experiment were conducted on five-weekold nude male BALB/c mice. Both control cells (at a concentration of 5 × 10 6 ) and treated cells (at a concentration of 5 × 10 6 ) were injected into the backs of the naked mice. All mice were executed 25 days following injection, and tumor weights were measured for IHC. The lung metastasis model was used in in vivo metastasis testing. Cells (1 × 10 6 ) were infused into mice via the tail veins. Mice were euthanized, and all lungs were retrieved for IHC examination after another 25 days.

Statistical Analysis
The GraphPad Prism 8 and R software (version 4.0.4) were employed for all statistical analyses. All trials were repeated three times, and the results are presented as the mean with the standard deviation. p values of less than 0.05 were considered significant.

CircRNA hsa_circ_0008234 Is Overexpressed in Colon Cancer Tissue and Cell Lines
Based on the circRNA throughput data of GSE172229, differentially expressed circR-NAs were evaluated between five colon cancer and adjacent normal tissues. Figure 1A depicts the top five increased and decreased circRNAs in tumor tissue. Of note, hsa_circ_0008234 was the most significantly upregulated circRNA ( Figure 1B). Meanwhile, hsa_circ_0008234 was highly expressed in colon cancer tissue ( Figure 1C). Hsa_circ_0008234 includes 587 nucleotides and is derived from the exons 8, 9, 10, and 11 of FOXP1 in chr3 (71090478-71102924), also known as circFOXP1 ( Figure 1D). As such, we performed Sanger sequencing of its PCR product to confirm that hsa_circ_0008234 had a covalently closed circular structure ( Figure 1E). Moreover, hsa_circ_0008234 was found to have a higher expression value in colon cancer cell lines ( Figure 2A).

CircRNA hsa_circ_0008234 Promote the Proliferation, Invasion, and Migration of Colo Cancer Cells
To examine the biological function of hsa_circ_0008234 in colon cancer, we kno it down and performed qRT-PCR to validate the effectiveness of this knockdown in c cancer cells. The results indicated that sh-circ#2 was the most efficient, so it was ch for future experiments (Figures 2B,C and S1A). The SW480, DLD-1, and HCT8 cell were used for further experiments due to their higher hsa_circ_0008234 expres Colony generation and CCK8 experiment demonstrated that hsa_circ_000 knockdown significantly reduced colon cell growth (Figures 2D-F and S1B,C). EdU a results suggested that inhibiting hsa_circ_0008234 significantly decreased the propo of EdU-positive cells ( Figure 2G). Transwell and wound healing tests revealed Hsa_circ_0008234 promotes colon cancer proliferation. Notes: (A) Hsa_circ_0008234 was increased in colon cancer cells, *** = p < 0.001; (B,C) qRT-PCR assay was conducted to assess the knockdown efficiency of hsa_circ_0008234, NS. = p > 0.05, * = p < 0.05, *** = p < 0.001; (D) Colony formation assay between hsa_circ_0008234 knockdown and control cells, *** p < 0.001; (E,F) CCK8 assay between hsa_circ_0008234 knockdown and control cells, *** = p < 0.001; and (G) EdU assay between hsa_circ_0008234 knockdown and control cells, *** = p < 0.001.

CircRNA hsa_circ_0008234 Promote the Proliferation, Invasion, and Migration of Colon Cancer Cells
To examine the biological function of hsa_circ_0008234 in colon cancer, we knocked it down and performed qRT-PCR to validate the effectiveness of this knockdown in colon cancer cells. The results indicated that sh-circ#2 was the most efficient, so it was chosen for future experiments (Figures 2B,C and S1A). The SW480, DLD-1, and HCT8 cell lines were used for further experiments due to their higher hsa_circ_0008234 expression. Colony generation and CCK8 experiment demonstrated that hsa_circ_0008234 knockdown significantly reduced colon cell growth (Figures 2D-F and S1B,C). EdU assay results suggested that inhibiting hsa_circ_0008234 significantly decreased the proportion of EdU-positive cells ( Figure 2G). Transwell and wound healing tests revealed that hsa_circ_0008234 knock-

CircRNA hsa_circ_0008234 Functions as a miR-338-3p Sponge in Colon Cancer Cells
CircRNA subcellular localization can reveal its function mechanism. As such employed the FISH test to determine the subcellular localization of hsa_circ_000 Hsa_circ_0008234 was found mostly in the cytoplasm, indicating that it may functio a ceRNA mechanism ( Figure 4A). Additionally, miRNAs with potential attachment for hsa_circ_000824 were discovered using the Circular RNA interactome and Sta databases ( Figure 4B). The miR-338-3p was selected for future research based on prev findings for colon cancer [19]. Figure 4C depicts the anticipated attaching positio hsa_circ_0008234 and hsa-miR-338-3p. Cell line studies revealed that hsa_miR-338-3p decreased in colon cancer cells ( Figure 4D). Furthermore, we used miR-338-3p mi and the qRT-PCR results showed a satisfactory overexpression efficiency (Figure Transfection of cells with miR-338-3p mimics significantly lowered wild hsa_circ_0008234 reporter gene activity while having no effect on hsa_circ_000 mutated-type reporter gene activity ( Figure 4F,G). CCK8 test revealed that miR-33 significantly inhibits colon cancer cell proliferation ( Figure 4H,I). Additionally transwell test result revealed that miR-338-3p significantly suppressed colon cance aggressiveness (Figure 4 J,K).

CircRNA hsa_circ_0008234 Functions as a miR-338-3p Sponge in Colon Cancer Cells
CircRNA subcellular localization can reveal its function mechanism. As such, we employed the FISH test to determine the subcellular localization of hsa_circ_0008234. Hsa_circ_0008234 was found mostly in the cytoplasm, indicating that it may function via a ceRNA mechanism ( Figure 4A). Additionally, miRNAs with potential attachment sites for hsa_circ_000824 were discovered using the Circular RNA interactome and Starbase databases ( Figure 4B). The miR-338-3p was selected for future research based on previous findings for colon cancer [19]. Figure 4C depicts the anticipated attaching positions of hsa_circ_0008234 and hsa-miR-338-3p. Cell line studies revealed that hsa_miR-338-3p was decreased in colon cancer cells ( Figure 4D). Furthermore, we used miR-338-3p mimics, and the qRT-PCR results showed a satisfactory overexpression efficiency ( Figure 4E). Transfection of cells with miR-338-3p mimics significantly lowered wild-type hsa_circ_0008234 reporter gene activity while having no effect on hsa_circ_0008234 mutated-type reporter gene activity ( Figure 4F,G). CCK8 test revealed that miR-338-3p significantly inhibits colon cancer cell proliferation ( Figure 4H,I). Additionally, the transwell test result revealed that miR-338-3p significantly suppressed colon cancer cell aggressiveness (Figure 4 J,K).

ETS1 Is the Targeted Gene of miR-338-3p
Three miRNA-mRNA databases ENCORI, miRDB and TargetScan were used to identify the target gene for miR-338-3p. miR-338-3p served as an oncogene in colon cancer ( Figure 5A) [20]. ETS1 was discovered as the targeted gene for miRNA. Moreover, miR-338-3p was found to be negatively correlated with ETS1 ( Figure 5B, r = −0.139, p = 0.003). We discovered that cells containing miR-338-3p mimics expressed lower ETS1 RNA values at the cellular level ( Figure 5C). The anticipated ETS1 and hsa-miR-338-3p attaching locations were shown in Figure 5D. The luciferase reporter experiment revealed that cells transfected with miR-338-3p mimics had significantly lower ETS1 reporter gene activity in wild-type cells than in ETS1 mutant cells ( Figure 5E,F). The association with the AGO2 protein was perceived as a significant indicator of ceRNA function. The RIP results demonstrated that the AGO2 antibody significantly increased hsa_circ_0008234 and miR-338-3p in comparison to the control IgG antibody ( Figure 5G,H). The inhibitor efficiency was validated using qRT-PCR ( Figure 5I). Moreover, cells treated with miR-338-3p inhibitor had higher ETS1 RNA expression than control cells, but hsa_circ_0008234 knockdown could reverse this effect ( Figure 5J). in wild-type cells than in ETS1 mutant cells ( Figure 5E,F). The association with the AGO2 protein was perceived as a significant indicator of ceRNA function. The RIP results demonstrated that the AGO2 antibody significantly increased hsa_circ_0008234 and miR-338-3p in comparison to the control IgG antibody ( Figure 5G,H). The inhibitor efficiency was validated using qRT-PCR ( Figure 5I). Moreover, cells treated with miR-338-3p inhibitor had higher ETS1 RNA expression than control cells, but hsa_circ_0008234 knockdown could reverse this effect ( Figure 5J).

CircRNA hsa_circ_0008234 Promotes Colon Cancer Proliferation and Metastasis in Vivo
The biological effect of hsa_circ0008234 was investigated in vivo. The nude mice tumorigenesis test revealed that hsa_circ_0008234 knockdown could significantly inhibit colon cancer cell growth in vivo ( Figure 7A,B). Ki67 staining showed that the sh-hsa_circ_0008234 xenograft tumors had a lower proportion of Ki67-positive cells than sh-NC cells ( Figure 7C). The lung metastasis model showed that inhibiting hsa_circ_0008234 could reduce the number of metastatic lung foci ( Figure 7D). The concept of our study was shown in Figure 8.

CircRNA hsa_circ_0008234 Promotes Colon Cancer Proliferation and Metastasis in Vivo
The biological effect of hsa_circ0008234 was investigated in vivo. The nude mice tumorigenesis test revealed that hsa_circ_0008234 knockdown could significantly inhibit colon cancer cell growth in vivo ( Figure 7A,B). Ki67 staining showed that the sh-hsa_circ_0008234 xenograft tumors had a lower proportion of Ki67-positive cells than sh-NC cells ( Figure 7C). The lung metastasis model showed that inhibiting hsa_circ_0008234 could reduce the number of metastatic lung foci ( Figure 7D). The concept of our study was shown in Figure 8.

Discussion
Colon cancer remains a major global public health concern, claiming approximately 600,000 death lives each year [21]. CircRNA has recently received attention due to its broad impact on disease. Meanwhile, the highly stable characteristics brought by its covalent closed-loop structure make circRNA an excellent biomarker for diagnosis and therapy [22]. Consequently, it is urgent and meaningful to explore novel circRNAs that have the potential to be new clinical biomarkers in colon cancer.
In this present investigation, we identified has_circ_0008234 as a potential circRNA for subsequent analyses. CircRNA hsa_circ_0008234 is derived by backward splicing of FOXP1 (forkhead box P1) exons 8, 9, 10, and 11. Elevated hsa_circ_0008234 levels were found in colon cancer cells. In vitro and in vivo tests revealed that hsa_circ_0008234 promoted colon cancer aggressiveness. In particular, hsa_circ_0008234 acts as a miRNAsponge for miR-338-3p to increase ETS1 RNA expression, which potentially influences AKT/mTOR pathway stimulation. Previous research investigated the biological effects of hsa_circ_0008234 on cancer. For instance, Cai et al. discovered that hsa_circ_0008234 promoted the growth of cutaneous squamous cell carcinoma via the miR-127-5p/ADCY7 axis [23]. Luo et al. demonstrated that has_circ_0008234 could be a unique predictive serum biomarker for non-small cell lung cancer [24]. To the best of our knowledge, this is a pioneer study of the effect of hsa_circ_0008234 in colon cancer.
Recent research suggests that the ceRNA is the primary function mechanism for circRNAs found in the cytosol [25]. According to the FISH results, we discovered that

Discussion
Colon cancer remains a major global public health concern, claiming approximately 600,000 death lives each year [21]. CircRNA has recently received attention due to its broad impact on disease. Meanwhile, the highly stable characteristics brought by its covalent closed-loop structure make circRNA an excellent biomarker for diagnosis and therapy [22]. Consequently, it is urgent and meaningful to explore novel circRNAs that have the potential to be new clinical biomarkers in colon cancer.
In this present investigation, we identified has_circ_0008234 as a potential circRNA for subsequent analyses. CircRNA hsa_circ_0008234 is derived by backward splicing of FOXP1 (forkhead box P1) exons 8, 9, 10, and 11. Elevated hsa_circ_0008234 levels were found in colon cancer cells. In vitro and in vivo tests revealed that hsa_circ_0008234 promoted colon cancer aggressiveness. In particular, hsa_circ_0008234 acts as a miRNA-sponge for miR-338-3p to increase ETS1 RNA expression, which potentially influences AKT/mTOR pathway stimulation. Previous research investigated the biological effects of hsa_circ_0008234 on cancer. For instance, Cai et al. discovered that hsa_circ_0008234 promoted the growth of cutaneous squamous cell carcinoma via the miR-127-5p/ADCY7 axis [23]. Luo et al. demonstrated that has_circ_0008234 could be a unique predictive serum biomarker for non-small cell lung cancer [24]. To the best of our knowledge, this is a pioneer study of the effect of hsa_circ_0008234 in colon cancer.
Recent research suggests that the ceRNA is the primary function mechanism for circRNAs found in the cytosol [25]. According to the FISH results, we discovered that hsa_circ_0008234 was largely localized in the cytosol, indicating that it potentially functions via a ceRNA mechanism. In this view, we searched three databases, ENCORI, miRDB, and TargetScan, to find the miRNAs of interest. Finally, we were intrigued by the miR-338-3p. MiR-338-3p has been shown to influence cancer progression in a variety of cancers. Luan et al. discovered that lncRNA XLOC 006390 could potentially promote tumorigenesis and metastasis of cervical cancer by acting as a ceRNA versus miR-338-3p [26]. In addition, Zou et al. revealed that miR-338-3p could hamper colon cancer by inhibiting MACC1 expression [19]. In our investigation, we found that miR-338-3p was potentially correlated with the cancer-boosting effect of hsa_circ_0008234, which refined the regulatory network of miR-338-3p.
ETS1 is a member of the ETS transcription factors that can recognize the core consensus DNA sequence of GGAA/T in specific genes (ETS-binding domain) [27]. The ETS transcription factor family, which includes ETS1, plays a role in stem cell generation, cell senescence, apoptosis, and tumorigenesis [27]. These ETS family members can either activate or inhibit the transcription process in several different genes. Chen et al. found that WTAP could accelerate the development of hepatocellular carcinoma by suppressing ETS1 via an m6A-HuR-dependent mechanism [28]. Furthermore, Rodgers et al. discovered that ETS1 may activate the transformed β signaling growth factor and promote epithelial-mesenchymal transition in prostate cancer [29]. Gu et al. found that miR-532-3p reduced the development of colorectal cancer by disrupting the ETS1/TGM2-axis-mediated Wnt/β-catenin signaling [30]. We performed several tests in the course of our research to investigate the existing has_circ_0008234 and the miR-338-3p/ETS1 axes relationship. The results of rescue studies suggested that has_circ_0008234 influences the malignancy of colon cancer by regulating ETS1 via miR-338-3p.
Research evidence suggests that ETS1 may accelerate the development of cancer by influencing the function of the PI3K/AKT/mTOR signaling pathway. Xu et al. discovered that miR-129 could inhibit prostate cancer and metastasis by blocking ETS1 and modulating the PI3K/AKT/mTOR pathway [31]. In addition, Tong et al. found that miR-365 inhibits lung cancer development by blocking ETS1 and inactivating the AKT/mTOR pathway [32]. In this present investigation, PI3K/AKT/mTOR signaling was found to be the downstream path of the hsa_circ_0008234/miR-338-3p/ETS1 axis, broadening the regulatory network of PI3K/AKT signaling in cancers. Our results also indicated that hsa_circ_0008234 could regulate the PI3K/AKT/mTOR signaling pathway. As a widespread regulator, circRNA seems reasonable to influence cancer progression by regulating the classic PI3K/AKT/mTOR signaling. Wang et al. discovered that circEPSTI1 could affect oral squamous cell carcinoma progression via PI3K/AKT/mTOR signaling, which is dependent on the mir-942-5p/LTBP2 axis [33]. Moreover, Ling et al. noticed that Circ-PRKDC could promote autophagy and apoptosis in T-cell acute lymphoblastic leukemia via the PI3K/AKT/mTOR signaling [34].
Some limitations should be noticed. Firstly, we only proved the in vivo cancerpromoting effect of hsa_circ_0008234.
However, the in vivo experiments of hsa_circ_0003596/miR-502-5p/IGF1R/PI3K/AKT axis have not been performed. Comprehensive in vivo experiments can enhance the stability of the conclusion. Secondly, the evaluation of the tissue level of hsa_circ_0008234 is limited in our study. In the future, a larger sample size of colon cancer is needed to support our conclusions. Thirdly, since circRNAs have a broad regulatory effect, including protein-protein interaction and protein encoding, other underlying mechanisms have not been identified.

Conclusions
In conclusion, this work revealed the tumor-promoting effect and potential molecular mechanisms of the circRNA hsa_circ_0008234 in colon cancer. Significantly elevated cir-cRNA hsa_circ_0008234 levels in colon cancer indicated that it could play a pivotal part in the diagnosis and prognosis prediction. Moreover, results from in vitro and in vivo investigations demonstrate that hsa_circ_0008234 may promote colon cancer aggressiveness. These findings suggest that hsa_circ_0008234 could be a therapeutic target for colon cancer.