【摘要】 背景与目的:mi R-21可能通过抑制PDCD4表达调控结肠癌浸润及转移等恶性行为。本研究通过下调mi R-21表达后,检测结肠癌HT-29细胞功能的变化,并观察PDCD4在蛋白及m RNA表达水平的改变,探讨mi R-21及PDCD4的表达在结肠癌恶性行为中的关系及机制。方法:构建靶向mi R-21的干扰质粒simi R-21,转染HT-29细胞后,以实时定量PCR(q RT-PCR)法检测转染效率,MTT法检测转染后细胞增殖变化,流式细胞术检测转染后细胞调亡变化,Transwell检测迁移及浸润能力改变,蛋白质印迹法(Western blot)及q RT-PCR法检测干扰后PDCD4表达水平变化。结果:经q RT-PCR检测simi R-21在HT-29细胞转染效率为60%~65%,转染效率佳;MTT显示转染后72、96和120 h,HT-29增殖能力减弱(t=1.276,P<0.05;t=3.276,P<0.01;t=4.523,P<0.01);流式细胞术结果显示,与si-negative control及mi R-21组对比转染后HT-29调亡率明显增加(t=2.132,P<0.05;t=3.524,P<0.05);Transwell结果显示,simi R-21转染细胞迁移能力降低(t=2.423,P<0.05;t=3.153,P<0.05),侵袭能力降低(t=3.245,P<0.05;t=5.236,P<0.05);Western blot检测结果显示,PDCD4蛋白在simi R-21细胞的表达水平明显上调(t=2.342,P<0.05;t=4.215,P<0.05);q RT-PCR检测结果显示,PDCD4 m RNA在simi R-21细胞的表达水平明显上调(t=2.261,P<0.05;t=3.492,P<0.05)。结论:simi R-21下调mi R-21表达后,结肠癌HT-29细胞增殖能力受抑制,并促进其调亡,抑制迁移及浸润能力,PDCD4上调。mi R-21可能通过下调PDCD4表达促进肿瘤细胞恶性行为,可作为结肠癌治疗新的靶向候选基因。更多还原

【Abstract】 Background and purpose: PDCD4 may be inhibited by mi R-21 to regulate the malignant behaviors of colon cancer such as invasion and migration. This study aimed to explore the function of colon cancer HT-29 cell lines by downregulating mi R-21 expression and discuss the mechanisms and relationship between mi R-21 and PDCD4 in colon cancer malignant behaviors. Methods: simi R-21 was transfected into colon cancer cell line HT-29 to downregulate the expression of mi R-21. Proliferation, apoptosis, migration and invasion were detected by MTT, flow cytometry and Transwell assay after transfection. PDCD4 expression was detected by Western blot and q RTPCR. Results: The q RT-PCR analysis result proved that the transfection efficiency was 60%-65%. MTT analysis result showed that the proliferations of HT-29 cells were inhibited after the transfection of mi R-21 for 72, 96, 120 h(t=1.276, P<0.05; t=3.276, P<0.01; t=4.523, P<0.01). Comparing with si-negative control and mi R-21 groups, flow cytometry result showed that the apoptosis rate was increased after mi R-21 expression downregulated(t=2.132, P<0.05; t=3.524, P<0.05). Transwell assay result showed that migration(t=2.423, P<0.05; t=3.153, P<0.05) and invasion(t=3.245, P<0.05; t=5.236, P<0.05) were inhibited; Western blot result showed that PDCD4 expression was up-regulated at protein level(t=2.342, P<0.05; t=4.215, P<0.05); q RT-PCR result showed that PDCD4 expression was up-regulated at m RNA level(t=2.261, P<0.05; t=3.492, P<0.05). Conclusion: The proliferation, migration and invasion are the inhibited, and apoptosis is attenuated after mi R-21 downregulated by simi R-21 transfection, PDCD4 expression is upregulated. mi R-21 may enhance the malignant behavior of cancer cells by downregulating the PDCD4 expression, mi R-21 might be a target gene for colon cancer therapy.更多还原