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. 2016 May 28;22(20):4881-90.
doi: 10.3748/wjg.v22.i20.4881.

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

Qiang Fu  1 , Tao Qin  1 , Lin Chen  1 , Chuan-Jiang Liu  1 , Xu Zhang  1 , Yu-Zhu Wang  1 , Ming-Xing Hu  1 , Hao-Yuan Chu  1 , Hong-Wei Zhang  1
Affiliations

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

Qiang Fu et al. World J Gastroenterol. .

Abstract

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.

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

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Figures

Figure 1
Figure 1
TUNEL staining of pancreatic tissue (× 400). TUNEL-positive cells displayed brown fluorescence. A: TUNEL staining was detected in control rats; B: The tissue of the L-arginine treated rats. The apoptosis increased significantly in Figure 1B.
Figure 2
Figure 2
Hierarchically clustered heat map illustrating the changes in miRNA expression profiles between the acute edematous pancreatitis groups and control groups. The significantly expressed miRNA clusters were identified using the Student’s t-test. The red and green sections represent an increase and a decrease in miRNA expression, respectively, between control group and AEP group. The expression of miR-29a was significantly up-regulated in the AEP group compared with the control group. AEP: Acute edematous pancreatitis.
Figure 3
Figure 3
Expression of amylase, activated caspase 3 protein, apoptosis rate of AR42J cells and miR-29a level increase in the experimental group compared with the control group. A: The expression of amylase analysis in the supernatant; B: Western blot analysis of activated caspase 3 in AR42J cells; C: The apoptosis rate of AR42J cells after the treatment with TNF-α for 24 h. Date were obtained from three independent experiments in triplicate and are shown as the mean ± SD. aP < 0.05 vs control group.
Figure 4
Figure 4
Quantitative real-time PCR analysis of miR-29a in AR42J cells at 3 h and 6 h. The expression of miR-29a was normalized to U6 expression using 2-ΔΔct. Data were obtained from three independent experiments in triplicate and are shown as the mean ± SD. aP < 0.05 vs control group.
Figure 5
Figure 5
Lentiviral transfection and miRNA expression after transfection. A: Cells were infected with 50 MOI of lentivirus, and imaged 72 h post-transfection. Comparison of bright field filter view to FITC filter view (GFP-expression cells) for the same fields of cells showed about 90% infection efficiency by 72 h; B: Quantitative real-time PCR analysis of miR-29a expression in the AR42J cells after transfection. Date are shown as a ratio of mi-29a mimic and AMO groups to vehicle groups using the 2-ΔΔct. Data are representative of three independent experiments. aP < 0.05 vs vehicle group.
Figure 6
Figure 6
miR-29a promotes the apoptosis of the AR42J cells. A: The amylase analysis in the supernatant increased obviously; B: Western blot analysis of activated caspase 3 in AR42J cells; C: The apoptosis rate of AR42J cells was determined by FACS analysis. Data are representative of mean ± SD from three independent experiments performed in triplicate. aP < 0.05 vs control or vehicle group.
Figure 7
Figure 7
miR-29a targets TNFRSF1A. A: The predicted miR-29a binding sites within the 3’UTR of TNFRSF1A and mutant version generated by site mutagenesis are shown; B: Luciferase activity was determined 48 h after transfection. The ratio of normalized sensor to control luciferase activity is shown. Data are shown as the mean ± SD and were obtained from three independent experiments performed in triplicate (aP < 0.05 vs control miR-transfected cells).
Figure 8
Figure 8
miR-29a promotes TNFRSF1A gene expression. A: Quantitative real-time RT-PCR analysis of TNFRSF1A expression in AR42J cells after transfection. Date are shown as a ratio of miR-29a mimic and AMO groups to vehicle group using the 2-ΔΔct. Data are representative of three independent experiments (aP < 0.05 vs vehicle group); B: Western blot analysis of TNFR1 protein in AR42J cells after transfection.
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