微RNA-21表达下调可抑制人结肠癌SW620细胞在裸鼠体内的生长

Down-regulation of microRNA-21 expression inhibits the growth of human colon carcinoma SW620 cells in nude mice

目的 :探讨反义寡核苷酸(antisense oligonucleotides,ASOs)下调微RNA-21(micro RNA-21,mi R-21)表达对高侵袭性人结肠癌SW620细胞在裸鼠体内生长的影响。方法 :首先建立人结肠癌SW620细胞的裸鼠荷瘤模型,并于肿瘤局部分别注射p-mi R-21-ASOs重组质粒或p-Cont对照质粒,然后观察裸鼠体内肿瘤的生长变化。末次注射5 d后,处死裸鼠,剥取肿瘤组织,测量各组肿瘤组织的大小和质量。采用HE染色法观察肿瘤组织形态学变化;TUNEL法检测肿瘤局部细胞凋亡情况;免疫组织荧光法检测肿瘤组织中增殖核抗原Ki-67蛋白的表达变化;实时荧光定量PCR法检测肿瘤组织中mi R-21成熟体的表达,以及细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK)2、CDK3、CDK4和CDK6的表达变化;蛋白质印迹法检测磷酸化蛋白激酶B(phosphorylated protein kinase B,p-PKB,又称为p-Akt)和磷酸化细胞外信号调节激酶(phosphorylated extracellular regulated protein kinase,p-ERK)的表达水平。结果 :与p-Cont对照组相比,p-mi R-21-ASOs质粒转染组的肿瘤生长缓慢(P<0.05),剥离肿瘤的质量明显减少(P<0.01),肿瘤组织中可见大片坏死区域,而且肿瘤细胞中mi R-21成熟体的表达水平明显下调(P<0.01)。mi R-21表达下调后,肿瘤组织中Ki-67的表达量明显减少(P<0.01),而相对凋亡细胞数明显增多(P<0.05);并且,细胞周期相关分子CDK2、CDK3、CDK4和CDK6的表达均明显下调(P值均<0.001),Akt和ERK蛋白的磷酸化水平也均明显下调(P值均<0.05)。结论 :ASOs下调mi R-21表达可显著抑制人高侵袭性结肠癌细胞在裸鼠体内的生长,提示mi R-21可作为结肠癌基因治疗的一个潜在新靶标。

Objective： To investigate the effect of microRNA-21 （miR-21） expression down-regulated by antisense oligonucleotides （ASOs） on the growth of human colon carcinoma SW620 cells in nude mice. Methods： Firstly, the nude mouse model of human colon carcinoma cell line SW620 was established. Then the recombinant plasmid p-miR-21-ASOs or the control plasmid p-Cont was locally injected into tumor tissues （once every 4 days）, and the tumor growth was observed. On the 5th day after the last injection, all of mice were sacrificed, and the tumor tissues were removed and measured. Then the histomorphology of tumor tissues was observed by HE staining assay. The apoptosis of tumor cells was analyzed by TUNEL assay. The expression of Ki-67 proliferation nuclear antigen was measured by immunofluorescence assay. Furthermore, the expression levels of mature miR-21, as well as cyclin-dependent kinase （CDK） 2, CDK3, CDK4 and CDK6 were detected by real-time fluorescent quantitative PCR. And the expressions of phosphorylated protein kinase B （p-Akt） and phosphorylated extracellular regulated protein kinase （p-ERK） were analyzed by Western blotting. Results： Compared with p-Cont group, the xenograft tumors in p-miR-21-ASOs group grew slowly （P〈0.05）, and the weight of tumor tissues was significantly reduced （P〈0.01）. Meanwhile, there were large areas of necrosis in tumor tissues in p-miR-21-ASOs group. The expression level of miR-21 in tumor tissues was significantly decreased after p-miR-21-ASOs injection （P〈0.01）. Moreover, the expression of Ki-67 in tumor tissues was significantly down-regulated （P〈0.01）, and the relative number of apoptosis cells increased obviously （P〈0.05） in p-miR-21-ASOs group. Furthermore, the relative expression levels of CDK2, CDK3, CDK4, CDK6, p-Akt and p-ERK were significantly down-regulated in p-miR-21-ASOs group as compared with p-Cont group （all P〈0.05）. Conclusion： Down-regulation of miR-21 expression by ASOs can significantly inhibit the growth of human colon carcinoma SW620 cells in nude mice, indicating that miR-21 may be a potential target for gene therapy of colon cancer.