摘要
目的探讨miR-195调控FOXK1基因及PI3K/Akt通路对胃癌细胞增殖侵袭和迁移能力的影响及机制。方法采用公共数据库样本对胃腺癌中miR-195的表达差异和预后意义进行分析。研究分为MGC803和AGS两种细胞系中过表达miR-195-5p实验组和cel-miR-67-3p mimic和cel-miR-239b-5p mimic两组阴性对照组。通过阿尔玛蓝、细胞划痕和Transwell实验检测细胞增殖、侵袭和迁移能力的改变。通过starbase v3.0对miR-195潜在的靶基因及结合位点进行预测。采用Western blot检测过表达miR-195-5p后靶基因FOXK1和PI3K/Akt通路磷酸化位点的表达水平,并采用双荧光素酶报告验证miR-195-5p和FOXK1的关系。统计分析采用IBM SPSS 22软件和R 4.0.3软件。结果miR-195在胃腺癌肿瘤标本中的表达水平相比于癌旁正常组织的表达显著降低(P<0.001),并与患者生存显著相关(HR:1.853,P=0.011)。多种表型实验证明miR-195表达水平的上调可明显抑制胃腺癌细胞的增殖和侵袭能力(MGC803侵袭:P<0.001,MGC803迁移:P<0.001;AGS侵袭:P<0.001,AGS迁移:P<0.001)(划痕:MGC803迁移:P<0.010,AGS迁移:P<0.010)。进一步研究证实FOXK1基因可能受到miR-195的调控,是miR-195的潜在靶点之一。而miR-195在调控FOXK1的同时能明显降低PI3K/Akt信号通路的激活程度,过表达miR-195之后,PI3K/Akt通路中的磷酸化Akt(S473位点)表达明显下降。结论本研究说明miR-195调控FOXK1抑制PI3K/Akt通路,从而抑制胃癌细胞增殖侵袭能力,进一步说明miR-195在胃癌的诊断和治疗中潜在的重要作用,对胃癌发生发展的分子机制及新靶点的发现具有重要的临床意义。
Objective To investigate the effect and mechanism of miR-195 regulating FOXK1 gene and PI3K/Akt pathway on stomach adenocarcinoma proliferation,invasion and migration ability.Methods Public database samples were employed to analyze the expression differences and prognostic significance of miR-195 in stomach adenocarcinoma.After overexpression of mir-195-5p in two cell lines,MGC803 and AGS,altered cell proliferation,invasion,and migration abilities were detected by Alamar Blue,Wound healing,and Transwell assays.The potential target genes and binding sites of miR-195 were predicted by the starBase.Western blot was used to detect the expression levels of foxk1 and phosphorylation sites in the PI3K/Akt pathway of target genes after overexpression of mir-195-5p.A Dual-luciferase reporter assay was used to verify the relationship between mir-195-5p and foxk1.Statistical analyses were performed with IBM SPSS 22 software and R 4.0.3.Results Our results showed a significant over-expression of miR-195 in the tumor tissues,compared with the paired normal tissues(P<0.001),which could inhibit the proliferation and invasion of stomach carcinoma cells and significantly correlated with survival(P=0.011).Moreover,our study indicated that miR-195 depressed the expression of FOXK1 and significantly reduced the activation of the PI3K/Akt pathway,which had a negative effect on the proliferation and invasion of stomach carcinoma cells.The phosphorylated Akt(s473 site)expression in the PI3K/Akt pathway was significantly decreased after overexpression of miR-195.Conclusion Overall,our studies clarify the important function of the miR-195 in the diagnosis and therapy of patients with stomach carcinoma and reveal the FOXK1 and PI3K/Akt pathway regulation by the miR-195,which are of important clinical significance in the differential diagnosis.
作者
范晓彬
宋峰峰
李晓青
李文星
范现英
胡延伟
宋志岗
王强
连彦军
Fan Xiaobin;Song Fengfeng;Li Xiaoqing;Li Wenxing;Fan Xianying;Hu Yanwei;Song Zhigang;Wang Qiang;Lian Yanjun(Department of Gastrointestinal Surgery,Xingtai Third Hospital,Xingtai 054099,China;The Second Division of General Surgery,People’s Hospital of Binzhou,Binzhou 256600,China;Department of Gastrointestinal Surgery,the Second Hospital of Shanxi Medical University,Taiyuan 030000,China;Department of Hepatobiliary Surgery,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400042,China)
出处
《中华内分泌外科杂志》
CAS
2022年第6期655-661,共7页
Chinese Journal of Endocrine Surgery
基金
邢台市重点研发计划项目(2020ZC232)。
