TIPE3干扰质粒对SW480结肠癌细胞生长的作用及相关机制探讨

发布时间：2018-10-12 10:25

【摘要】：目的:通过TIPE3干扰质粒转染SW480结肠癌细胞,验证干扰TIPE3表达对SW480结肠癌细胞生长的影响并探讨相关机制。方法:构建TIPE3-shRNA-p SIREN-Retro Q干扰质粒,通过脂质体转染法成功将干扰质粒导入SW480细胞,通过RT-PCR、Western blot检测重组质粒的干扰效率。应用CCK-8方法检测SW480细胞生存率。Annexin V-FITC/PI流式细胞法检测细胞凋亡。使用Western blot检测细胞增殖、凋亡相关分子的表达情况。结果:成功设计、构建和筛选具有生物活性且干扰效率最佳的TIPE3-shRNA-p SIREN-Retro Q干扰质粒。CCK-8检测证实干扰SW480结肠癌细胞TIPE3表达可以抑制细胞生长。流式结果显示,TIPE3-shRNA3干扰组的凋亡率为(27.99±1.087)%,显著高于正常细胞组(12.10±2.213)%及转染空载质粒组(11.44±0.277 0)%。证实了降低TIPE3表达可以增加SW480对a DR5Sc Fv所诱导的细胞凋亡敏感性。Western blot结果显示干扰TIPE3表达可以活化caspase3蛋白,降低p-AKT、p-PDK1、PCNA等分子的表达。结论:干扰TIPE3的表达对SW480结肠癌细胞具有促进凋亡,抑制增殖的作用。
[Abstract]:Aim: to investigate the effect of interfering TIPE3 expression on the growth of SW480 colon cancer cells by transfection of SW480 colon cancer cells with TIPE3 interference plasmid. Methods: the TIPE3-shRNA-p SIREN-Retro Q interference plasmid was constructed, and the interference plasmid was successfully transfected into SW480 cells by liposome transfection. The interference efficiency of the recombinant plasmid was detected by RT-PCR,Western blot. The survival rate of SW480 cells was detected by CCK-8 and apoptosis was detected by Annexin V-FITC/PI flow cytometry. Western blot was used to detect the expression of cell proliferation and apoptosis-related molecules. Results: TIPE3-shRNA-p SIREN-Retro Q interference plasmids with biological activity and best interference efficiency were successfully designed, constructed and screened. CCK-8 analysis showed that interfering TIPE3 expression in SW480 colon cancer cells could inhibit cell growth. The apoptotic rate in TIPE3-shRNA3 interference group was (27.99 卤1.087)%, which was significantly higher than that in normal cell group (12.10 卤2.213)% and no-load plasmid transfection group (11.44 卤0.277)%. It was confirmed that decreasing the expression of TIPE3 could increase the sensitivity of SW480 to apoptosis induced by a DR5Sc Fv. The results showed that interfering with TIPE3 expression could activate caspase3 protein and decrease the expression of p-AKTU p-PDK1 protein. Conclusion: interfering with the expression of TIPE3 can promote apoptosis and inhibit proliferation of SW480 colon cancer cells.
【作者单位】：福建医科大学基础医学院;同济大学第十人民医院普通外科;厦门大学抗癌研究中心器官移植研究所;厦门大学附属中山医院病理科;
【基金】：国家自然科学基金(81272720) 福建省属公益类科研院所基本科研专项项目(2016R1102)资助
【分类号】：R735.35

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