CircSEC24A upregulates TGFBR2 expression to accelerate pancreatic cancer proliferation and migration via sponging to miR-606

Background Circular RNA (circRNA), producing by special selective splicing, was widely expressed in the cytoplasm of eukaryotic cells as a newly non-coding RNAs. It played different roles in a variety of diseases including cancer and performed different functions. Nonetheless, reports on the specific function of circRNA in pancreatic cancer (PC) were still rarely so far. In particular, the role of circSEC24A in PC remains unclear. Methods Real-time fluorescent quantitative PCR was used to evaluate the expression level of circSEC24A in pancreatic cancer tissues and cell lines. Furthermore, we used some functional experiments, such as EDU and Transwell assays, to explore the effects of circSEC24A on the proliferation and invasiveness of pancreatic cancer. Finally, the corresponding relationship among circSEC24A, miR-606 and TGFBR2 was explored by dual luciferase reporter and other mechanism studies. Results The expression of circSEC24A in both pancreatic cancer tissues and cell lines was evidently up-regulated. Furthermore, knockdown of circSEC24A significantly inhibited the proliferative, migration and invasive capacity of pancreatic cancer cells, whereas miR-606 inhibitor obviously counteracted these effects. Further study confirmed that circSEC24A alleviated suppression on target TGFBR2 expression by directly sponging miR-606 and then influenced the tumorigenesis of pancreatic cancer. Conclusions These findings indicated that the progression of pancreatic cancer can be driven by circSEC24A influencing miR-606/TGFBR2 axis. Therefore, circSEC24A might be used as a critical biomarker influencing the early diagnosis and prognosis of pancreatic cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-02392-y.

biomarkers for early diagnosis of pancreatic cancer and seek potential therapeutic targets for pancreatic cancer.
Circular RNAs (circRNAs) can be covalently linked at the 5′ and 3′ ends to form a circular structure. As a new type of non-coding RNA, it has not been found to have protein-coding ability [5]. CircRNAs are highly conserved and abundant with closed-loop molecular structures, which are not affected by exonuclease and are not easy to be degraded [6]. There is increasing evidence that circRNAs participate in multiple biological functions in tumors, including cell proliferation, apoptosis, differentiation, metastasis and regulation of gene expression [7]. CircRNAs have miRNA response elements (MREs), hence, circRNAs can be utilized as competing endogenous RNA (ceRNA) to indirectly modulate gene expression in posttranscriptional level via specially binding to miRNAs [8]. Zhou et al. [9] reported that circFAT1 promoted malignant progression of cervical cancer by binding miR-409-3p to upregulate CDK8 and activating ERK1/2 signaling. Gu et al. [10] found that circHIPK3 resulted in activation of AKT/mTOR signaling to facilitate lung cancer cell growth, metastasis and glycolysis via serving as sponge for miR-381-3p. Previous study suggested that circCDR1as was overexpressed in pancreatic cancer and promoted the proliferation, migration and invasion of pancreatic cancer cells by regulating E2F3 expression via functioning as sponge for miR-432-5p [11]. In addition, inhibitory expression of circ_0013912 significantly suppressed pancreatic cancer cell proliferation and metastasis via sponging miR-7-5p [12]. Zhang et al. declared that circ_001587 elevated SLC4A4 expression to suppress malignant phenotype of pancreatic cancer, including angiogenesis and metastasis. The underlying mechanism was that circ_001587 competitively sponged to miR-223, thereby increased the expression of SLC4A4, which was a tumor suppressor [13]. Therefore, deep exploration of the circRNA-miRNA-mRNA network for pancreatic cancer is benefited for excavating novel biomarkers for diagnosis and treatment of pancreatic cancer.
In this study, we identified a novel circRNA hsa_ circ_0003180 (circSEC24A) with 252 nucleotides in length, which was derived from back-splicing the SEC24A mRNA and located on chromosome 5: 134002512-134007576. Interestingly, circSEC24A was overexpression in pancreatic cancer tissues and high expression levels was positively correlation to malignant phenotype, including cell proliferation and metastasis. Mechanistically, circSEC24A acted as a ceRNA of TGFBR2 to bind miR-606, blocking the inhibitory function of miR-606 on TGFBR2. Therefore, circSEC24A/ miR-606/TGFBR2 signaling axis might be one of the indispensable factors affecting the therapeutic effect of pancreatic cancer.

Clinical tissue specimens
All clinical specimens including pancreatic cancer patients' tissues and matched adjacent normal tissues were obtained from 20 patients with pancreatic cancer surgically resected in the Renmin Hospital of Wuhan University. The patients without radiotherapy, chemotherapy or other neoadjuvant treatments were selected for our study. The group selected as normal tissues showed no cancer cells in pathological examination. After surgical resection, the specimens from those patients were collected immediately. This research gained permission from the Ethics Committee of Renmin Hospital of Wuhan University.

RNase R treatment
RNase R was an exonuclease that primarily acted on and removed linear RNA. To digest the linear RNA from PANC-1 and MIA PaCa-2, total isolated RNA was mixed with RNase R (30 units, Shanghai Hengfei Biotechnology Co., Ltd.) for 30 min at 37 °C. Then, real-time PCR was performed to quantitative the expression of circSEC24A and SEC24A, respectively.

Fluorescence in situ hybridization
FISH, an important non-radioactive in situ hybridization technique, was used in our study to explore the location of circSEC24A in pancreatic cancer cells. The special circSEC24A probes, which working concentration was 0.5 µM, were purchased from Ruibo Biotechnology (Guangzhou, China). Then, the hybridization was executed all night with the probes. Finally, we utilized a fluorescence microscope (Olympus, Japan) to capture the images and selected some representative images for analysis.

Subcellular fractionation
About 2 × 10 6 PANC-1 cells were collected and rinsed with cold PBS. Nuclear and cytoplasmic RNA were collected respectively using the PARIS ™ kit (Invitrogen, USA) according to the manufacturer's manual. Subsequently, circSEC24A expression was analyzed by qRT-PCR, meanwhile, 18S rRNA and U6 detected as control of cell cytoplasm and cell nuclei, respectively.

Cell culture and transfection
Normal pancreatic duct epithelial cell line HPDE, and 4 types of pancreatic cancer cell lines (PANC-1, MIA PaCa-2, SW1990, BxPC-3) obtained from American Type Culture Collection (ATCC) were indispensable members of our various experiments. HPDE and BxPC-3 were maintained in RPMI 1640 (No. A1049101, Gibco, USA); while other cell lines including PANC-1, MIA PaCa-2 and SW1990 were maintained in high glucose DMEM (No. 11965092, Gibco, USA). To maintain cells viability, not only 10% fetal bovine serum (FBS, No. 10099141, Gibco, USA) but 1% penicillin-streptomycin solution (No. 15070063, Gibco, USA) should be added to the medium. The medium was exposed to UV radiation for 30 min before we used. In conclusion, all cells we used were maintained and stored following the instructions provided by ATCC. Moreover, the appropriate temperature (37 °C) and the suitable concentration of carbon dioxide(5% CO 2 )can effectively promoted cell growth rapidly, so all cells need to be grown in special incubators that meet these conditions. Firstly, we seeded PANC-1 and MIA PaCa-2 cells into 6-well plates. Transfection can be carried out when cells attached to the bottom of the hole and 60-70% of the total area is reached. Different reagents were subsequently transfected into different cell lines, respectively. These reagents were synthesized by Ruibo Biotechnology (Guangzhou, China) and included si-circSEC24A, miR-606 mimics, miR-606 inhibitors, and negative controls. Appropriate amount of Lipofectamine ™ 3000 reagent (No. L3000015, Invitrogen, USA) was added during the transfection process to improve the transfection efficiency. Additionally, we purchased sh-circSEC24A and sh-NC lentivirus vector from Ruibo Biotechnology (Guangzhou, China), then transfected into PANC-1 cells for lentiviral packaging according to the manufacturer's protocol.

Cell counting Kit8 assays
About 2000 transfected cells were plated and cultured until adherent. Then, original CCK-8 solution (Boster, China) was mixed with culture medium according to the rate of 1:9 and added directly into each well at the indicated time. After 2 h of incubation, we utilized the optional density (OD) value measured by microplate reader (BioTek, USA) to evaluated cell proliferation ability.

Colony formation assays
About 500 transfected cells were seeded in 6-well plates. Two weeks later, the clones visible to the naked eye appeared. After washing with PBS (Gibco, USA) for three times, we used 1 mL 4% paraformaldehyde (Vazyme Biotech Co., Ltd) to fix cells for 30 min. In order to stain the cells, it was subsequently maintained in 0.1% crystal violet solution (Vazyme Biotech Co., Ltd) for another 30 min. Excess staining was removed by washing with PBS solution and cell counts were performed with a clear field of vision.

EDU assays
The EDU cell Proliferation Kit (No. C0075S, Beyotime Biotechnology, China) was used to assess the proliferation ability of cells. Cover glass slides were added into the six-well plate and pancreatic cancer cells with different treated were planted. Before starting the experiment, it is a priority to configure the relevant working solution including EDU working solution. The cells in each well were then immersed in a new EDU working solution to allow the EDU to penetrate the DNA while it was being replicated. Then, the cells were fixed and stained according to the manufacturer's protocol. Subsequently, we utilized a fluorescence microscope (Olympus, Japan) to acquire the images. The data obtained above are processed by Photoshop software (version 21.0).

Flow cytometry analysis
Transduced cells (at least 1 × 10 6 ) were trypsinized and then washed in pre-cooled PBS. Cell fixation was conducted using 70% ethanol. Then, the cells were stained using PI and RNase reagent (Si Nan Biotechnology Services Co., LTD, China) and incubated at 37 °C. Cell cycle distribution analysis was performed using a flow cytometry device (BD Biosciences, USA).

Wound healing assays
Pancreatic cancer cells were cultured in 6-well plates until to 100% confluence. Next, a straight line of scars was generated via a tool such as sterile pipette device used to scratch the cell monolayer. The cell scratch wound was washed with PBS and treated with serum depleted media. The random injury filed of cell layer was photographed by a fluorescence microscope at 0 and 24 h. Representative images we screened were analyzed via GraphPad and then calculated the relative migration rate.

Transwell migration and invasion assays
The matrigel mix (BD Biosciences, USA) need first paved at the surface of upper chamber for invasion assays. In contrast, the migration experiment did not require the application of matrigel mix. After the relevant items have been prepared, 200 µL serum-free medium containing 5 × 10 4 pancreatic cancer cells was added slowly into upper chamber (Corning, USA). Then, 700 µL of the culture medium with 10% FBS was added into the bottom chamber. The chambers were fixed and then stained after migration for 28 h or invasion for 36 h. Residual staining solution and unpenetrated cells were removed with PBS and cotton swabs, respectively. After nature air dries, the migratory or invasive cells were photographed and counted via fluorescence microscope.

Western blot
The protein for subsequent detection was extracted from the previously transfected cells under the action of Radio Immunoprecipitation Assays lysis buffer (RIPA, Boster, China). The amounts of protein were assessed by bicinchoninic acid (BCA, Boster, China). Owing to the different molecular weights of the proteins being measured, they can be isolated and formed into bands in the 10% SDS-PAGE. The isolated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Boster, China). The PVDF membranes was incubated with 5% skim milk. The cut membranes were then placed in the primary antibodies, respectively. The primary antibodies purchased from Abcam (MA, USA) were as following: anti-CDK6, anti-cyclin D1, anti-CDK4, anti-GAPDH, anti-ZEB2, anti-Snail, anti-Twist, anti-TGFBR2, anti-AKT, anti-p-AKT, anti-ERK, anti-p-ERK, anti-smad2, anti-p-smad2. All antibodies were diluted in skim milk at the concentration of 1:1000. The membranes were immersed completely in secondary antibody (abcam, USA) at room temperature. The blots acquired in our study were dripped with ECL chemiluminescent reagent (Servicebio, Wuhan, China) to produce a light-emitting reaction that can be captured and visualized by the ChemiDoc XRS+ (Bio-Rad), allowing us to analyze each blot with Image Lab Software.

Bioinformatics analysis
GEO database (GSE69362) was performed to screen for significantly differentially expressed circRNAs in pancreatic cancer. This dataset containing circRNA profiling in six pairs of human PDAC and adjacent normal tissue was conducted by microarray. The CSCD database, TargetScan database and other databases were used to explore the possible role network of circSEC24A in pancreatic cancer.

Luciferase reporter assay
About 3 × 10 5 cells were seeded into each well of 24-well plates. To construct respectively the special plasmids, the WT and Mut 3′UTRs of circSEC24A or TGFBR2 were synthesized by Ruibo Biotechnology (Guangzhou, China), termed as circSEC24A-WT, circSEC24A-Mut, TGFBR2-WT and TGFBR2-Mut. Then, the plasmids obtained above were co-transfected into cells in pairs using Lipofectamine TM 3000 reagent (Invitrogen). 48 h later, the luciferase activities were examined in line with the manufacturer's manual.

Xenograft tumorigenesis
The PANC-1 cells used in our experiment were treated with sh-circSEC24A or sh-NC lentivirus. Next, 5 × 10 6 treated PANC-1 cells suspension was prepared and then subcutaneously injected into 6-week-old female BALB/c nude mice (Beijing Laboratory Animal Center, Beijing, China). Next, we randomly divided those mice into circ-SEC24A silenced group and control group. Tumor volume in each mouse was monitored weekly. Finally, mice were sacrificed and subcutaneous tumor were detected for tumor weight and IHC staining.

Statistical analysis
GraphPad Prism (version 8.0.1) was used for statistical analysis in our study. All Data we acquired in these experiments were presented as the mean ± SD. Only P < 0.05 indicates that the difference is statistically significant.

CircSEC24A is highly expressed in pancreatic cancer
To explore the expression profiles of circRNA in pancreatic cancer, we performed bioinformatics methods to screen differentially expressed circRNA based on GEO database (https:// www. ncbi. nlm. nih. gov/ geo/, GSE69362) via R software using "limma" and "heatmap" packages. Top 20 differentially expressed circRNA was list in Fig. 1A (Additional file 1). Through reviewing literature and measuring the expression in pancreatic cancer tissues, we filtered out hsa_circ_0003180 (circSEC24A) as our candidate circRNA for subsequent research. Firstly, PCR analysis suggested that circSEC24A was significantly highly expressed in pancreatic cancer tissues compared with their paired adjacent normal tissues (Fig. 1B). Next, we measured the expression of circSEC24A in different pancreatic cancer cell lines compared with human pancreatic duct epithelial cells (HPDE). Surprisingly, four different pancreatic cancer cell lines were all higher expression than HPDE (Fig. 1C). To detect the stability of circSEC24A, we utilized RNase R treatment to digest circSEC24A and the mRNA of SEC24A respectively. The results revealed that circSEC24A was not able to be digested by RNase R, while the SEC24A mRNA expression dramatically decreased after treating with RNase R (Fig. 1D, E). FISH and subcellular fraction assays were performed to measure the subcellular localization of circ-SEC24A, the results confirmed that circSEC24A was predominantly located in the cytoplasm, which supported the hypothesis that circSEC24A might serve as molecular sponge for miRNA to indirectly regulate gene expression in post-transcriptional levels ( Fig. 1F-I).

CircSEC24A facilitates pancreatic cancer cell proliferation
To detect the proliferated ability of circSEC24A in pancreatic cancer cells, PANC-1 and MIA PaCa-2 were transfected with silencing sequences and negative control sequence, respectively. PCR analysis was performed to evaluate the effectiveness of transfection. The results indicated that silencing sequences was successfully transfected into pancreatic cancer cells and downregulated the circSEC24A expression effectively ( Fig. 2A). CCK-8, EDU and colony formation assays were respectively evaluated the proliferation ability from different angles. CCK-8 assays suggested that silencing circSEC24A expression dramatically suppressed cell ability compared with control groups (Fig. 2B, C). In addition, EDU assays illustrated that silencing circSEC24A expression significantly inhibited cell growth (Fig. 2D-F). Moreover, colony formation assays indicated that negative control groups displayed more numbers of colony formations than silence groups (Fig. 2G, H). Eventually, we utilized western blot to detect expression of CDK4, CDK6 and Cyclin D1, which were key proteins in transition of cell cycle from G1 to S phase. The results demonstrated that silencing circSEC24A expression resulted in protein downregulation of CDK4, CDK6 and Cyclin D1, indicating that inhibited circSEC24A could negatively modulate pancreatic cancer cell proliferation (Fig. 2I). In PANC-1 and MIA CaPa-2 cells with low circSEC24A expression level, the proportion of G0/G1 phase cells was increased, and the proportion of S and G2/M phase cells was decreased (Fig. 2J).

CircSEC24A facilitates pancreatic cancer migration and invasion
To further confirm the effect of circSEC24A on migration and invasion in pancreatic cancer cells, transwell assays and wound healing assays were performed to measure the migrated and invaded ability. Wound healing assays illustrated that the migrated ability of silencing circSEC24A expression groups was constraint compared with control group (Fig. 3A-D). Transwell assays further verified that the cell migration and invasion was also constraint with or without Matrigel in silencing circSEC24A expression groups (Fig. 3E-H). Subsequently, we performed western blot to measure the protein markers of epithelial to mesenchymal transition (EMT), including Twist1, Snail1 and ZEB2. Interestingly, knockdown expression of circ-SEC24A obviously inhibited the protein levels of Twist1, Snail1 and ZEB2, suggesting circSEC24A had the ability to promote EMT transition (Fig. 3I).
To further confirm whether circSEC24A could exert as a sponge for miR-606, dual luciferase reporter assay was performed to measure the binding between circSEC24A and miR-606. The prediction binding site was displayed in Fig. 4D. Dual luciferase reporter assay suggested that miR-606 overexpressed significantly inhibited wild type circSEC24A luciferase reporter activity. However, mutant type circSEC24A luciferase reporter activity seemed to change little (Fig. 4E). PCR analysis indicated silencing circSEC24A expression dramatically increased expression of miR-606 (Fig. 4F). Additionally, we detected miR-606 expression in pancreatic cancer tissues and adjacent normal tissues by using PCR analysis, revealing that miR-606 was low expression in tumor tissues (Fig. 4G).
Pearson correlation analysis showed that circSEC24A was negatively correlated with miR-606 (Fig. 4H). Subsequently, rescuing experiments were performed to further evaluate the effect of miR-606 on proliferation, migration and invasion in pancreatic cancer cells. The results illustrated that inhibited expression of miR-606 in silencing circSEC24A groups resulted in accelerating effect on proliferation and migration, partly reversing the silencing circSEC24A expression mediated inhibitory function ( Fig. 4I-O).

TGFBR2 is target of miR-606 and promotes pancreatic cancer cell proliferation and migration
Previous prediction results indicated miR-606 had three potential targets (DUSP16, TGFBR2 and CORO7) acquired by Venn analysis from miRDB, miRTarbase and Targetscan database. Firstly, PCR analysis was performed to measure the expression changes of these three potential targets in miR-606 overexpression groups and control groups, respectively. The results indicated that only TGFBR2 was downregulated in miR-606 overexpression group, suggesting that TGFBR2 might be a downstream target of miR-606 (Fig. 5A). Subsequently, we utilized dual luciferase reporter assay to further verify the interaction between miR-606 and TGFBR2. The wild type and mutant type sequences of TGFBR2 were shown in Fig. 5B. Dual luciferase reporter assay illustrated that miR-606 overexpression dramatically decreased the luciferase activity in wild type TGFBR2 but not changes in mutant type TGFBR2, suggesting that miR-606 could directly bind to the 3'UTR of TGFBR2 mRNA (Fig. 5C).
In addition, PCR analysis and western blot both confirmed that upregulated or downregulated miR-606 expression resulted in inhibitory or facilitating TGFBR2 expression in mRNA levels or protein levels, respectively (Fig. 5D, E). Gain-function experiments were performed to evaluate the effect of TGFBR2 on miR-606 mediated inhibitory function on proliferation, migration and invasion. As expected, the increasing TGFBR2 expression in miR-606 overexpression groups could partly rescue the miR-606 overexpression mediated inhibitory function on proliferation, migration and invasion ( Fig. 5F-L). These results further confirmed that TGFBR2 was the target of miR-606.

CircSEC24A promotes TGFBR2 expression via sponging to miR-606 and activates AKT signaling
To further explore the underlying molecular mechanism how circSEC24A promotes malignant phenotype, we utilized the top 200 circSEC24A-related genes for GO and KEGG enrichment analysis via R software using "clusterProfilerGO" and "clusterProfilerKEGG" packages. Interestingly, GO enrichment analysis illustrated that circSEC24A was significantly associated with cell adhesion, receptor-mediated endocytosis, actin filament binding and MAPK phosphatase activity, which all supported for cell proliferation, metastasis and signaling transduction (Fig. 6A). KEGG enrichment analysis also illustrated that circSEC24A was obviously correlated with PI3K-AKT signaling pathway, endocytosis and cell cycle et al. (Fig. 6B). Subsequently, we performed western blot assay to confirm the enrichment results. First, we detected expression of TGFBR2 in silencing circSEC24A expression groups, indicating TGFBR2 was significantly downregulated in silencing groups (Fig. 6C). Meanwhile, total AKT and phosphorylated AKT were also downregulated in silencing circSEC4A expression groups (Fig. 6C). Additionally, we added TGFBR2 overexpressed vectors or miR-606 inhibitors to circSEC4A silencing group and measured these protein expression levels. The results indicated that silencing circSEC4A expression led to downregulation of these protein, however, TGFBR2 overexpression or miR-606 downregulation could partly increase the protein expression of circSEC24A mediated inhibitory effect (Fig. 6D). These results suggested circSEC24A could activate AKT signaling and at least these effects partly depended on circSEC24A sponging to miR-606 to modulate TGFBR2 expression.

CircSEC24A promotes the growth of pancreatic cancer cell in vivo
To further investigate the regulatory effect of circSEC24A on tumor growth in vivo. We firstly constructed the xenograft tumor model in nude mice with or without subcutaneous injection of the treated cells. Next, to compare the proliferation rate of circSEC24A silenced group with control group, the volume of tumors developed in all nude mice was measured and recorded weekly. After 4 weeks, the tumor was removed and weighed. Our data showed that tumor weight in the circSEC24A silenced group was significantly lower than that in the control group ( Fig. 7A-C). HE staining showed large and hyperchromatic nuclei in the control group compared with the circSEC24A knockdown group. The control cells were significantly more malignant than circSEC24A knockdown group. Furthermore, IHC staining demonstrated that knockdown of circSEC24A remarkably reduced the expression of Ki-67, PCNA and TGFBR2 in xenograft tumor tissues (Fig. 7D). Taken together, these results suggest that circSEC24A can promote the expression of TGFBR2 through miRNA sponge effect, thereby enhancing the tumorigenicity of pancreatic cancer (Fig. 7E).

Discussion
Pancreatic cancer is one of digestive cancers with the highest mortality rate [14]. Most cases of pancreatic cancer are generally diagnosed in advanced stages, with poor treatment outcomes and poor prognosis [15]. Emerging evidence has confirmed that circRNAs participated in irreplaceable regulatory functions involved in multiple biological processes, especially in the occurrence, development and metastasis of cancers [16]. CircRNAs have potential to become biomarkers for cancer treatment and diagnosis. Compared with other non-coding RNAs, circRNAs show their unique characteristics, relatively stable and specific expression in cells and tissues [17]. Different circRNAa may function as tumor suppressors or promotors in different cancers. Han et al. reported that circ_0071036 was significantly correlated with unfavorable characteristics and prognosis in pancreatic cancer, and highly expressed circ_0071036 facilitated tumourigenesis [18]. In addition, circBFAR accelerated cell proliferation and invasion of pancreatic cancer via upregulating mesenchymal-epithelial transition factor in pancreatic cancer [19]. Conversely, circNFIB1 suppressed lymphangiogenesis and lymphatic metastasis through inhibiting the PI3K/AKT pathway in pancreatic cancer [20].
In this study, we identified an unreported circular RNA in pancreatic cancer, circSEC24A, with 252 nucleotides in length, which was derived from back-splicing the SEC24A mRNA. Previous studies on SEC24A showed that it exerted as an essential mediator to promote endoplasmic reticulum (ER) stress-induced cell death  [21]. Moreover, phosphorylated and stabilized SEC23B interacted with SEC24A and localized to the ER-Golgi intermediate compartment to control autophagy [22]. However, circSEC24A lacked the capacity to encode protein and was reported to function as an oncogene in cutaneous squamous cell carcinoma. The underlying molecular mechanism was that circSEC24A competitively binding miR-1193 to regulate MAP3K9 [23].
In addition, circSEC24A also induced apoptosis and inflammation in chondrocytes [24]. In our data, we found circSEC24A was dramatically high expression in pancreatic cancer tissues and cells. In addition, overexpressed circSEC24A promoted pancreatic cell proliferation, migration and invasion. Subsequently, we utilized bioinformatic method combined with experiments, revealing that circSEC24A exerted as a sponge for miR-606 to modulate TGFBR2, which further activated AKT signaling pathway. These results suggested that circSEC24A Fig. 6 CircSEC24A promotes TGFBR2 expression via sponging to miR-606 and activates AKT signaling. A Go enrichment analysis was performed to explore the potential function on cellular component (CC), molecular function (MF) and biological process (BP), respectively. B KEGG enrichment analysis was performed to explore the potential signaling pathway correlated with CircSEC24A. C Western blot was performed to confirm the result of KEGG enrichment (PI3K/AKT signaling pathway). D Western blot was performed to further evaluate the key protein expression levels of AKT signaling pathway. Then miR-606 inhibitor or TGFBR2 vector was respectively cultured with si-circSEC24A pancreatic cancer cells. The signaling pathway markers was measured by western blot served as potential oncogene to facilitate pancreatic cancer progression. The transcripts of CircRNAs and mRNAs have the same miRNA binding sites, they form a complex to participate in regulate post-transcriptional levels of each other by functioning as ceRNA [25]. The researches about circRNAs mainly focused on their roles in cytoplasm, where circRNAs exerted as sponges for miRNAs, interacted with RNA binding protein to participate in regulatory function in protein translation [26]. Accumulated evidence has reported that most of circRNAs contained MREs and ceRNA mechanism was the prominent route to exert their regulatory functions in biological processes [27]. For example, silencing circFOXK2 significantly inhibited pancreatic cancer progression and elevated miR-942 expression, eventually indirectly suppressed expression of PAX6 and GDNF [28].
TGFBR2, namely TGF-β type II receptor, belongs to a member of the TGF-β signaling, which is involved in tumorigenesis and tumor procession. The TGFBR2 signals participate in cell growth, differentiation, angiogenesis and metastasis [29]. In addition, TGFBR2 binds with ligand and interacts with TGFBR1 to construct a hetero-tetrameric complex, activating smad2 and smad3 to interact with smad4. Eventually, the smad members translocate into nucleus to associate with transcription factors to regulate target gene expression [30]. Moreover, TGF-β signals exist crosstalk with AKT signaling pathway, thereby activated TGF-β could exert promoting function of cancer. Moreover, Previous study has reported TGFBR2 inhibitor as a potent drug for pancreatic cancer treatment, the TGFBR2 inhibitor, Galunisertib significantly improved overall survival in patients with unresectable pancreatic cancer [31]. Our study revealed that circSEC24A could regulate the progression of pancreatic cancer in a TGFBR2-dependent way. However, there are several limitations to our results. Firstly, even if our study confirms the ability of circSEC24A to bind miR-606, it cannot be ruled out that other miRNAs may also bind circSEC24A to regulate the progression of pancreatic cancer. In addition, whether circSEC24A regulates the progression of pancreatic cancer through other mechanisms, such as its interaction with RNA binding proteins, needs further study. Therefore, it is necessary to further understand the therapeutic potential of circ-SEC24A in pancreatic cancer.