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目的构建稳定低表达Runt相关转录因子2(Runx2)的HCT-8结肠癌细胞,检测对结肠癌细胞生长和迁移等生物学行为的影响。方法首先采用Western blot法检测Runx2在不同结肠癌细胞系中表达,确定HCT-8结肠癌细胞高表达Runx2;将含有Runx2的慢病毒短发卡RNA(shRNA)的干扰载体与慢病毒包装辅助质粒共转染人胚肾HEK293FT细胞后,收集病毒上清,感染HCT-8结肠癌细胞。经嘌呤霉素筛选后,获得慢病毒介导的Runx2 shRNA的稳定表达细胞,利用实时定量PCR和Western blot法检测Runx2的shRNA的干涉效果;用CCK-8法检测细胞增殖活性、平板克隆形成实验检测癌细胞生长、Transwell~(TM)侵袭实验和划痕愈合实验检测Runx2 shRNA转染对癌细胞侵袭和迁移的影响。结果建立了稳定敲低Runx2水平的HCT-8结肠癌细胞;敲低HCT-8细胞Runx2表达水平后,癌细胞的生长、侵袭和迁移受到明显抑制。结论敲低Runx2抑制结肠癌细胞的生长、侵袭和迁移。
Objective To construct HCT-8 colon cancer cells stably expressing low Runt-associated transcription factor 2 (Runx2) and investigate the effects on the biological behavior of colon cancer cells such as growth and migration. Methods The expression of Runx2 in different colon cancer cell lines was determined by Western blot. Runx2 was identified in colon cancer cells of HCT-8 cells. The interference vectors of Runx2-containing short hairpin RNA (shRNA) and lentivirus packaging helper plasmid After transfecting human embryonic kidney HEK293FT cells, the virus supernatant was collected and infected with HCT-8 colon cancer cells. The lentivirus-mediated expression of Runx2 shRNA was confirmed by puromycin screening. The interference effect of Runx2 shRNA was detected by real-time PCR and Western blot. The cell proliferation activity and plate clone formation assay Detection of cancer cell growth, Transwell ~ (TM) invasion assay and scratch healing assay Runx2 shRNA transfection on cancer cell invasion and migration. Results Runx2 knockdown of HCT-8 colon cancer cells was established. After knockdown of Runx2 expression in HCT-8 cells, the growth, invasion and migration of cancer cells were significantly inhibited. Conclusion Knockdown of Runx2 inhibits the growth, invasion and migration of colon cancer cells.