miR-326靶向MAPK/MEK信号通路抑制食管鳞状细胞癌恶性生物学行为  

miR-326 inhibiting esophageal squamous cell carcinoma by targeting MAPK/MEK signaling pathway

作　　者：李娜[1] 许春蕾[1] 唐勇[1] 汤旭山[1] 马兰英[1] LI Na;XU Chunlei;TANG Yong;TANG Xushan;MA Lanying(Department of Gastroenterology,Cancer Hospital Affiliated to Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学附属肿瘤医院消化内科,乌鲁木齐830011

出　　处：《中华实用诊断与治疗杂志》2020年第9期878-883,共6页Journal of Chinese Practical Diagnosis and Therapy

基　　金：新疆维吾尔自治区自然科学基金青年基金(2017D01C413)。

摘　　要：目的探讨miR-326靶向MAPK/MEK信号通路对食管鳞状细胞癌细胞增殖、迁移、侵袭和凋亡的影响。方法 20例食管鳞状细胞癌患者,取手术切除食管癌组织和癌旁正常组织,采用实时荧光定量PCR法检测miR-326mRNA和MAPK mRNA相对表达量,采用免疫组织化学法检测MAPK蛋白表达。3种食管癌细胞系(KYSE150、EC9706、TE-9)和人正常食管上皮细胞系Het-1A,采用实时荧光定量PCR法检测细胞miR-326 mRNA和MAPK mRNA相对表达量,Western blot法检测细胞MAPK蛋白相对表达量。将对数生长期EC9706细胞随机分为miR-326组、转染对照组和空白对照组,miR-326组和转染对照组分别转染miR-326 mimic和mimic NC,空白对照组细胞不作任何处理,采用Western blot法检测细胞MAPK、p-MEK、p-ERK1、p-ERK2和p-C-Jun蛋白相对表达量,克隆形成试验检测细胞克隆形成率,划痕试验检测细胞划痕闭合率,Transwell小室试验检测细胞侵袭,流式细胞术检测细胞周期和细胞凋亡,荧光素酶活性检测试剂盒检测细胞荧光素酶活性。结果食管癌组织miR-326 mRNA相对表达量低于癌旁正常组织(P<0.05),MAPK mRNA相对表达量高于癌旁正常组织(P<0.05)。KYSE150、EC9706和TE-9细胞miR-326 mRNA相对表达量均低于Het-1A细胞(P<0.05),MAPK mRNA和MAPK蛋白相对表达量高于Het-1A细胞(P<0.05),且EC9706细胞MAPK蛋白相对表达量最高。miR-326组细胞MAPK 3’UTR WT荧光素酶活性(0.53±0.05)明显低于转染对照组(1.22±0.14)(P<0.05),MAPK 3’UTR MUT荧光素酶活性(0.86±0.06)与转染对照组(0.91±0.11)比较差异无统计学意义(P>0.05)。miR-326组细胞MAPK蛋白相对表达量(0.32±0.15)低于转染对照组(0.88±0.07)和空白对照组(0.91±0.06)(P<0.05),细胞克隆形成率[(21.30±0.55)%]、划痕闭合率[(29.47±4.12)%]和侵袭细胞数[(113.86±3.15)个]低于转染对照组[(75.43±0.81)%、(80.52±2.51)%、(427.27±4.25)个]和空白对照组[(79.13±0.65)%、(83.44±1.87)%、(468.10±3.38)个](P<0.05),G0/Objective To investigate the influences of miR-326 targeting mitogen-activated protein kinase/mitogen-activated extracellular signal-regulated kinase(MAPK/MEK) signaling pathway on the proliferation, migration, invasion and apoptosis of esophageal squamous cell carcinoma(ESCC) cells. Methods The relative expressions of miR-326 and MAPK mRNAs were detected by real-time fluorescence quantitative PCR and MAPK protein was detected by immunohistochemistry method in ESCC samples and adjacent normal tissues from 20 ESCC patients. The relative expressions of miR-326 and MAPK mRNAs were detected by real-time fluorescence quantitative PCR and the relative expression MAPK protein was detected by Western blot in three esophageal cancer cell lines(KYSE150, EC9706, TE-9) and human normal esophageal epithelial cell line Het-1 A. The EC9706 cells in logarithmic growth phase were randomly divided into miR-326 group transfected with miR-326 mimic, transfection control group transfected with mimic NC and blank control group with no treatment. Western blot was used to detect the relative expressions of MAPK, p-MEK, p-ERK1, p-ERK2 and p-C-Jun proteins, clone formation experiment was used to detect the rate of clone formation, scratch test was used to detect the rate of scratch closure, Transwell chamber test was used to detect the number of invading cells,flow cytometry was used to detect the cell cycle and apoptotic rate,and luciferase activity detection kit was used to detect the luciferase activity.Results The relative expression of miR-326 mRNA was lower and the relative expression of MAPK mRNA was higher in esophageal cancer tissues than that in adjacent normal tissues(P<0.05).The relative expression of miR-326 mRNA was lower while the relative expressions of MAPK mRNA and protein were higher in KYSE150,EC9706 and TE-9 cells than those in Het-1 Acells(P<0.05),and the relative expression of MAPK protein was the highest in EC9706 cells.The luciferase activity of MAPK 3\UTR WT was lower in miR-326 group(0.53±0.05)than that in tran

关 键 词：食管鳞状细胞癌 miR-326 MAPK/MEK 细胞增殖 细胞迁移 细胞侵袭 细胞凋亡 

分 类 号：R735.1[医药卫生—肿瘤]