摘要
目的探讨长链非编码RNA(lncRNA)生长阻滞特异性转录因子5(GAS5)对雨蛙肽(caerulein)诱导的急性胰腺炎腺泡细胞凋亡的影响及机制。方法取对数生长期胰腺腺泡细胞AR42J,分为Control组(正常培养)、Caerulein组(caerulein诱导)、Caerulein+Vector组(转染阴性对照载体,caerulein诱导)及Caerulein+GAS5组(转染GAS5过表达对照载体,caerulein诱导),Caerulein+GAS5+miR-NC组(转染GAS5过表达对照载体、mimics control,caerulein诱导)及Caerulein+GAS5+miR-135a组(转染GAS5过表达对照载体、miR-135a mimics,caerulein诱导),分别采用细胞计数试剂盒8(CCK-8)实验、实时聚合酶链反应(Realtime PCR)、流式细胞术及Western blot检测各组细胞增殖、miR-135a表达、细胞凋亡水平及剪切的半胱氨酸天冬氨酸蛋白酶-3(C-Caspase-3)、C-Caspase-9表达;将miR-135a mimics(miR-135a组)、mimics control(miR-NC组)和含GAS5结合位点的野生型萤光素酶报告载体(WT,WT组)、含突变以后GAS5结合位点的突变型萤光素酶报告载体(MUT,MUT组)共转染至胰腺腺泡细胞,采用萤光素酶活性测定试剂盒分析各组胰腺炎腺泡细胞萤光素酶活性变化。结果与Control组比较,Caerulein组胰腺腺泡细胞存活率降低、凋亡率升高,细胞中C-Caspase-3、C-Caspase-9蛋白表达增加,差异均有统计学意义(P<0.05);与Caerulein+Vector组比较,Caerulein+GAS5组caerulein诱导的胰腺腺泡细胞的存活率升高、凋亡率降低,C-Caspase-3、C-Caspase-9蛋白表达减少,差异均有统计学意义(P<0.05);与Caerulein+GAS5+miR-NC组比较,Caerulein+GAS5+miR-135a组胰腺腺泡细胞存活率降低、凋亡率升高,差异均有统计学意义(P<0.001);与miR-NC+WT组比较,miR-135a+WT组细胞萤光素酶活性降低,差异有统计学意义(P<0.05);与miR-NC+MUT组比较,miR-135a+MUT组细胞萤光素酶活性无变化,差异无统计学意义(P>0.05)。结论过表达lncRNA GAS5可减少caerulein诱导的急性胰腺炎腺泡细胞凋亡,其机制可能与下调miR-135a表达有关。
Objective To investigate the effect and mechanism of long noncoding RNA(lncRNA)-growth arrest-specific 5(GAS5)on caerulein-induced acinar cell apoptosis in acute pancreatitis.Methods Pancreatic acinar cell line AR42J at logarithmic growth phase were divided into Control group(normal culture),Caerulein group(Caerulein induction),Caerulein+Vector group(transfected with negative control vector,Caerulein induction),Caerulein+GAS5 group(transfected with GAS5 overexpression control vector,Caerulein induction),Caerulein+GAS5+miR-NC group(transfected with GAS5 overexpression control vector,mimics control,Caerulein induction),and Caerulein+GAS5+miR-135a group(transfected with GAS5 overexpression control vector,miR-135a mimics,caerulein induction).Cell counting 8(CCK-8)assay,real-time PCR,flow cytometry,and Western blot were used to detect cell proliferation,miR-135a expression,cellular apoptosis and the protein expression levels of cleaved caspase-3(C-Caspase-3)and cleaved caspase-9(C-Caspase-9)in each group,respectively.miR-135a mimics(miR-135a group),mimics control(miR-NC group),WT(luciferase reporter vector containing GAS5 binding site),MUT(luciferase reporter vector containing mutated GAS5 binding site)were co-transfected into pancreatic acinar cells.Luciferase activity assay was used to analyze the changes in cellular luciferase activity in each group.Results When compared with Control group,Caerulein group had reduced survival rate of pancreatic acinar cells,increased apoptotic rate and elevated expression levels of C-Caspase-3 and C-Caspase-9 proteins(P<0.05).When compared with Caerulein+Vector group,Caerulein+GAS5 group had increased survival rate of caerulein-treated pancreatic acinar cells decreased apoptotic rate and reduced expression levels of C-Caspase-3 and C-Caspase-9 proteins(P<0.05).When compared with Caerulein+GAS5+miR-NC group,Caerulein+GAS5+miR-135a group had decreased survival rate and increased apoptotic rate of pancreatic acinar cells(P<0.001).When compared with miR-NC+WT group,miR-135a+WT group had decreased cellular luciferase activity(P<0.05).There was not statistically significant difference in the cellular luciferase activity between miR-NC+MUT and miR-135a+MUT groups(P>0.05).Conclusion Overexpression of lncRNA GAS5 can reduce the apoptosis of acinar cells in acute pancreatitis induced by caerulein,and its mechanism may be related to the downregulation of miR-135a expression.
作者
许冀
佘秋芳
彭金娥
汤瑜
杨亚东
XU Ji;SHE Qiufang;PENG Jin'e;TANG Yu;YANG Yadong(Department of Critical Care Medicine,Li Shizhen Hospital of Traditional Chinese Medicine,Qichun 435300,Hubei,China;Department of Critical Care Medicine,Huanggang Central Hospital,Huanggang 438000,Hubei,China)
出处
《贵州医科大学学报》
CAS
2022年第2期184-190,共7页
Journal of Guizhou Medical University
基金
中华国际医学交流基金会项目(Z-2017-24-2028-29)。
