miR-9-5p通过SIRT1/NF-κB通路促进胰腺癌细胞凋亡的实验研究  

Experimental study on miR-9-5p role in promoting apoptosis of pancreatic cancer cells through SIRT1/NF-κB pathway

作　　者：李雪颀 周青宏 潘春风 王语阳 苏拾香 岑兰英 潘路娟 覃月秋[1] Li Xueqi;Zhou Qinghong;Pan Chunfeng;Wang Yuyang;Su Shixiang;Cen Lanying;Pan Lujuan;Qin Yueqiu(The Affiliated Hospital of Youjiang Medical University for Nationalities,Baise 533000,Guangxi,China;Graduate School,Youjiang Medical University for Nationalities,Baise 533000,Guangxi,China)

机构地区：[1]右江民族医学院附属医院,广西百色533000 [2]右江民族医学院研究生学院,广西百色533000

出　　处：《右江民族医学院学报》2023年第2期195-201,共7页Journal of Youjiang Medical University for Nationalities

基　　金：国家自然科学基金项目(82260134);右江民族医学院附属医院高层次人才科研项目(R202011702)。

摘　　要：目的探讨miR-9-5p通过调控SIRT1及NF-κB表达对胰腺癌细胞(PANC-1)凋亡相关基因Caspase-3表达的影响。方法使用miR-9-5p mimic慢病毒转染人胰腺癌PANC-1细胞建立稳转株(mimic组),空载体转染作为阴性对照组(NC组),以未处理的PANC-1细胞作为空白对照组(空白组)。采用qRT-PCR检测PANC-1细胞中miR-9-5p、SIRT1及Caspase-3 mRNA的表达水平;Western Blot检测SIRT1、NF-κB通路相关蛋白及Caspase-3蛋白表达量;ELISA检测细胞裂解液中SIRT1及Caspase-3蛋白表达;双荧光素酶报告基因实验明确miR-9-5p和SIRT1的靶向关系;Pearson相关分析确定miR-9-5p、SIRT1及Caspase-3 mRNA表达的相互关系。结果mimic组miR-9-5p mRNA表达较对照组明显升高,结果有统计学意义,提示转染成功。与空白组及NC组相比,mimic组SIRT1 mRNA及蛋白表达水平下调(P<0.01),p-NF-κB p65与NF-κB p65上调(P<0.01),Caspase-3 mRNA及蛋白表达水平上调(P<0.01)。双荧光素酶报告基因显示miR-9-5p可抑制野生型SIRT1细胞的荧光活性,miR-9-5p可靶向调控SIRT1的表达;Pearson相关分析发现miR-9-5p和SIRT1的表达水平呈负相关(r=-0.849,P<0.05);miR-9-5p和Caspase-3的表达水平呈正相关(r=0.796,P<0.05)。结论过表达miR-9-5p可促进PANC-1细胞凋亡相关基因Caspase-3的表达,其机制可能与靶向调控SIRT1继而调节NF-κB通路有关,这将为胰腺癌的诊断及治疗提供新的思路。Objective To investigate the effects of miR-9-5p on the expression of apoptosis-related gene Caspase-3 in pancreatic cancer cells(PANC-1)by regulating the expression of SIRT1 and NF-κB.Methods Human pancreatic cancer cells(PANC-1)were transfected with miR-9-5p mimic lentivirus to establish stable transfected cells as mimic group,while empty vectors were transfected as negative control group(NC group),and untreated PANC-1 cells were used as blank control group(blank group).qRT-PCR was used to detect the expression levels of miR-9-5p,SIRT1 and Caspase-3 mRNA in PANC-1 cells;Western Blot was used to detect the protein expressions of SIRT1,NF-κB pathway related proteins and Caspase-3 in each group;ELISA was used to detect the protein expressions of SIRT1 and Caspase-3 in cell lysate;Dual luciferase reporter gene assay was performed to confirm the targeted relationship between miR-9-5p and SIRT1;Pearson correlation analysis was used to determine the correlation among the expressions of miR-9-5p,SIRT1 and Caspase-3 mRNA.Results The expression of miR-9-5p mRNA in the mimic group was significantly higher than that in the control group,and the results had statistical differences,indicating the success of transfection(P<0.05).Compared with the blank group and NC group,the expression levels of SIRT1 mRNA and protein in the mimic group were down-regulated(P<0.01),p-NF-κB p65 and NF-κB p65 were up-regulated(P<0.01),The expression levels of Caspase-3 mRNA and protein were up-regulated(P<0.01).Dual luciferase reporter gene assay showed that miR-9-5p could inhibit the fluorescence activity of wild-type SIRT1 cells,and miR-9-5p could make target regulation of the expression of SIRT1;Pearson correlation analysis found the expression levels of miR-9-5p were negatively correlated with those of SIRT1(r=-0.849,P<0.05);The expression levels of miR-9-5p were positively correlated with those of Caspase-3(r=0.796,P<0.05).Conclusion Overexpression of miR-9-5p can promote the expression of the apoptosis-related gene Caspase-3 in PANC-1

关 键 词：miR-9-5p 沉默调节蛋白1 半胱氨酸蛋白酶-3 NF-ΚB 胰腺肿瘤 

分 类 号：R735.9[医药卫生—肿瘤]