miR-7靶向Sp3对急性胰腺炎腺泡细胞增殖和凋亡的影响及机制研究

MiR-7 regulates proliferation and apoptosis of acinar cells in acute pancreatitis by targeting Sp3

背景重症急性胰腺炎(acute pancreatitis, AP)是一种发病急、进展快、死亡率高的严重疾病,其病因复杂,近年来研究发现, miRNA在AP的发生和发展中发挥重要作用,研究miRNA在AP中的机制对于AP的治疗、预防等有一定的帮助.而miR-7与胃癌、肺癌、乳腺癌、胶质瘤等多种肿瘤的进展相关,但在AP中研究鲜有报道.目的研究miR-7对AP腺泡细胞增殖、凋亡的影响及潜在的作用机制.方法用雨蛙素处理大鼠胰腺腺泡AR42J构建AP模型,设置NC组、Cerulein组、miR-con组(转染miR-con)、miR-7组(转染miR-7 mimics)、anti-miR-con组(转染anti-miR-con)、anti-miR-7组(转染anti-miR-7)、Cerulein+anti-miR-con组(Cerulein处理后转染antimiR-con)、Cerulein+anti-miR-7组(Cerulein处理后转染anti-miR-7)、Cerulein+pcDNA组(Cerulein处理后转染pcDNA)、Cerulein+pcDNA-特异性蛋白3(specificprotein 3, Sp3)组(Cerulein处理后转染pcDNA-Sp3)、Cerulein+anti-miR-7+si-con组(Cerulein处理后共转染anti-miR-7和si-con)、Cerulein+anti-miR-7+si-Sp3组(Cerulein处理后共转染anti-miR-7和si-Sp3),用脂质体法转染至AR42J细胞. ELISA法检测雨蛙素处理AR42J细胞的淀粉酶(Amylase, AMY)、肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)和白细胞介素-6(Interleukin-6, IL-6)的表达;qRT-PCR检测雨蛙素处理AR42J细胞中miR-7、Sp3 mRNA的表达水平;Western Blot检测Sp3蛋白表达;流式细胞术检测细胞凋亡;双荧光素酶报告基因检测实验检测荧光活性.结果雨蛙素处理AR42J细胞后, AMY、TNF-α、IL-6的表达均呈现先升高后下降的趋势,且均在8 h时达到最大,建模成功.相较于NC组, Cerulein组miR-7的表达水平显著升高;Sp3 mRNA和蛋白的表达水平显著降低(P <0.01).相较于Cerulein+anti-miR-con组,Cerulein+anti-miR-7组miR-7的表达水平显著下降,细胞凋亡率显著降低(P <0.01).相较于Cerulein+pcDNA组, Cerulein+pcDNA-Sp3组Sp3蛋白的表达水平显著升高,细胞凋亡率显著降低(P <0.01). miR-7靶向负调控Sp3;抑制Sp3表达逆转了敲减miR-7对雨蛙素处理AR42J细胞的凋亡抑制作用.结论敲减miR-7表达可以抑制AP腺泡细胞凋亡,其机制可能与靶向调控SP3有关.可为AP诊断和治疗提供新靶点和新思路.

BACKGROUND Severe acute pancreatitis (AP) is a serious disease with acute onset, rapid progression, and high mortality. The etiology of AP is complicated. In recent years, studies have found that miRNAs play an important role in the occurrence and development of AP. Elucidating the mechanisms of miRNAs in AP is helpful for the treatment and prevention of AP. MiR-7 is associated with the progression of various tumors such as gastric cancer, lung cancer, breast cancer, and glioma, but few studies have been performed in AP. AIM To investigate the effect of miR-7 on proliferation and apoptosis of AP acinar cells and the potential mechanism involved. METHODS An AP model was constructed by treating pancreatic acinar AR42J cells with cerulein. AR42J cells were divided into different groups: normal control (NC) group, cerulein group, miR-con group (transfected withmiR-con), miR-7 group (transfected with miR-7 mimics), anti-miR-con group (transfected with anti-miR-con), anti-miR-7 group (transfected with anti-miR-7), cerulein + anti-miR-con group (transfected anti-miR-con after cerulein treatment), cerulein + anti-miR-7 group (transfected with anti-miR-7 after cerulein treatment), cerulein + pcDNA group (transfected with pcDNA after cerulein treatment), cerulein + pcDNA-specific protein 3 (Sp3) group (transfected with pcDNA-Sp3 after cerulein treatment), cerulein + anti-miR-7 + si-con group (co-transfected with anti-miR-7 and si-con after cerulein treatment), and cerulein + anti-miR-7 + si-Sp3 group (co-transfected with anti-miR-7 and si-Sp3 after cerulein treatment). Transfections were performed using the liposome method. The levels of amylase (AMY), tumor necrosis factor-α(TNF-α), and interleukin-6 (IL-6) in AR42J cells treated with cerulein were detected by ELISA. qRT-PCR was used to detect miR-7 and Sp3 mRNA expression in AR42J cells treated with cerulein. Western blot was used to detect Sp3 protein expression. Flow cytometry was used to detect apoptosis, and dual luciferase reporter gene assay was used to detect fluorescence activity. RESULTS After treatment of AR42J cells with cerulein, the levels of AMY, TNF-α, and IL-6 all increased first, reaching the peak at 8 h, and then decreased, suggesting that the modeling was successful. Compared with the NC group, the expression of miR-7 in the cerulein group was significantly increased, while the expression of Sp3 mRNA and protein was significantly decreased (P < 0.05). Compared with the cerulein + anti-miR-con group, the expression of miR-7 in the cerulein + antimiR- 7 group was significantly decreased, and the apoptosis rate was also significantly decreased (P < 0.05). Compared with the cerulein + pcDNA group, the expression of Sp3 protein in the cerulein + pcDNA-Sp3 group was significantly increased, and the apoptosis rate was significantly decreased (P < 0.05). MiR-7 negatively regulated the expression of Sp3, and inhibition of Sp3 expression reversed the inhibitory effect of miR-7 knockdown on the apoptosis of AR42J cells treated with cerulein. CONCLUSION MiR-7 knockdown can inhibit apoptosis of acinar cells in AP via mechanisms possibly related to targeted regulation of SP3. These findings can provide new targets and ideas for the diagnosis and treatment of AP.