AR42J细胞中miR-29a的上调通过靶向TNFRSF1A基因促进细胞凋亡。
miR-29a up-regulation in AR42J cells contributes to apoptosis via targeting TNFRSF1A gene.
作者信息
Fu Qiang, Qin Tao, Chen Lin, Liu Chuan-Jiang, Zhang Xu, Wang Yu-Zhu, Hu Ming-Xing, Chu Hao-Yuan, Zhang Hong-Wei
机构信息
Qiang Fu, Tao Qin, Lin Chen, Chuan-Jiang Liu, Xu Zhang, Yu-Zhu Wang, Ming-Xing Hu, Hao-Yuan Chu, Hong-Wei Zhang, Department of Hepatobiliary Pancreatic Surgery, People's Hospital of Zhengzhou University, School of Medicine, Zhengzhou University, Zhengzhou 450003, Henan Province, China.
出版信息
World J Gastroenterol. 2016 May 28;22(20):4881-90. doi: 10.3748/wjg.v22.i20.4881.
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.
目的
研究miR-29a在大鼠急性胰腺炎中的表达及其在AR42J细胞凋亡中的功能作用。
方法
将12只SD大鼠随机分为对照组和急性水肿性胰腺炎（AEP）组。AEP组腹腔注射L-精氨酸（150 mg/kg）诱导AEP，对照组注射等体积的0.9%氯化钠。采用TUNEL法检测胰腺组织腺泡细胞凋亡情况。进行miRNA芯片检测以分析两组中miRNA的表达。此外，为进一步探究miR-29a在体外凋亡中的作用，用重组大鼠TNF-α（50 ng/mL）处理大鼠胰腺腺泡细胞系AR42J以诱导AR42J细胞凋亡。采用定量实时PCR（qRT-PCR）检测miR-29a表达。然后，用miRNA模拟物、miRNA反义寡核苷酸（AMO）和对照载体转染AR42J细胞。通过qRT-PCR确认miR-29a的表达，并通过流式细胞术分析检测AR42J细胞的凋亡率。采用蛋白质免疫印迹法检测活化的caspase3的表达。此外，我们使用生物信息学软件和荧光素酶报告基因检测法来检测TNFRSF1A是否为miR-29a的靶基因。转染后，用qRT-PCR和蛋白质免疫印迹法检测转染后AR42J细胞中TNFRSF1A的表达。
结果
miRNA芯片检测显示，AEP组中miR-29a的表达明显高于对照组。体外诱导AR42J细胞凋亡后，与对照组相比，miR-29a的表达显著增加了1.49±0.04倍。qRT-PCR检测显示，miR-29a模拟物组中miR-29a的表达相对于对照载体组高2.68±0.56倍，同时腺泡细胞凋亡率明显升高（42.83±1.25对24.97±0.15，P<0.05）。此外，miRNA AMO组中miR-29a的表达比对照载体组低0.46±0.05倍，相应地细胞凋亡率也低得多（17.27±1.36对24.97±0.15，P<0.05）。生物信息学软件和荧光素酶报告基因检测法的结果表明，TNFRSF1A可能是miR-29a的靶基因。miR-29a模拟物组中TNFRSF1A表达上调，而miR-29a AMO组则呈现相反趋势。
结论
miR-29a可能通过上调其靶基因TNFRSF1A的表达促进AR42J细胞凋亡。
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