miR-339-3p regulated acute pancreatitis induced by caerulein through targeting TNF receptor-associated factor 3 in AR42J cells

Abstract Acute pancreatitis (AP) is an inflammatory disease with high morbidity and mortality. The regulation mechanism of miRNA is involved in the production and development of various diseases, but the regulation mechanism of miRNA in AP is still not fully elucidated. The expression of miR-339-3p was detected using quantitative real-time PCR. The levels of TNF-α, IL-1β, and IL-6 were detected using enzyme-linked immunosorbent assay. Cell apoptosis was measured using flow cytometry. The protein expressions of TNF receptor-associated factor 3 (TRAF3), Bcl-2, C-caspase 3, Bax, p-p38, and p38 were measured using western blot. Luciferase reporter assay and RNA immunoprecipitation assay were applied to ensure that miR-399-3p targeted TRAF3. Caerulein promoted the expression of TNF-α, IL-1β, and IL-6, enhanced the expression of C-caspase 3 and Bax while inhibited Bcl-2 protein expression. Meanwhile, caerulein also reduced the expression of miR-339-3p and induced the expression of TRAF3 in rat pancreatic acinar cells. miR-399-3p transfection inhibited the levels of TNF-α, IL-1β, and IL-6 and C-caspase 3 and Bax protein expression as well as suppressed cell apoptosis, while increased Bcl-2 protein expression in caerulein-induced AP. TRAF3 has been verified as a target of miR-339-3p. Interestingly, the reduction of miR-399-3p inhibited the p38 pathway, which was impaired by the upregulation of TRAF3. In addition, the suppression effects of miR-339-3p on cell inflammation and apoptosis in caerulein-induced AP were reversed by enhancing TRAF3 expression. In this study, in vitro model of AP was characterized by strong inflammation and cell apoptosis. We have first demonstrated the regulatory network of miR-339-3p and TRAF3. Overexpression of miR-339-3p inhibited cell inflammation and cell apoptosis in caerulein-induced AP through modulating TRAF3 expression via the p38 pathway, providing a new therapeutic target in the treatment of AP.


Introduction
Acute pancreatitis (AP) is a sudden pancreatic inflammatory response accompanied by strong pain, mainly caused by necrosis of pancreatic acinar cells. AP is still fatal despite intensive treatment [1][2][3]. With the development of gene sequencing and biotechnology, the research on the mechanism of AP has also been greatly developed. It also provides an important theoretical basis for improving treatment methods and developing molecular therapy. However, the pathogenesis of AP has not yet been fully explained.
miRNA-related regulatory networks and mechanisms have been reported to play a crucial role in the mechanisms of diseases [4]. miRNA, as a non-coding small RNA, plays a major role in mRNA degradation or translational inhibition by binding to the mRNA 3′-UTR [5]. Various studies have shown that miRNA, as an important regulator, is involved in cellular processes in a variety of diseases, including GC, breast cancer, HCC, NCSLC, and pancreatitis [6][7][8][9]. In AP, different miRNAs have different expression patterns. In example, the high expression of miR-21 in AP promoted apoptotic activity, and the promotion of miR-22 and miR-135a may induce the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression [10,11]. Growing evidence determined that miRNAs took part in cell progression widely, including cell proliferation, migration, inflammatory response, apoptosis and necrosis, and autophagy [12][13][14].
A variety of miRNA regulatory networks interact to jointly regulate the occurrence, immune response and inflammation of AP [15][16][17][18]. In addition, miR-216a, miR-7, miR-9, miR-122, and miR-141 could act as potential markers of AP [19,20]. Interestingly, Wu et al. reported that miR-339-3p decreased cell inflammation and cell apoptosis in mice with AP through Anxa3 by activation of the Akt/mTOR signaling pathway [21]. However, the regulatory mechanism of miR-339-3p has not yet been fully discovered. Therefore, we investigated the role and regulatory mechanism of miR-339-3p in AP.
TNF receptor-associated factor 3 (TRAF3) is an important signal transducer in intracellular signaling pathways and plays a crucial role in the immune system. Similarly, TRAF3 can also be involved in cell immunity, inflammation, and growth as a target gene in the miRNA regulatory pathway. It has been shown that TRAF3 was a target mRNA of miR-29b-3p and interacted with miR-29b-3p to affect ischemia/reperfusion injury and breast cancer [22,23]. Additionally, TRAF3 was identified to be a target mRNA of miR-455, miR-322, miR-17-92, and miR-188-3p in cerebral ischemic stroke, ischemia/reperfusion injury, and gastric cancer [24][25][26][27]. However, the function of TRAF3 in AP has not been fully clarified.
In this study, we also constructed this model to observe the regulation mechanism of miR-339-3p on inflammatory response and cell necrosis in AP. Finally, we found that miR-399-3p affected cell inflammation and cell apoptosis through targeting TRAF3 via the p38 pathway in caerulein-induced AP. This paper will present a new regulatory pathway for pancreatitis and provide new therapeutic targets for the treatment of pancreatitis.

Cell culture and treatment
In this experiment, a rat pancreatic acinar cell line, AR42J, was stimulated by caerulein to construct an AP model. The rat pancreatic acinar cell line AR42J was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were cultured in F12-K medium containing 10% fetal bovine serum and 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C, 5% carbon dioxide, and 95% air and humidity in an incubator. Then, the AR42J cells were treated with 0, 25, 50, and 100 nM caerulein, and after 8 h of culture, the samples were collected and stored at −80°C. In addition, the AR42J cells were treated with 100 nM caerulein, and collected at different times, respectively, at 0, 2, 4, 6, and 8 h and then stored at −80°C for subsequent experiments.

Luciferase reporter assay
To explore whether miR-339-3p could directly target TRAF3, a luciferase reporter assay was performed. The fragments containing the wild-type and mutant-type TRAF3 were amplified from 3′-UTR full-length TRAF3 and then were inserted into the luciferase reporter vector psiCHECK-2 (Promega, Madison, WI, USA). Next, the TRAF3-wt and TRAF3-mut were co-transfected with NC and miR-339-3p into the cells using Lipofectamine 2000 reagent (Invitrogen). After transfection for 48 h, firefly and Renilla luciferase activities were analyzed using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). All transfection experiments were applied at least three times.

Enzyme-linked immunosorbent assay (ELISA)
According to the instructions, the levels of TNF-α, IL-1β, and IL-6 were detected using an Enzyme Immunoassay Kit (Boster, Inc., Wuhan, China). Briefly, when the macrophages reached 70% fusion, the cell supernatant was collected, the antigen was added and kept at 4°C overnight, and then enzyme-labeled secondary antibody (Abcam Inc., Cambridge, MA, USA) was added and incubated at room temperature for 1 h. Finally, the optical density of each sample at 450 nm was measured, and the levels of TNF-α, IL-1β, and IL-6 were calculated according to the regression equation of the standard curve.

RNA immunoprecipitation (RIP) assay
An RIP experiment was performed using a Magna RNAbinding protein immunoprecipitation kit (Millipore, Billerica, MA, USA). The cells were co-transfected with NC, miR-339-3p, or TRAF3 and then incubated with Proteinase K to isolate the immunoprecipitated RNA. After all, the cells were incubated with negative control normal mouse IgG or human anti-Ago2 antibody, RNA was extracted, and the expression of TRAF3 was detected using qRT-PCR.

Cell apoptosis
An Annexin-V-PE/7-AAD double staining apoptosis kit (BD, Franklin Lakes, NJ, USA) was applied to detect cell apoptosis according to the manufacturer's instructions. Briefly, the cells were resuspended in Annexin V-FITC binding buffer and mixed with Annexin V-FITC and propidium iodide and incubated for 30 min. Then, cell apoptosis rates were analyzed using flow cytometry.

Statistical analysis
All values were expressed as mean ± SD. All data were analyzed using SPSS software and GraphPad Prism 7.0. The statistical significance of differences between groups was determined by Student's t-test. P < 0.05 was considered as statistically significant.

Caerulein induced inflammation and apoptosis in AR42J cells
First, we determined the cellular inflammatory factor content using ELISA kits. Cell apoptosis and apoptotic factor protein expression were determined using flow cytometry and western blot, respectively. ELISA results displayed that the expression of TNF-α, IL-6, and IL-1β showed similar changes under different concentrations. As the treatment concentration of caerulein increases, their content continued to increase significantly (Figure 1a-c). Meanwhile, flow cytometry analysis showed that cell apoptosis was obviously induced by caerulein (Figure 1d). In addition, the results of western blot showed that the protein expression of Bcl-2 and Bcl-2/Bax was decreased gradually with the increase of the concentration of caerulein, while the protein expression of C-caspase 3/caspase 3 and Bax was enhanced gradually (Figure 1e). The above data indicated that caerulein induced inflammatory response and apoptosis in AR42J cells suggesting that in vitro model of AP was characterized by a strong inflammatory response and high cell apoptosis.

miR-339-3p expression was inhibited while TRAF3 expression was increased in AR42J cells induced by caerulein
Next, we constructed a model of AP by treating AR42J cells with different concentrations of caerulein. As shown in Figure 2a and c, qRT-PCR analysis showed that the expression level of miR-339-3p decreased significantly as caerulein concentration increased. In contrast, western blot analysis showed that the protein expression of TRAF3 was obviously induced with increasing caerulein concentration. In the 100 nM caerulein treatment, the expression of miR-339-3p was significantly lower than that under other concentrations, and the expression of TRAF3 was significantly higher than in other treatments, so we chose 100 nM caerulein treatment for the next experiment. Next, we found that the expression of miR-339-3p was significantly reduced with increasing the processing time, while the expression of TRAF3 was significantly promoted (Figure 2b and d). These results indicate that miR-339-3p and TRAF3 may play an important role in the pathogenesis of AP. To further explore the function of miR-339-3p in AP, miR-339-3p was transfected into 100 nM caeruleininduced AR42J cells, which increased the expression of miR-339-3p (Figure 3a). We discovered that the levels of TNF-α, IL-6, and IL-1β were lower in the 100 nM caerulein + miR-339-3p group than those in 100 nM caerulein + NC and 100 nM caerulein groups (Figures  3b-d). Additionally, as shown in Figure 3e, we found that cell apoptosis was remarkably inhibited by miR-339-3p transfection in 100 nM caerulein-induced AR42J cells. Moreover, compared with 100 nM caerulein + NC and 100 nM caerulein groups, the protein levels of Bcl-2 and Bcl-2/Bax were significantly improved while the protein levels of C-caspase 3/caspase 3 and Bax were significantly decreased in the 100 nM caerulein + miR-339-3p group (Figure 3f). These results indicated that overexpression of miR-339-3p weakened the effects of caerulein on cell inflammation and apoptosis in AR42J cells, implying that the upregulation of miR-339-3p might inhibit AP.

TRAF3 is a target of miR-339-3p
To further explore the regulatory network of miR-339-3p, we successfully found that TRAF3 is a target of miR-339-3p, and miR-339-3p targeted the TRAF3 3′-UTR (Figure 4a). We then used the luciferase reporter assay to analyze this result and constructed luciferase reporters containing the wild-type TRAF3 or mutant sites of TRAF3. As shown in Figure 4b, luciferase activity was significantly reduced when miR-339-3p was bound to TRAF3-wt, whereas the activity was not changed when miR-339-3p was bound to TRAF3-mut (Figure 4b).
Otherwise, the RIP analysis showed that the endogenous TRAF3 was particularly enriched in the cells of the miR-339-3p transfected group compared with the NC group ( Figure 4c). Furthermore, the protein expression of TRAF3 was measured in the NC group, the miR-339-3p group, the anti-NC group, and the anti-miR-399-3p group using western blot. The results showed that overexpression of miR-399-3p could inhibit the expression of TRAF3, while suppression of miR-399-3p expression could enhance the expression of TRAF3 (Figure 4d). Therefore, these results determined that TRAF3 is a target of miR-399-3p.

Promotion of TRAF3 could reverse the effects of miR-339-3p on cell inflammation and apoptosis in AR42J cells induced by caerulein
To further explore the role of the regulatory networks of TRAF3 and miR-339-3p in AP, we treated AR42J cells with 100 nM caerulein to construct an AP model, and miR-339-3p, NC, miR-399-3p + vector, and miR-339-3p + TRAF3 were transfected into these cells and divided into 100 nM caerulein + miR-339-3p, 100 nM caerulein + NC, 100 nM caerulein + miR-339-3p + vector, and 100 nM caerulein + miR-339-3p + TRAF3 groups. Western blot analysis indicated that overexpression of miR-339-3p reduced TRAF3 protein expression, but this effect was reversed by the promotion of TRAF3 in AR42J cells induced by caerulein (Figure 5a). Otherwise, we found that the levels of TNF-α, IL-6, and IL-1β were lower in the 100 nM caerulein + miR-339-3p group than in the 100 nM caerulein + NC group. Compared with the 100 nM caerulein + miR-339-3p + TRAF3 group, the levels of TNF-α, IL-6, and IL-1β were decreased significantly in the 100 nM caerulein + miR-339-3p + vector group. These data indicated that the induction of miR-339-3p inhibited inflammation in AR42J cells induced by caerulein, which was impaired by the overexpression of TRAF3 (Figure  5b-d). In addition, high expression of miR-399-3p inhibited cell apoptosis, which was impaired by upregulating TRAF3 (Figure 5e). As shown in Figure 5f, Bcl-2 and Bcl-2/Bax protein levels were induced and C-caspase 3/caspase, Bax, and RIP3 protein levels were reduced by the overexpression of miR-339-3p. However, the transfection of TRAF3 reversed its effect in AR42J cells induced by caerulein. As a result, the promotion of TRAF3 could reverse the effects of miR-399-3p on cell inflammation and apoptosis in caerulein-induced AR42J cells.

Discussion
AP is a common gastrointestinal disease with high morbidity and mortality, and its symptoms are mainly pancreatic necrosis and the inflammatory response. TNF-α, IL-6, and IL-1β are important pro-inflammatory factors. They activate the secretion of macrophages and T-cells in inflammatory signaling and accumulate leukocytes, which are key indicators of inflammatory response [28][29][30]. Bcl-2, Ccaspase 3, and Bax proteins are important indicators of cell apoptosis in diseases. Bcl-2 is an important anti-apoptotic protein, and the increase of Bcl-2 can effectively inhibit apoptosis, while C-caspase 3 and Bax protein expressions are significantly elevated in apoptotic cells [31][32][33]. In this study, we constructed an in vitro model of AP by inducing rat pancreatic acinar cells with caerulein. We found that, consistent with previous studies, the levels of TNF-α, IL-6, and IL-1β were significantly elevated, and cell apoptosis was promoted in the AP model. The protein expression of C-caspase 3 and Bax was significantly increased, and the  protein expression of Bcl-2 was significantly decreased, indicating that AP involves a severe inflammatory reaction and induces apoptosis and necrosis. More than that, we found that the expression of miR-399-3p was significantly decreased, while the expression of TRAF3 was significantly increased in caerulein-induced in vitro model of AP. In this study, the promotion of miR-399-3p significantly inhibited the expression of TNF-α, IL-6, and IL-1β and cell apoptosis and decreased the protein expression of C-caspase 3 and Bax. It is shown that inducing the expression of miR-339-3p can effectively inhibit cellular inflammatory response and apoptosis, and alleviate the symptoms of AP.
The RIP analysis and luciferase reporter assays demonstrated that TRAF3 is a target gene for miR-339-3p, and miR-339-3p transfection significantly inhibited TRAF3 expression. TRAF3 protein encoded by the TRAF3 gene is associated with cell inflammation, immune response, and signal transduction of CD40 [34][35][36]. Interestingly, TRAF3 has been reported to be highly expressed and play an important role in human diseases, such as breast cancer, gastric cancer, and cerebral ischemic stroke [37][38][39]. Moreover, in AP, TRAF3 has been shown to be involved in p38 activation to influence cell inflammation [40,41]. In our study, the promotion of TRAF3 could alleviate the effects of high miR-399-3p expression on pro-inflammatory factors and cell apoptosis in the in vitro model of AP. Also, we found that the relative protein expression of caeruleininduced p-p38/p38 was inhibited by overexpression of miR-399-3p, which was impaired by the induction of TRAF3 in the in vitro model of AP. Thus, these results indicated that the effect of miR-399-3p on inflammation and apoptosis in the in vitro model of AP was mediated by regulating the p38 pathway via TRAF3.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.