摘要
目的检测结肠癌组织及细胞中miR-346及人分泌型卷曲相关蛋白4(SFRP4)的表达,观察其对结肠癌细胞增殖的作用及可能的机制。方法选取30例经手术切除的结肠癌组织及其相应的癌旁正常组织(距离肿瘤边缘5cm),另选取结肠癌细胞株及正常结肠上皮细胞株,采用实时定量聚合酶链反应(qRT-PCR)及免疫印迹(Western blot)方法检测组织及细胞中的miR-346、SFRP4mRNA及蛋白的表达,采用Pearson rank检验miR-346与SFRP4的相关性,采用Target Scan及Find Tar软件预测两者的靶向调节作用。转染miR-346拟似物(mimics)或抑制物(inhibitor)以上调或下调miR-346的表达,MTT法检测结肠癌细胞的增殖,qRT-PCR及Western blot检测SFRP4mRNA和蛋白的表达。结果 miR-346在结肠癌组织中的表达高于癌旁正常组织(t=12.871,P<0.001)。SFRP4mRNA及蛋白在结肠癌组织中的表达低于癌旁正常组织(t=8.609,P<0.001;t=20.892,P<0.001)。miR-346在结肠癌细胞株(SW480、SW620、HCT116、CaCo-2、LoVo、HT29)中的表达高于正常结肠上皮细胞株FHC(F=10.17,P<0.001)。SFRP4mRNA及蛋白在结肠癌细胞株中的表达低于正常结肠上皮细胞株(F=39.01,P<0.001;F=16.25,P<0.001)。结肠癌组织中miR-346与SFRP4mRNA的表达呈负相关(r=-0.55,95%CI:-0.757～-0.230,P=0.001 8)。与miRNA阴性对照(miR-NC)组比较,给予miR-346mimics后,MTT显示在72h、96h时间点miR-346组细胞存活率升高(t=3.484,P<0.05;t=5.868,P<0.001),SFRP4mRNA及蛋白表达下降(t=2.267,P<0.05;t=5.889,P<0.01);给予miR-346inhibitor后,MTT显示在72h、96h时间点miR-346组细胞存活率下降(t=4.400,P<0.001;t=3.591,P<0.001),SFRP4mRNA及蛋白表达升高(t=4.495,P<0.01;t=7.130,P<0.01)。结论 miR-346在结肠癌中高表达,SFRP4低表达,miR-346通过靶向作用于SFRP4来调控结肠癌细胞的增殖。
Objective This paper intends to detect the expression of miR-346 and human secretory frizzled protein 4 (SFRP4) in colon cancer tissues and cells in order to observe its effect on colon cancer cell proliferation and its possible mechanisms. Methods Thirty paired samples of colon cancer tissues, paracancer normal tissues (5 cm from the edge of the tumor), colon cancer cell lines and normal colon epithelium cells were selected, whose miR-346, SFRP4 mRNA, and protein expression were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assay. The correlation between miR-346 and SFRP4 was observed by Pearson rank test. The target regulation of miR-346 and SFRP4 mRNA was predicted by Target Scan and Find Tar. Mimics or inhibitor were transfected to up-regulate or down-regulate miR-346 expression. The proliferation was detected by MTT assay while the SFRP4 mRNA and protein expression were detected by qRT-PCR and Western blot assay. Results The expression of miR-346 in colon cancer tissues was higher than that in paracancerous normal tissues ( t =12.871, P <0.001). The expression of SFRP4 mRNA and protein in colon cancer tissues was lower than that in paraancerous normal tissues ( t =8.609, P <0.001;t =20.892, P <0.001). The expression of miR-346 in colon cancer cell lines (SW480, SW620, HCT116, CaCo-2, LoVo, HT29) was higher than that in normal colonic epithelial cell line FHC ( F =10.17, P <0.001). The expression of SFRP4 mRNA and protein in colon cancer cell lines was lower than that in normal colonic epithelial cells ( F =39.01, P <0.001;F =16.25, P <0.001). There was a negative correlation between miR-346 and SFRP4 mRNA expression in colon cancer tissues ( r =-0.55, 95% CI :-0.757 to -0.230, P = 0.001 8 ). Compared with the miR-negative control (miR-NC) group, after administration of miR-346 mimics, there was an increase in cell viability in the miR-346 group at 72 h and 96 h ( t =3.484, P < 0.05;t = 5.868 , P < 0.001 ). There was a decrease in expression of SFRP4 mRNA and protein
作者
朱恩宇
郑斯卓
高凯
石刚
张睿
ZHU Enyu;ZHENG Sizhuo;GAO Kai;SHI Gang;ZHANG Rui(Department of Surgery,the Second Hospital Affiliated to Liaoning University of Traditional Chinese Medicine,Shenyang 110042,China;Department of Internal Medicine,General Hospital of Liaoning Provincial Bureau of Prison,Shenyang 110042,China;Department of Colorectal Surgery,Cancer Hospital of China Medical University,Shenyang 110042,China)
出处
《国际消化病杂志》
CAS
2019年第1期40-45,共6页
International Journal of Digestive Diseases
基金
国家自然基金资助项目(81672427)
辽宁省博士科研启动基金(201601417)
沈阳市科技局计划项目(18-014-4-75)
