50 SD rats with a body weight of (200 ± 20) g were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. After free feeding for one week, they were used to build a model of pancreatic cancer. The models, mainly based on previous research, were also known as DMBA induction method [14–16]. The methods were as follows: the rats were anesthetized using pentobarbital sodium, and their abdomen was depilated and disinfected. A 1–2 cm incision was made in the abdomen to expose the pancreas. DMBA (9 mg) was implanted between pancreatic tissues and parenchyma, and then the pancreatic parenchyma and abdominal incision were sutured in order to prevent infection. 4 weeks later, the rats were sacrificed, and tissues were collected to determine whether the model was successfully built.

The model rats were randomly divided into 4 groups and named as model group (Model), BA (Sigma-Aldrich) low-dose group (Low, 50 mg/kg), medium-dose group (Medium, 100 mg/kg) and high-dose group (High, 150 mg/kg), respectively. The rats were euthanized 15 days after administration, and the samples were collected for subsequent analysis.

The rats in the blank control group only had their wounds heal and did not undergo DMBA transplantation. All animal experiments were carried out under the supervision and guidance of the Animal Ethics Committee of Lanzhou University and the First Affiliated Hospital. All animals were handled according to the animal welfare guidelines and approved by the Ethics Committee of the First Hospital of Lanzhou University (LDYYLL2021.09).

After the frozen sections were fixed and sealed, Akt and STAT3 specific primary antibodies were added and incubated overnight at 4°C. Subsequently, the biotinylated secondary antibody was incubated at 37°C for 30 min. Streptomycin-labeled HRP was added and incubated for 30 min. Finally, the slices were colored and counterstained with DAB, sealed with neutral gum, and photographed under a microscope for observation and analysis.

Paraffin-embedded tissue sections were deparaffinized and hydrated, the sample was stained with hematoxylin and let stand at room temperature for 5 min. Eosin was added after washing, and the sample was then incubated for about 2 min at room temperature. After being rinsed with gradient ethanol, a neutral gel was then used for sealing. The sections were observed and photographed under a microscope. 3 experienced pathologists analyzed the results.

IL-6 assay was performed according to the instructions of the manufacturer of IL-6 kit (Beyotime, PI328). The serum to be tested and the standards with different concentrations were added to the corresponding wells and incubated for 2 h at 37°C. Biotinylated antibody and HRP-Streptavidin were then added and incubated, respectively, and TMB was added to colorate. A450 absorbance was measured.

PANC-1 cells were derived from ATCC. The cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillom-ycin. BxPC-3 cells were derived from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillom-ycin. The cells were cultured in an incubator with 5% CO₂ at 37°C.

miR-365 inhibitor was purchased from Shanghai GenePharma. Lipofectamine 3000 was adopted for transfection experiment. The cells were subcultured in a 6-well plate and cultured for 24 h. The inhibitor was mixed with the transfection reagent, placed at 37°C for 15 min, and then added to cells. After 48 h, the cells were collected and the expression level of miR-365 was tested by RT-PCR.

The cell viability was determined by MTT assay. The cells were subcultured in a 96-well plate and cultured for 24 h for different treatments. MTT solution was added and incubated for 4 h, and then DMSO was added to terminate the reaction. The absorbance was measured at 450 nm under a microplate reader.

The cells were treated with 0, 10, 20 and 30 µM BA [2,15], and the treatment lasted 24, 48 and 72 h, respectively. Each treatment was repeated for 6 times, and the concentration of BA IC50 was screened for subsequent experiment. The experiment was divided into Control group, Inhibitor-NC (miR-365 inhibitor NC) group, Inhibitor group (miR-365 inhibitor), BA group (IC50 concentration) and BA+ Inhibitor group. The cells were subjected to different treatments and the cell viability was determined 48 h later.

The invasion assay was performed in a Transwell chamber, with the upper surface of the 8-μm (pore size) membrane being coated with Matrigel. The cells undergoing different treatments were inoculated in a Transwell chamber, and a complete medium with 10% FBS was added to the outer cavity of chamber. After incubating at 37°C for 24 h, the chamber was removed and cleaned with PBS. After that, 4% paraformaldehyde was fixed at 37°C for 30 min, and crystal violet staining was performed. After excessive cells in the upper layer of chamber were removed, the number of migrated cells was observed under a microscope. 3 fields were randomly selected as replicates in each sample.

The cells were inoculated in a 6-well plate. After the adhesion and fusion of cells, different concentrations of BA (0, 10, 20, 30 µM) were added, and the cells were collected for 48 h. Annexin V-FITC was added to the suspended cells and incubated for 15 min, followed by PI for 5 min. FACS C6 was adopted to detect cell apoptosis and FlowJo was used for data analysis.

The cells undergoing different treatments were centrifuged and collected, supplemented with RIPA lysate, and placed on ice for 30 min to extract total protein. The proteins were separated by SDS-PAGE and transferred to an activated PVDF membrane.

After blocking with 5%BSA, specific primary antibodies were added, and incubated overnight at 4°C. After washing, HRP-labeled secondary antibodies were added and incubated at 37°C for 1 h. The target protein was colorated with ECL method, and the images obtained by the gel imaging system were analyzed semi-quantitatively with Image J software. The antibodies used in the experiment were BTG2 (Abcam, AB244260), IL-6 (Abcam, AB233706), p-AKT (CST, 4060S), AKT (CST, 4685S), p-STAT3 (CST, 9145S) and STAT3 (CST, 9139S), respectively.

Total RNA was extracted from the samples with Trizol. After determining the concentration and quality of RNA, cDNA was synthesized according to the instructions of reverse transcription kit (QIAGEN, 205411), TaKaRa’s commercial instructions. Taking 2 µL cDNA product as the template, the relative expression levels of target genes were analyzed by QuantiNova SYBR Green PCR Kit (QIAGEN, 208054). The primer sequences are shown in Table 1.

miRNA-specific TaqMan miRNA assay Kit (Thermo Fisher Scientific Waltham, MA, USA) was used to analyze the expression level of miRNA. U6 was used for gene normalization. The relative expression value was expressed by the value of 2^−∆∆Ct.

SPSS 20.0 and GraphPad Prism 7.0 were adopted for statistical analysis and figure rendering. All data were expressed as Mean ± SD. The comparison between multiple groups was done with ANOVA and the comparison between two groups was done with t-test. p < 0.05 indicated the difference was statistically significant.

We investigated the effect of BA on the biological behavior of pancreatic cancer cells, so as to better understand the role of BA in pancreatic cancer. PANC-1 and BXPC-3 cells were selected as pancreatic cancer cell lines. First of all, the MTT assay was used to assess changes in the proliferation ability of pancreatic cancer cells after BA treatment. BA had a concentration-dependent inhibitory effect on the proliferation of pancreatic cancer cells, as shown in Fig. 1A, and with the increase of concentration, cell proliferation was inhibited at different time points. Transwell experiments were then carried out to examine the changes in the migration ability of pancreatic cancer cells after BA treatment. As shown in Fig. 1B, as the concentration of BA increased, pancreatic cancer cells’ ability to migrate weakened. Finally, flow cytometry was adopted to investigate the effect of BA on the apoptosis of pancreatic cancer cells. The degree of apoptosis of pancreatic cancer cells increased with the increase of the concentration of BA, as shown in Fig. 1C.

In an effort to study the mechanism of the effect of BA on pancreatic cancer, a rat model of pancreatic cancer was built and BA with different concentrations, that is, low, medium and high, was used. H&E analysis showed that in the model group, the tumor cells were densely arranged, inflammatory cells were infiltrated, and vascular hyperplasia occurred. After BA treatment, the number of cancer cells decreased, which was negatively correlated with the dose of BA (Fig. 2A). Following that, we discovered that miR-365 was overexpressed in the pancreatic cancer model group, compared with the control group. BA significantly inhibited the level of miR-365 in a dose-dependent manner when compared with the model group (Fig. 2B). Then through the Targetscan database, we discovered that miR-365a-3p and BTG2 had binding sites (Fig. 2C). On this basis, we evaluated the changes in the level of BTG2 mRNA in the BA-mediated pancreatic cancer animal model. The expression of BTG2 mRNA in the model group was lower than that in the blank group, and BA can up-regulate the level of BTG2 mRNA in tumor tissues, compared with the model group (Fig. 2D). Subsequently, the model group and the BA group were compared in terms of the expression levels of IL-6, AKT, and STAT3. As expected, the secretion level of IL-6 was significantly reduced in the BA high level group, and the immunohistochemical results indicated that the proteins of AKT and STAT3 were inhibited by BA (Figs. 2E and 2F). In this regard, we preliminarily speculated that BA may inhibit the expression of IL-6/AKT/STAT3 by mediating the expression of miR-365/BTG2.

According to two previous cell MTT experiments, the 48-h IC50 of BA in PANC-1 was 27.57 and 22.47 μM in BxPC-3. Therefore, the following experiment selected the condition that BA was 28 μM in PANC-1 and 23 μM in BxPC-3. To investigate whether BA played a role in pancreatic cancer by mediating miR-365, we introduced the inhibitor of miR-365 into the experiment to verify whether the regulatory effect of BA on miR-365 was similar to that of the inhibitors. As shown in the figure, the inhibitor significantly down-regulated the level of miR-365, which was consistent with the effect of BA (Figs. 3A and 3B). At the same time, both BA and inhibitor had an inhibitory effect on the proliferation and invasion ability of pancreatic cancer cells, and the combined effect of the two further weakened the proliferation and invasion ability of cancer cells (Figs. 3C and 3D).

To further investigate the mechanism of the inhibition of progression of pancreatic cancer by miR-365/BTG2 mediated by BA, we first measured the expression of BA-mediated miR-365/BTG2 in two pancreatic cancer cell lines. We discovered that both BA and miR-365 inhibitors can promote BTG2 expression (Figs. 4A–4C), which was consistent with previous studies. Following that, we measured IL-6/AKT/STAT3 expression and discovered that BA and miR-365 inhibitors can inhibit IL-6/AKT/STAT3 expression and AKT/STAT3 phosphorylation (Figs. 4A–4C). We concluded from the above experimental results that BA ultimately inhibited progression of pancreatic cancer by mediating miR-365/BTG2 and then inhibiting IL-6/AKT/STAT3 expression.