miR-29a up-regulation in AR42J cells contributes to apoptosis via targeting TNFRSF1A gene

Aim: To investigate the expression of miR-29a in rat acute pancreatitis and its functional role in AR42J cell Apoptosis.

Methods: Twelve SD rats were divided into a control group and an acute edematous pancreatitis (AEP) group randomly. AEP was induced by intraperitoneal injection of L-arginine (150 mg/kg) in the AEP group and equal volume of 0.9% NaCl was injected in the control group. The Apoptosis of acinar cells in pancreatic tissue was determined by TUNEL assay. miRNA chip assay was performed to examine the expression of miRNAs in two groups. Besides, to further explore the role of miR-29a in Apoptosis in vitro, recombinant rat TNF-α (50 ng/mL) was administered to treat the rat pancreatic acinar cell line AR42J for inducing AR42J cell Apoptosis. Quantitative Real-Time PCR (qRT-PCR) was adopted to measure miR-29a expression. Then, miRNA mimic, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells. The expression of miR-29a was confirmed by qRT-PCR and the Apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of activated caspase3. Moreover, we used bioinformatics software and luciferase assay to test whether TNFRSF1A was the target gene of miR-29a. After transfection, qRT-PCR and Western blot was used to detect the expression of TNFRSF1A in AR42J cells after transfection.

Results: The expression of miR-29a was much higher in the AEP group compared with the control group as displayed by the miRNA chip assay. After inducing Apoptosis of AR42J cells in vitro, the expression of miR-29a was significantly increased by 1.49 ± 0.04 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-29a was 2.68 ± 0.56 times higher in the miR-29a mimic group relative to the control vector group, accompanied with an obviously increased acinar cell Apoptosis rate (42.83 ± 1.25 vs 24.97 ± 0.15, P < 0.05). Moreover, the expression of miR-29a in the miRNA AMO group was 0.46 ± 0.05 times lower than the control vector group, and the cell Apoptosis rate was much lower accordingly (17.27 ± 1.36 vs 24.97 ± 0.15, P < 0.05). The results of bioinformatics software and luciferase assay showed that TNFRSF1A might be a target gene of miR-29a. TNFRSF1A expression was up-regulated in the miR-29a mimic group, while the miR-29a AMO group showed the reverse trend.

Conclusion: miR-29a might promote the Apoptosis of AR42J cells via up-regulating the expression of its target gene TNFRSF1A.

AR42J; Acute edematous pancreatitis; Apoptosis; TNFRSF1A; Target gene; miR-29a.