Circ_0075829 facilitates the progression of pancreatic carcinoma by sponging miR‐1287‐5p and activating LAMTOR3 signalling

Abstract Pancreatic cancer (PC) is a leading cause of cancer‐related mortality globally. Though increasing evidence has demonstrated that circular RNAs (circRNAs) are linked to the development and progression of cancers, the biological functions of circRNAs in PC remain largely unexplored so far. Based on previous studies, Hsc_circ_0075829 (circ_0075829) was screened out and then further identified in PC clinical specimens and cell lines by real‐time PCR. After the stability tests, a series of in vitro and in vivo functional experiments were performed to investigate the role of circ_0075829 in PC development. Furthermore, fluorescent in situ hybridization (FISH), bioinformatics tools, dual‐luciferase assays and rescue experiments were conducted to clarify the regulatory mechanisms of circ_0075829 in SW1990 and BxPC‐3 cells. Compared with paracancerous tissues, the expression of circ_0075829 was increased in PC tissues, which was positively correlated with the clinical features of PC. Knockdown of circ_0075829 significantly suppressed the proliferative, migratory and invasive rates of SW1990 and BxPC‐3 cells both in vitro and in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assay indicated that circ_0075829 could bind to miR‐1287‐5p. Mechanism research and rescue experiments demonstrated that circ_0075829 could regulate the LAMTOR3/p‐ERK signalling pathway via sponging miR‐1287‐5p in PC cell lines. Our data reveal that the circ_0075829 could facilitate the proliferation and metastasis of PC through circ_0075829/miR‐1287‐5p/LAMTOR3 axis.


| BACKG ROU N D
Pancreatic cancer (PC) is the fourth cause of cancer-associated mortality with a lowest overall 5-year survival rate (<9%) globally. 1 So far, there have been no clinically sensitive and effective screening services for early-stage PC. The reality is that more than 80% newly diagnosed patients have already developed locally advanced or metastatic tumours, and surgery is still the first option for treatment. 2 Despite the significant advances in surgery, chemotherapy and radiotherapy, the therapeutic effects are not ideal for patients due to the chemosensitivity and complex genetic mutations of PC. 3 Therefore, it is necessary to understand the key molecular mechanisms of this disease and develop new treatment strategies.
Lysosomal adaptor, MAPK and MTOR activator 3 (LAMTOR3, also known as MAPKSP1 or MP1), initially identified as a MEK1/ERK1 scaffolding protein, can regulate ERK1 activation. 4 Now, LAMTOR3 was recognized as a crucial link in MEK-ERK interaction and hyperphosphorylation of MEK and ERK. Recent research has demonstrated that LAMTOR3 could activate MAPK and mTOR signalling to improve pancreatic tumorigenesis, 5,6 making it an important regulatory site in PC.
Circular RNAs (circRNAs), a special type of non-coding RNAs, are formed by a covalently closed cyclic structure lacking 5′ cap or a 3′ poly A tail. Initially considered as the by-products of splicing, 7 cir-cRNAs have been found to play a series of important biological roles such as competing endogenous RNAs (ceRNAs) via sponging microR-NAs (miRNAs), protein/peptide transcripts, gene transcription and RNA splicing factors. 8 In recent years, emerging evidence shows that dysregulated circRNAs could disturb the balance of ceRNA network, and further promote the tumorigenesis of different cancers, including PC. 9 For example, circ_0061825 facilitates breast cancer progression through targeting miR-326 and the signal path of its host gene TFF1. 10 And, circ-ASH2L is proved to facilitate tumour progression by sponging miR-34a and affecting the expression of Notch1 in PC. 11 Meanwhile, some circRNAs may serve as potential biomarkers for cancer diagnosis. 12 These results indicate that circRNAs are a new category of potential biomarkers or therapeutic targets for PC. Elevated circ_0075829 (Hsa_circ_0075829) has been identified in PC tissues according to circRNA microarray data. 13,14 In the present study, we further confirmed the clinical relevance of circ_0075829 expression in PC and discovered that circ_0075829 could promote PC cell proliferation and metastases as a ceRNA to sponge miR-1287-5p targeting LAMTOR3. Particularly, the regulation of miR-1287-5p/ LAMTOR3 by circ-0075829 has never been demonstrated before. This article further reveals one regulatory network mechanism of circRNAs in PC, and broadens the horizon for early diagnosis of PC.

| Real-time PCR
Total RNA extracted from tissues and cells was prepared by using RNA-Quick Purification Kit (ES Science) according to the manufacturer's instructions. Extracted RNA was used for reverse transcription to obtain cDNA (Vazyme, USA) (reverse transcription of circRNAs used random primers), followed by RT-PCR analysis through SYBR Premix Ex Taq™ Kit (Takara). The relative circRNA or mRNA expression level was normalized with β-actin. U6 was used as internal reference for miRNAs. The microRNA measure kit for miR-1287-5p was purchased from Takara (Mir-X miRNA qRT-PCR TB Green Kit), and the level of miRNA was tested according to the manufacturer's instructions. The 2 -ΔΔCt method was applied to evaluate the expression of target gene. The sequences of primers used in this study are listed in Table S1.

| RNase R treatment
Total RNA (2 μg) after being extracted by TRIzol reagent was incubated with 10 U RNase R (Epicentre Technologies, USA) for 20 minutes at 37°C, followed by 70°C for 5 minutes to deactivate the RNase R. After enzyme inactivation, reverse transcription reaction and RT-PCR can be performed. For controls, the RNA was mocktreated without the enzyme.
After treatment, cells were collected and tested for circ_0075829 and β-actin expression through RT-PCR.
All shRNA and miRNA inhibitor sequences are presented in Table S2.
Transfection was conducted according to the manufacturer's instructions.

| RNA fluorescence in situ hybridization (FISH)
To detect the subcellular location of circ_0075829, FISH was performed with the fluorescent in situ hybridization kit, according to the manufacturer's protocol (RiboBio Biotechnology). The nucleus was counterstained with DAPI. Cy3-labelled circ_0075829 probes and positive control RNA probes were incubated. The images were obtained with a confocal microscope (Olympus).
After incubation for different times (24, 48 or 72 hours), the cells were treated with CCK-8 reagent for 2 hours. The absorbance of each well (450 nm) was measured in microplate reader. As EdU assay, cells were seeded into 96-well plates (1-5 × 10 4 per well) and then cultured overnight. After 4 hours of incubation in EdU medium, the cells were disposed according to the manufacturer's instructions, and the number of EdU staining cells was counted under the fluorescence microscopy. For colony formation, cells were counted and plated in 6-well plates (400-800 per well) and cultured in medium with 10% foetal bovine serum. After 2-3 weeks, colonies were stained with 0.5% crystal violet. The number of colonies was integrated with ImageJ software to assess cell proliferation.

| Wound healing experiment
The cells in active growth phase were seeded in a 6-well plate.
When the cells reached 80% to 90% fusion, a wound was made with a small sterile pipette, and cells were washed lightly with PBS. The scratch width was observed and recorded. After that, the cells were cultured with 2.5% FBS (minimizing the effects of cell proliferation) for 24 hours, the condition of scratch healing was observed and recorded again.

| Western blot
Cells were lysed in RIPA lysis buffer with PMSF and protease inhibitor cocktail, the protein concentration was analysed by a bicinchoninic acid kit (Beyotime). Equal amounts of protein were loaded on SDS-PAGE and transferred to PVDF membrane (Millipore). The membranes were blocked with 5% BSA (Beyotime) and incubated

| Immunohistochemistry (IHC)
For tissue immunohistochemistry, paraffin-embedded tissues were prepared as stated above, and primary antibody dilutions were as

| Statistical analysis
The statistical analyses were performed with Prism 7 software (GraphPad Software, USA). The results in the bar graphs were presented as mean ± SD. The P-values were calculated by Student's t test or ANOVA (analysis of variance), and statistical difference was defined as P < .05.

| Circ_0075829 was significantly up-regulated in PC tissues and cell lines
According to the results of the microarray assay and GEO database in other research, the expression of circ_0075829 was found significantly increased (fold > 2.5, P < .001) in PC tissues compared with that in paratumour tissues. Circ_0075829 is derived from exons 7-8 of the CASC15 gene (chr6:22020567 to 22056919) ( Figure 1A).
We used 38 pairs of PC and paracancer specimens to examine the expression of circ_0075829. RT-PCR analysis showed that the expression of circ_0075829 was up-regulated in PC tumour tissues compared with the adjacent non-cancerous tissues ( Figure 1B).
Then, we grouped patients based on their circ_0075829 expression and explored the relevance between the clinicopathological characteristics and increased circ_0075829 expression. The details are summarized in Table 1. Circ_0075829 expression was associated with tumour size (P = .017) and regional lymphatic metastasis (P = .013). No significant correlation was found between circ_0075829 expression and the patient's age, gender, As circRNAs are characterized by the covalently linked ends, we extracted total RNA from SW1990 and BxPC-3 cells respectively using RNase R to identify the circular structure of circ_0075829.
The β-actin mRNA expression markedly reduced whereas the circ_0075829 was not, which proved circ_0075829 was resistant to RNase R treatment ( Figure 1D,E). We also discovered that unlike linear mRNA, the expression of circ_0075829 was not reduced time-dependently by actinomycin D treatment in SW1990 and BxPC-3 cells (Figure 1F,G).

| Circ_0075829 bound directly to miR-1287-5p in PC cells
As for subcellular localization, FISH results showed that most fluorescence signals of circ_0075829 were located in the cytoplasm of SW1990 and BxPC-3 cells (Figure 3A,B). Previous evidence suggests that circRNAs in the cytoplasm have the potential of sponging miRNAs to regulate gene expression. Two web databases, CircInteractome (https://circi ntera ctome.nia.nih.gov) 15 and mi-Randa (http://www.micro rna.org/micro rna/home.do), were used to predict the specific miRNAs interacting with circ_0075829. The top 3 intersectional miRNAs, miR-1287-5p, miR-576-3p and miR-326, were identified by RT-PCR ( Figure S1B,C). Then, miR-1287-5p was selected among the candidate miRNAs, for its low expression in PC and the negative correlation with the circ_0075829 expression as depicted by Spearman's correlation curve ( Figure 3C,D). miR-1287-5p was highly expressed in both SW1990 and BxPC-3 cells following the down-regulation of circ_0075829 ( Figure 3E). Thus, we suggested that miR-1287-5p was most likely the miRNA sponged by circ_0075829.

| Circ_0075829 regulated LAMTOR3 expression by sponging miR-1287-5p
As miR-1287-5p has been proved to regulate tumour progression in multiple cancers, 16 we investigated whether circ_0075829 could promote PC development via miR-1287-5p. Through colony formation ( Figure 4A,B), EdU ( Figure 4C,D), wound healing ( Figure 4E,F) and transwell assays ( Figure 4G,H), we found the Sh-circ_0075829induced suppression of tumour invasion and proliferation could be blocked by miR-1287-5p inhibitor in both SW1990 and BxPC-3 cells, and the same trend was found in proteins related to proliferation and metastasis, such as cyclin D, E-cadherin and N-cadherin ( Figure 4I).
Utilizing four bioinformatics tools including TargetScan Figure 5A, Figure S1C). Among them, LAMTOR3 was of particular interest for its high expression in PC and roles regulated by miR-1287-5p inhibitor ( Figure 5B, Figure S1D). We found the miR-1287-5p levels in PC tissue specimens were negatively associated with the expression of LAMTOR3, a gene involved in the MAPK signalling pathway ( Figure 5C). Therefore, we speculated that circ_0075829 might regulate LAMTOR3 expression via miR-1287-5p. RT-PCR illustrated that the miR-1287-5p inhibitor could increase the LAMTOR3 mRNA expression, whereas the co-transfection of the miR-1287-5p inhibitor and circ_0075829 shRNA could rescue the LAMTOR3 expression both in SW1990 and in BxPC-3 cells ( Figure 5D). A dual-luciferase reporter assay confirmed that LAMTOR3 was a downstream target of miR-1287-5p in 293T cells ( Figure S1E, Figure 5E). Western blot assay results showed the same tendency of LAMTOR3 and downstream protein factors such as p-ERK and p-AKT ( Figure 5F). These findings illustrated that circ_0075829 could participate in PC development by activating LAMTOR3 via miR-1287-5p.

| Circ_0075829 promoted tumorigenicity and metastasis in vivo
The  Figure 6D, Figure S1F). Furthermore, for pancreatic tail injection with SW1990, gross specimens and HE staining showed that circ_0075829 knockdown both decreased the size of in situ implantation and ameliorated liver metastasis compared with the control group ( Figure 6E, Figure S1G). Hence, all the above observations indicated that circ_0075829 promoted PC growth and metastasis in vivo. Meanwhile, exosome-delivered circRNA_0005963, as a sponge for the PKM2-targeted miR-122, was positively correlated with chemoresistance in colorectal cancer. 20 Identified through circRNA pulldown and mass spectrometry, circRNAs could also bind to and then sequester proteins, serving as protein decoys or scaffolds to regulate gene expression. 21 For example, circFOXK2 was found to complete F I G U R E 4 The oncogenic role of circ_0075829 was partly based on its regulation of miR-1287-5p. A, B, The cell vitality of NC-, Sh-circ_0075829 + inhibitor NC-, miR-1287-5p-inhibitor-and Sh-circ_0075829 + miR-1287-5p-inhibitor-treated SW1990 and BxPC-3 cells measured by colony formation assay. C, D, Cell proliferation of SW1990 and BxPC-3 cells treated as indicated tested by EdU assay. E, F, Cell migration abilities of the above cells measured by wound healing assay. G, H, Cell invasion abilities of the above cells detected by transwell assay. I, Western blot analysis of cyclin D, E-cadherin and N-cadherin proteins in the above cell lines, normalized by β-actin expression. Scale bar: 100μm. Data are from at least three independent experiments and expressed as mean ± SD (*P < .05) In the current study, by integrating two previous studies, we screened out circ_0075829, which was highly expressed in PC.

F I G U R E 3
Clinically, the overexpression of circ_0075829 was correlated with the lymphatic metastasis and tumour size of PC, which arised our interest in its role in PC development. After shRNA interference, circ_0075829 was found to promote PC cell proliferation, migration and invasion. Considering that most circRNAs in the cytoplasm were functioning as miRNA sponges, we used bioinformatics analyses to predict the potential RNAs that might bind to the 3′UTR of circ_0075829, and proved circ_0075829 containing the binding sites for miR-1287-5p using luciferase reporter assays. Then, RT-PCR results also showed the negative correlation between the expression of circ_0075829 and miR-1287-5p in PC tissues. Therefore, we confirmed that circ_0075829 reduced miR-1287-5p expression by serving as a ceRNA. miR-1287-5p could suppress the tumour by targeting various genes in different cancers including breast, lung and cervical cancer, [24][25][26] and was also found to be a target of circRNAs. dysregulation was not only found in PC, 28 glioma, 29 breast cancer 30 and gastric cancer, 31 but also linked to proliferation, metastasis and cell differentiation. 32 We found that LAMTOR3, as a scaffold protein between MEK1 and ERK1, was the target of circ_0075829/ miR-1287-5p in PC progression, because the inhibition of miR-1287-5p could partially reverse the decline in LAMTOR3/p-ERK caused by the knockdown of circ_0075829. Combining with subsequent in vivo experiments of circ_0075829, we manifested that circ_0075829 could affect the proliferation and metastasis of PC cells by targeting miR-1287-5p to regulate LAMTOR3/p-ERK expression.
For the first time, the functions and interactions of circ_0075829 and miR-1287-5p in PC were uncovered. However, the studies of circRNAs in PC are still at initial stage. PC progression essentially depends on the crosstalk with the tumour microenvironment.
With the help of genetic mouse models of PC, further circRNA research may focus on the PC tumour microenvironment, such as macrophages and NK cells. 33 Relying only on bioinformatics analysis to further excavate downstream pathways has certain limitations. It has also been reported that single circRNA can sponge up multiple types of miRNAs. 34 Using single-cell sequencing or RNA pull-down assay, more multivariate and in-depth circRNA/miRNA regulatory mechanisms in tumour microenvironment would be discovered. Based on the high stability of circRNA, non-invasive detection in body fluids may be the future direction of their clinical transformation. 35 For the correlation between circRNA and PC survival, more long-term follow-up data and more accurate clinical feature grouping are needed. A better understanding of circRNAs is momentous for a better comprehension of PC pathological process. As LAMTOR3-guided ERK and AKT are both important downstream signal pathways of K-Ras, the oncogenic mutation is present in 90% of pancreatic neoplasms, as well as a research hot spot of drug therapeutic target. 36,37 Research into the convergent roles of circRNAs in K-Ras signalling pathway might propose a new insight into PC treatment. However, there is still a long way to go before the clinical application of circRNAs.

| CON CLUS ION
Taken together, this study unveiled the critical function of circ_0075829 in PC progression. CircRNA_0075829/miRNA-1287-5p/LAMTOR3 regulates PC tumorigenesis. Therefore, our data suggest the considering circRNA-based diagnostic strategies for PC.