circSLC6A6 Sponges miR-497-5p to Promote Endometrial Cancer Progression via the PI4KB/Hedgehog Axis

Background As a new kind of noncoding RNAs, circular RNAs (circRNAs) have been substantiated to be involved in multiple biological processes. Accumulating studies indicate that circular RNAs (circRNAs) regulate the development of cancers by acting as miRNA sponges. However, the role of circRNAs in endometrial cancer (EC) is rarely reported. This study was aimed at investigating the functional roles of circSLC6A6 in EC. Methods The qRT-PCR assay was performed to detect the circSLC6A6 expression in EC tissues and cell lines. The luciferase reporter assay was performed to explore the connection between circSLC6A6 and miR-497-5p as well as the connection between miR-497-5p and PI4KB. The colony formation assay, EdU assay, wound healing assay, and transwell assay were performed to examine the proliferation, migration, and invasion of EC cells. The in vivo assay was performed to reveal the function of circSLC6A6 in tumorigenesis. Results We found that circSLC6A6 was highly expressed in both EC tissues and cells. And circSLC6A6 promoted the proliferation, migration, and invasion of EC cells in vitro. In vivo, circSLC6A6 promoted tumor growth. Besides, a mechanistic study demonstrated that circSLC6A6 could regulate tumor-associated signaling PI4KB/hedgehog pathway by sponging miR-497-5p. Conclusion This study illustrates that circSLC6A6 plays a role in promoting EC progression via the miR-497-5p-mediated PI4KB/hedgehog pathway. Our study may provide a potential novel biomarker for EC diagnosis or treatment.


Introduction
Endometrial cancer (EC) is the fourth most common cancer in women in industrialized country, being classified into many subtypes containing endometrioid endometrial cancer, serous endometrial cancer, clear cell endometrial cancer, mixed endometrial cancer, and uterine carcinosarcoma [1,2]. For patients diagnosed at early stages, the 5-year survival rate of them is up to 95% [3]. However, the 5-year survival rate of patients at advanced stages is less than 30% [4]. Although there are many therapies, such as surgery, chemotherapy, radiotherapy, and hormone therapy [5], the problems of chemotherapeutic resistance and poor response rate bring obstacles for the treatment [6,7]. Therefore, it is urgent to explore the underlying molecular mechanism of EC pathogenesis.
Circular RNAs (circRNAs), a subclass of noncoding RNAs with high stability and sequence conservation, are widely expressed in cells and tissues [8,9]. It has been reported that circRNAs are involved in the development of multiple tumors, such as gastric cancer, breast cancer, hepatocellular cancer, and endometrial cancer [10][11][12][13][14]. But the function of circRNAs in EC is still not clarified clearly.
Hedgehog (Hh) signaling, firstly discovered in Drosophila by genetic analysis, is highly conserved in evolution and function and essential in the development of normal embryos [15,16]. And the abnormal activation of Hh signaling promotes the cancer progression [17][18][19]. Phosphatidylinositol 4-kinase IIIβ (PIK4B), regulating cellular physiological functions that contain cell morphology, metabolic regulation, and signal transduction, can produce PI4P that participates in Hh signaling in the dictyosome [17]. And the disruption of signaling transduction from PTC to SMO that controls PI4P is considered the most common feature in the tumor [20]. Consequently, the PIK4B/hedgehog pathway might be involved in the pathogenesis of EC.
In our study, we explored the function and regulatory mechanism of circSLC6A6 in EC. Firstly, it was reported that circSLC6A6 was upregulated in EC and promoted EC cell proliferation, migration, and invasion. In addition, we found that the function of circSLC6A6 in EC was associated with the miR-497-5p-mediated PIK4B/hedgehog pathway. These findings indicate that circSLC6A6 might serve as a potential biomarker for EC.

RNA Extraction and qRT-PCR.
Total RNA was extracted from EC tissues and cell lines by using the Trizol reagent (Invitrogen, USA). The concentration of RNA was measured applying NanoDrop 2000 (Thermo, USA). Reversetranscription PCR was performed using PrimeScript RT Master Mix (Takara, Japan). The qRT-PCR was performed using SYBR Premix Ex Taq and SYBR PrimeScript miRNA RT-PCR Kit (Takara, Japan). For circRNA and mRNA, GAPDH was used as an internal control. For miRNA, U6 was applied as an internal control. The reactions were run on the ABI 7500 Real-Time PCR System (Life Technologies, USA). The relative expression levels of RNAs were calculated with the 2 −ΔΔCT algorithm. The primers used in this study are as follows:

circSLC6A6 Is Upregulated in Endometrial Cancer.
We screened the expression profiles of circRNAs taken from 5 EC and adjacent noncancerous endometrial tissues using cir-cRNA sequencing. The significantly dysregulated circRNAs were presented in the heat map (Figure 1(a)). It was found that circ_0064428 (termed circSLC6A6 in the remainder of the article) was the most upregulated circRNA. To verify whether circSLC6A6 expression was upregulated in EC tissues, we detected the expression of circSLC6A6 in 30 pairs of EC tissues using qRT-PCR; the results in Figure 1(b) showed that circSLC6A6 levels were significantly increased in EC tissues. Moreover, circSLC6A6 expressions in EC cell lines were significantly higher than those in the normal cell line (Figure 1(c)). HEC-1A and HEC-1B were the two cell lines with the lowest and largest change of circSLC6A6 expression separately and then were selected for subsequent studies. To verify the circular feature of circSLC6A6, random hexamer and oligo(dt)18 were used to amplify circSLC6A6   (Figure 1(d)). The RNase R treatment assay indicated that circSLC6A6 was resistant to RNase R, while linear LDLR mRNA was distinctly digested by RNase R, which confirmed the circular features of cir-cSLC6A6 (Figure 1(e)). These results showed that cir-cSLC6A6 is an upregulated circular RNA in EC.

circSLC6A6
Promotes EC Cell Proliferation, Migration, and Invasion In Vitro. To explore the function of circSLC6A6 in EC cells, two siRNAs against circSLC6A6 and the overexpression vector of circSLC6A6 were constructed. It showed that si-circSLC6A6-1 has higher inhibitory efficiency in the HEC-1B cell and was selected for the subsequent experiments in Figure 2(a). The overexpression vector of cir-cSLC6A6 in HEC-1A cells significantly induced the expression of circSLC6A6 (Figure 2(b)). The colony formation assay showed that circSLC6A6 knockdown significantly decreased the colonies of HEC-1B cell, whereas circSLC6A6 upregulation in the HEC-1A cell exerted opposite effects (Figure 2(c)). The EdU assay demonstrated that the EdU-positive cells were obviously reduced by the circSLC6A6 downregulation and greatly increased by circSLC6A6 overexpression (Figure 2(d)). In addition, the wound healing assay (Figure 2(e)) and transwell assay (Figure 2(f)) revealed that circSLC6A6 downregulation suppressed the migration and invasion of EC cells, whereas the impairment of migration and invasion was blocked by circSLC6A6 upregulation. These data indicate that circSLC6A6 promotes the proliferation, migration, and invasion of EC cells.

circSLC6A6
Serves as an Efficient miR-497-5p Sponge in EC Cells. It was reported that circRNAs can abrogate the function of miRNAs via acting as miRNA sponges [22]. To further explore the downstream pathway of circSLC6A6 in EC, it was predicted that miR-497-5p was the potential target miRNA of circSLC6A6 by CircInteractome (Figure 3(a)). The RIP assay was performed to pull down the RNA transcripts that are bound to AGO2 in HEC-1B cells transfected with miR-497-5p mimics or NC mimics. The results showed that cir-cSLC6A6 and miR-497-5p were pulled down by anti-AGO2  Journal of Immunology Research antibodies, suggesting that circSLC6A6 interacted with miR-497-5p through AGO2 protein in EC cells (Figures 3(b) and 3(c)). Next, the luciferase report assay showed that luciferase activity was significantly reduced in circSLC6A6 wild-type and miR-497-5p mimic groups, indicating that circSLC6A6 can directly interact with miR-497-5p in Figure 3(d). In addition, the expressions of miR-497-5p were significantly upregulated in EC cells transfected with si-circSLC6A6 but downregulated in EC cells transfected with oe-circSLC6A6 (Figures 3(e) and 3(f)). These results demonstrate that cir-cSLC6A6 can serve as a miR-497-5p sponge in EC cells.

miR-497-5p
Mediates the PI4KB/Hedgehog Pathway in EC Cells. The potential target genes of miR-497-5p were pre-dicted using TargetScan and Starbase. Among them, PI4KB was reported to be involved in the progression of cancers [23]. In this study, PI4KB was selected as the target gene of miR-497-5p for further exploration. Hedgehog signaling is upregulated in basal cell carcinoma and medulloblastoma, and PI4KB may be a relevant target protein for pharmacological inhibition of Hh signaling [17]. The binding sites of miR-497-5p and PI4KB are shown in Figure 4(a). The luciferase report assay in Figure 4(b) verified that miR-497-5p mimics reduced luciferase activity in the PI4KB wild-type group, whereas no suppression was observed in the PI4KB mutant group. In addition, the qRT-PCR assay showed that cir-cSLC6A6 knockdown would significantly reduce the expressions of PI4KB and hedgehog in HEC-1B cells, but    and 4(f) showed that miR-497-5p mimics remarkably decreased the expression of PI4KB and hedgehog in HEC-1B cells, while miR-497-5p inhibitors enhanced the expression of PI4KB and hedgehog in HEC-1A cells. These data reveal that miR-497-5p mediates the PI4KB/hedgehog pathway in EC cells.

circSLC6A6
Promotes EC Cell Proliferation, Migration, and Invasion via miR-497-5p/PIK4B/Hedgehog. Then, the results in Figure 5(a) revealed that the downregulation of circSLC6A6 significantly increased miR-497-5p expression and decreased the expressions of PIK4B/hedgehog, and the upregulation of circSLC6A6 markedly suppressed miR-497-5p expression and enhanced PIK4B/hedgehog expression, whereas miR-497-5p inhibitors or mimics reversed the above effects, respectively. The colony formation assay ( Figure 5(b)) and EdU assay ( Figure 5(c)) showed that cir-cSLC6A6 inhibition reduced HEC-1B cell proliferation and circSLC6A6 overexpression promoted HEC-1A cell proliferation, but extra supplement of miR-497-5p inhibitors or mimics could block the effects induced by circSLC6A6 inhibition or overexpression. The wound healing assay ( Figure 5(d)) and transwell assay ( Figure 5(e)) indicated that circSLC6A6 advanced the migration and invasion of EC cells, while miR-497-5p could attenuate the circSLC6A6induced effects. And western blotting showed that miR-497-5p inhibition in EC cells would promote the expressions of downstream targets GLI1, PTCH, and SHH in the hedgehog pathway, but extra supplement of si-PI4KB in EC cells transfected with the miR-497-5p inhibitor would in turn decrease the expressions of GLI1, PTCH, and SHH ( Figure 5(f)). These data suggest that circSLC6A6 promotes EC cell proliferation, migration, and invasion via miR-497-5p/PIK4B/hedgehog.

Silencing circSLC6A6 Inhibits the Growth of Tumors In
Vivo. To further investigate the effects of circSLC6A6 on tumor growth in vivo, HEC-1B cells stably transfected with si-circSLC6A6 or si-NC were subcutaneously injected into BALB/c nude mice. The image of xenograft tumors is shown in Figure 6(a). It was shown that tumor volumes were significantly decreased via circSLC6A6 inhibition with the growth of days (Figure 6(b)). Tumor weights of the si-circSLC6A6 group are significantly reduced in Figure 6(c). The IHC assay showed that the PIK4B and hedgehog expressions were downregulated in the tumor of the si-circSLC6A6 group, as shown in Figure 6(d). The results reveal that the inhibition of circSLC6A6 suppressed the tumor growth of EC in vivo.

Discussion
Increasing studies have shown that circRNAs act as regulators of multiple biological processes, especially the cancer progression [24]. However, studies focusing on circRNAs in EC are little to understand. In the present study, we analyzed the circRNA expression profiles using circRNA sequencing and found that circSLC6A6 was the most upregulated cir-cRNA in EC. Then, it was found that the expression levels of circSLC6A6 in EC tissues and cell lines were significantly increased, which was consistent with the data analysis. In addition, functional experiments showed that circSLC6A6 promoted the cell proliferation, migration, and invasion in vitro as well as tumor growth in vivo. Currently, cancer-related studies are focusing on the regulatory role of "circRNA-miRNA-mRNA" axis; many researches have reported that circRNAs can regulate gene expression by sequestering miRNAs that mediate the metastasis, metabolism, and proliferation of tumor cells [24,25]. Therefore, the potential miRNA target miR-497-5p of cir-cSLC6A6 was predicted by CircInteractome. The results of the RIP assay and luciferase report assay indicated that cir-cSLC6A6 sponged miR-497-5p in EC cells. In addition, PI4KB was predicted to be the target gene of miR-497-5p by Starbase. PI4KB is reported to be involved in the activation of Hh signaling, which is closely associated with cancer development [15]. Subsequently, the mechanistic study confirmed that miR-497-5p mediated the PI4KB/Hh pathway in EC cells. In this study, we first revealed the oncogenic effects of the PI4KB/Hh pathway in EC. We demonstrated that cir-cSLC6A6 promoted EC cell proliferation, migration, and invasion by regulating the PI4KB/Hh pathway through miR-497-5p.
However, there are several limitations in this study. The premise of noncoding RNA as an essential biomarker is that it can be stably detected in body fluids such as plasma [21]; therefore, whether circSLC6A6 can be detected in body fluids needs further investigation. In addition, the connection between circSLC6A6 and EC stages also needs further study and we will continue to investigate these issues.
In conclusion, the present research provides the evidence that circSLC6A6 contributes to EC development. cir-cSLC6A6/miR-497-5p/PI4KB/Hh signaling might be a potential direction for EC diagnosis and therapy.

Data Availability
The data of this study are available from the corresponding author on reasonable request.

Ethical Approval
Our work was approved by the Ethics Committee of the Central Hospital of Wuhan.

Consent
All participants had signed the written informed consent form before this study.