Hsa_circ_0050102 regulates the pancreatic cancer development via miR‐218‐5p/PPME1 axis

Abstract Background Pancreatic cancer (PC) is a malignancy worldwide. Circular RNAs (circRNAs) affects the growth of PC, nonetheless the mechanism is blurry. Here, we reconnoitered the parts of hsa_circ_0050102 in PC. Methods Hsa_circ_0050102, microRNA‐218‐5p (miR‐218‐5p) and protein phosphatase methylesterase 1 (PPME1) abundances were indicated by quantitative RT‐PCR or Western blot. Moreover, the cell functions were uncovered. Additionally, the relation of miR‐218‐5p and hsa_circ_0050102 or PPME1 was identified by dual‐luciferase reporter assay. Ultimately, the mice teats were utilized to quantity the part of hsa_circ_0050102. Results Hsa_circ_0050102 and PPME1 contents were increased, and the miR‐218‐5p was dwindled in PC. Hsa_circ_0050102 lack subdued cell vitality, colony formation, cell migration and invasion, and angiogenesis, but endorsed cell apoptosis in PC cells. Furthermore, miR‐218‐5p was established to block the development of PC cells via PPME1. Hsa_circ_0050102 bound to miR‐218‐5p to adjust the content of PPME1. Conclusion Hsa_circ_0050102 expedited the expansion of PC through growing PPME1 abundance by adjusting miR‐218‐5p.

progression of PC. 23 In addition, PPME1 promoted choriocarcinoma cell invasion. 24 However, the behaviour of PPME1 in PC is still blurry.
Here, we revealed the utility of hsa_circ_0050102 in PC cells.
Hsa_circ_0050102 might expedite PC by binging miR-218-5p and growing the PPME1 content. Our conclusions may be an innovative perception for PC.

| Cell lines
The human PC cell lines (SW1990 and Capan-1) with HPDE as control. HUVEC cells were selected for the tube formation assay. All cells were gathered from Cell Bank, Chinese Academy of Sciences (CAS, Shanghai, China) and cultured with 5% CO 2 .

| Quantitative RT-PCR and RNase R assay
The tangible method of RNA abstraction and qRT-PCR were performed conforming to the erstwhile commentary. 25 The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were engaged as controls. The gene contents were estimated by 2 −ΔΔCt mode. As well, the RNase R (Solarbio) was enforced to estimate the structure of hsa_circ_0050102. The primers as Table 1.
The absorbance at 450 nm was measured.

| Colony formation assay
SW1990 and Capan-1 (1 × 10 6 ) cells were plated in 6-well plates and then preserved for 10 days. Then, colony was stained and photographed under a microscope. The same method was enforced to detect the invasion ability, but the chamber with matrigel (BD Biosciences). Eventually, a light microscope was performed to validate the count of cells.

| Matrigel tube formation assay
HUVEC cells were implanted into 96-well plat at 4 × 10 4 cells/well in Matrigel-coated wells. Meanwhile, the tube formation rate was examined after 24 h. Afterwards, ImageJ software (NIH, Bethesda) was utilized to observe the number of tubes and the count of branches.
The elongated multi-cellular structures were considered tube-like structures. The intersecting points of two or more tubes were considered branches.

| Dual-luciferase reporter assay
The binding sites between miR-218-5p and hsa_circ_0050102 or PPME1 were foretold by starbase and targetscan. At that time, the hsa_circ_0050102 and PPME1 wild and mutant were produced by . Finally, the luciferase activity was scrutinized.

| RNA Immunoprecipitation (RIP) assay
SW1990 and Capan-1 were engaged with a RIP kit (Solarbio) in accordance with the guide to implement RIP assay. Succeeding, the abundances of miR-218-5p, hsa_circ_0050102 and PPME1 were illustrated.

| Xenograft models
The test was noticed by the Animal Care and Use Committee of Fourth Hospital of Hebei Medical University. All mice were acquired from Shanghai Laboratory Animal Company (SLAC). Capan-1 cells (5 × 10 6 ) with sh-hsa_circ_0050102 or sh-NC were vaccinated into mice (n = 6/group; female; 6 weeks; 18-22 g). To end, tumour volume = length × width 2 × 0.5. After 4 weeks, the tumours were carved for extra test.

| IHC assay
The Ki67 (ab92742; 1:1,000; Abcam) abundance in tumour were evaluated by IHC assay. The exhaustive mode is in the company of the explanation of Ma et al. 27 Eventually, the tumour slides were photographed.

| Statistical assay
The statistics were collected from no less than 3 recurrences.
Student's t test and ANOVA were employed in SPSS 17.0 to evaluate the difference. p < 0.05 was significant.

| Hsa_circ_0050102 was enhanced in PC
The abundance of hsa_circ_0050102 in PC tissues was augmented ( Figure 1A). And, high hsa_circ_0050102 was frequently observed in PC patients with the advanced clinical stage ( Figure 1B) and lymph node metastasis ( Figure 1C). Additionally, hsa_circ_0050102 was impelled in PC cell lines (SW1990 and Capan-1) versus HPDE cells ( Figure 1D).
As exposed in Figure 1E,F, the abundance of PGPEP1 mRNA was significantly abridged after RNase R treatment, but hsa_circ_0050102 was not altered. The consequence illustrated the cyclical construction

| Hsa_circ_0050102 lack promoted cell apoptosis, while restrained cell proliferation, cell migration and invasion and angiogenesis in PC cells
The hsa_circ_0050102 content was delimited in SW1990 and Capan-1 cells by si-hsa_circ_0050102 (Figure 2A,B). The hsa_ circ_0050102 lack diminished the cell proliferation ( Figure 2C-E).
Furthermore, anti-miR-218-5p reserved the influences of hsa_ circ_0050102 lack on augmented the abundance of Bax and decreased the contents of PCNA and Bcl-2 in SW1990 and Capan-1 cells ( Figure 4H,I).

| Hsa_circ_0050102 lack hampered tumour growth in vivo
As revealed in Figure 8A-C, sh-hsa_circ_0050102 dramatically inhibited tumour volume and weight. Additionally, the abundances of hsa_circ_0050102 and PPME1 were abridged, while the miR-218-5p content was enlarged in the sh-hsa_circ_0050102 group ( Figure 8D,E). The content of Ki67 was lesser in the sh-hsa_ circ_0050102 group, which signposted that hsa_circ_0050102's lack subdued tumour growth ( Figure 8F). These upshots approved that hsa_circ_0050102 lack repressed xenograft tumour growth via miR-218-5p/PPME1 axis in vivo.

| DISCUSS ION
Pancreatic cancer has a very high mortality rate and a very poor prognosis. 1 There is no valid screening method for PC, so primary prevention of the disease is crucial. The best prevention strategy  In brief, hsa_circ_0050102 and PPME1 were upregulated and miR-218-5p was downregulated in PC. Additionally, our article firstly established that hsa_circ_0050102 lack promoted cell apoptosis, but blocked PC cells growth via miR-218-5p/PPME1 axis. This mechanism might be extra verified by clinical tests someday. We had faith in that this data provided a new mode for the improvement of PC.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

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