Chen Fang, Zhou Chang, Lu Yanxia, Yuan Li, Peng Fanli, Zheng Lin, Li Xuenong
Department of Pathology, Southern Medical University, Guangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2013 May;33(5):654-60.
To explore the expression of hsa-mir-186 in colorectal cancer and study its role in regulating the biological behaviors of human colorectal cancer SW620 cells in vitro.
The expression of hsa-miR-186 in colon cancer tissue and the adjacent tissues as well as 5 colon carcinoma cells were analyzed using real-time quantitative RT-PCR. The precursor sequence of miR-186 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. The colorectal cancer cell line SW620 was transfected with PLVTHM-miR186 vector and the lentivirus-infected cells were sorted with flow cytometry. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. The migration and invasion of SW620 cells were investigated using Transwell assay and scratch test. Western blotting was used to detect the expression of YY1 protein in SW620 cell lines.
The relative expression of miR-186 in the cancer tissues was 0.0024∓0.0027, significantly lower than that in the adjacent tissues (0.066∓0.068, P=0.008); the relative expression level of hsa-miR-186 in SW620 and LoVo cells with a high metastatic potential was 0.118∓0.138 and 0.157∓0.001, respectively, significantly lower than that in HT-29 cells with a low metastatic potential (1.000∓0.00, P<0.05). The recombinant lentiviral vector PLVTHM-miR186, verified by enzyme digestion, sequencing and qPCR, caused significant inhibition of cell proliferation, migration and invasion and suppressed the expression of YY1 protein in SW620 cells.
As a tumor suppressor gene, Hsa-miR-186 is down-regulated in colon carcinoma tissues and in highly metastatic SW620 and LoVo cells. Has-miR-186 can inhibit the cell proliferation, migration and invasion of colon carcinoma cells in vitro possibly by suppressing YY1 expression.
探讨hsa - mir - 186在结直肠癌中的表达，并研究其在体外调节人结直肠癌SW620细胞生物学行为中的作用。
采用实时定量RT - PCR分析hsa - miR - 186在结肠癌组织及其癌旁组织以及5种结肠癌细胞中的表达。通过PCR从基因组DNA中扩增miR - 186基因的前体序列，并克隆到标记有绿色荧光蛋白的慢病毒载体PLVTHM中。用PLVTHM - miR186载体转染结直肠癌细胞系SW620，并用流式细胞术分选慢病毒感染的细胞。使用细胞计数试剂盒 - 8（CCK - 8）检测细胞增殖情况。采用Transwell实验和划痕实验研究SW620细胞的迁移和侵袭能力。用蛋白质免疫印迹法检测SW620细胞系中YY1蛋白的表达。
miR - 186在癌组织中的相对表达量为0.0024±0.0027，显著低于癌旁组织（0.066±0.068，P = 0.008）；具有高转移潜能的SW620和LoVo细胞中hsa - miR - 186的相对表达水平分别为0.118±0.138和0.157±0.001，显著低于具有低转移潜能的HT - 29细胞（1.000±0.00，P < 0.05）。经酶切、测序和qPCR验证的重组慢病毒载体PLVTHM - miR186可显著抑制SW620细胞的增殖、迁移和侵袭，并抑制YY1蛋白的表达。
作为一种肿瘤抑制基因，Hsa - miR - 186在结肠癌组织以及高转移的SW620和LoVo细胞中表达下调。Has - miR - 186可能通过抑制YY1表达来体外抑制结肠癌细胞的增殖、迁移和侵袭。
