靶向RECK基因的miR-374b-5p对裸鼠人胃癌移植瘤生长的影响  

Effect of mi R-374b-5p targeting RECK gene on xenogenic human gastric cancer in nude mice

作　　者：朱蔚远 李国庆[1] 周角凡 谢娟[4] 刘艳萍[1] 

机构地区：[1]南华大学附属第二医院消化内科,湖南衡阳421001 [2]邵阳市第一人民医院消化内科,湖南邵阳422001 [3]深圳罗湖区人民医院消化内科,广东深圳518000 [4]南华大学医学院诊断学教研室,湖南衡阳421001

出　　处：《肿瘤》2014年第11期975-981,共7页Tumor

基　　金：国家自然科学基金资助项目(编号:81071965/H1617);湖南省科技厅资助项目(编号:2013FJ3130)

摘　　要：目的 :探讨微RNA-374b-5p(micro RNA-374b-5p,miR-374b-5p)及其靶基因反转录富含半胱氨酸蛋白(reversion-inducing-cysteine-rich protein with Kazal motifs,RECK)在胃癌发生和发展中的作用。方法 :采用人胃癌MGC-803细胞建立BABL/c裸鼠移植瘤模型,当移植瘤体积达到60 mm3左右时,将小鼠随机分为3组(每组5只),即空白对照组、阴性对照组[体内转染无意义微RNA序列(miR-374b-5p negative control inhibitor)]和miR-374b-5p抑制组(体内转染miR-374b-5p inhibitor),并观察各组移植瘤的生长情况。在接种MGC-803细胞后第35天时(最后一次治疗3 d后)处死各组小鼠,苏木精-伊红(hematoxylin-eosin,HE)染色观察移植瘤标本的病理学特征;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测移植瘤组织中miR-374b-5p的表达水平;荧光素酶活性检测验证miR-374b-5p是否靶向作用于RECK基因;分别采用RFQPCR和免疫组织化学法检测移植瘤中RECK mRNA和蛋白的表达情况。结果 :miR-374b-5p抑制组移植瘤体积在转染miR-374b-5p inhibitor 9 d后(即接种细胞的第23天后),开始明显小于空白对照组和阴性对照组(P<0.05),而空白对照组和阴性对照组小鼠的移植瘤体积差异无统计学意义。病理学检测结果提示,miR-374b-5p抑制组移植瘤的恶性程度较空白对照组和阴性对照组明显下降。miR-374b-5p抑制组移植瘤中miR-374b-5p的表达水平均较空白对照组和阴性对照组明显下调(P<0.05)。荧光素酶报告载体系统证实,RECK是miR-374b-5p直接调控的靶基因;miR-374b-5p抑制组移植瘤中RECK m RNA及蛋白的表达水平均显著高于空白对照组和阴性对照组(P均<0.05)。结论 :miR-374b-5p可能通过RECK通路在胃癌的发生和发展中扮演着癌基因的角色。Objective：To investigate the role of micro RNA-374b-5p（miR-374b-5p） and its target gene reversion-inducing-cysteine-rich protein with Kazal motifs（RECK） in occurrence and development of gastric cancer.Methods：The human gastric cancer MGC-803 cells were used to establish BABL/c nude mice model bearing xenografts of MGC-803 cells.The mice were randomly divided into three groups when the volume of the xenograft reaching about 60 mm3：blank control group（n =5）,negative control（NC） group（transfecion with miR-374b-5p NC-inhibitor every four days）（n =5） and miR-374b-5p inhibitor group（transfecion with miR-374b-5p inhibitor every four days）（n =5）.The growth of the xenografts was observed.The mice in each group were sacrifi ced three days after the end of last transfection（on the 35 th day after transplantation of MGC-803 cells）.The pathological features of the xenografts were observed by hematoxylin-eosin（HE） staining.The expression level of miR-374b-5p in xenografts in nude mice after transfection was determined by real-time fluorogenic quantitative-PCR（RFQ-PCR）.The Luciferase Activity Assay was used to determine whether miR-374b-5p was targeting RECK gene.The expressions of RECK m RNA and protein were detected with RFQ-PCR and immunohistochemistry,respectively.Results：The tumor xenograft volume of miR-374b-5p inhibitor group was significantly smaller than those of the blank control group and NC group since the 9th day after transfection（since the 23 th day after the beginning of transplantation）（P 〈 0.05）,but the xenograft volume of the blank control group and the NC group had no signifi cant difference.Pathological results indicated that the degree of malignancy of xenografts in miR-374b-5p inhibitor group was lower than those of the other two groups.The expression level of miR-374b-5p in miR-374b-5p inhibitor group was signifi cantly lower than those in the other two groups（both P 〈 0.05）.The miR-374b-5p directly targeting RECK gene was proved by Luci

关 键 词：胃肿瘤 微RNAS 小鼠 裸 RECK基因 

分 类 号：R735.7[医药卫生—肿瘤]