MiR-106a作用于TIMP2诱导人胃癌BGC-823细胞的腹腔种植转移
MiR-106a induces peritoneal metastasis through acting on TIMP2 in human gastric cancer cell BGC-823

摘要：目的 探讨微小RNA(microRNA-106a, miR-106a)通过调节基质金属蛋白酶抑制物2(tissue inhibitor of metalloproteinases 2, TIMP2)的表达而诱导人胃癌BGC-823细胞的腹腔种植转移。方法 人胃癌细胞株BGC-823传代培养至对数生长期。细胞分3组：胃癌细胞BGC-823组、BGC-823/anti-miR-106a(拮抗剂)组和BGC-823/negative control组。Real-time PCR鉴定拮抗效果。Transwell法检测细胞体外迁移侵袭能力。以小切口注射方式制备裸鼠腹腔内移植瘤模型，动物分2组：miR-antagomir组和miR-NC组。大体观察裸鼠胃癌移植瘤生长情况。免疫组化法和Western印迹法联合检测TIMP2在腹腔内各器官上的表达。结果 BGC-823/anti-miR-106a组miR-106a表达下调，降低倍数2-△△Ct=0.05±0.01，与NC组相比具有统计学差异(t=－18.001，P<0.001)。细胞水平外源性沉默miR-106a基因，BGC-823/anti-miR-106a组迁移、侵袭的细胞数量均低于BGC-823和BGC-823/negative control组，差异均有统计学意义(P<0.001)。移植瘤体内实验显示miR-106a表达下调削弱BGC-823细胞在裸鼠腹腔内的种植转移能力，表现为种植瘤结节数量和体积的减少；当miR-106a表达受抑时，免疫组化法和印迹法均显示miR-antagomir组TIMP2蛋白表达量高于miR-NC组，差异有统计学意义(P<0.05)。结论 BGC-823细胞株具备裸鼠成瘤能力，沉默miR-106a抑制胃癌细胞的侵袭转移提示其具有癌基因样作用；miR-106a可能通过作用于TIMP2诱导BGC-823细胞腹腔种植转移能力增强。
ABSTRACT: Objective To investigate the induction of microRNA (microRNA-106a, miR-106a) on the peritoneal metastasis of human gastric cancer cell BGC-823 by regulating matrix metalloproteinase inhibitor 2 (tissue inhibitor of metalloproteinases 2, TIMP2). Methods Human gastric cancer cell line BGC-823 was cultured to the logarithmic growth phase. The cells were divided into three groups: BGC-823, BGC-823/anti-miR-106a (antagomir) and BGC-823/negative control. Real-time PCR was used to identify the effect of antagomir. Transwell assay was used to detect the cell migratory and invasive abilities of these three groups in vitro. With small incision, the cells were injected into the abdominal cavity of nude mice to prepare a xenograft model. The animals were divided into two groups: miR-antagomir and miR-NC. The tumor growth in the nude mice was generally observed and estimated. Immunohistochemistry and Western blot methods were used to detect the expression of metastasis-associated protein TIMP2 on the several abdominal organs. Results The expression level of miR-106a was down-regulated in BGC-823/anti-miR-106a group, with the fold change of 0.05±0.01, which was significantly different from that in NC group (t=－18.001，P<0.001). In vitro exogenously silencing of miR-106a gene, the numbers of invasive and migratory cells in BGC-823/anti-miR-106a group were both significantly lower than those in BGC-823 and BGC-823/negative control groups (P<0.001). In vivo xenograft model showed that the down-regulation of miR-106a weakened the peritoneal metastasis ability of BGC-823 cells in nude mice abdominal cavity, which was reflected by the decrease of tumor number and tumor size. With the inhibition of miR-106a, the expression of TIMP2 in