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目的通过sh RNA干扰技术,探讨靶向沉默RNA解旋酶DDX46基因对食管鳞癌TE-1细胞增殖及凋亡的影响。方法以人永生化食管鳞状上皮细胞Het-1A为对照,实时定量聚合酶链反应(q RT-PCR)检测DDX46基因m RNA在TE-1细胞中的相对表达;DDX46基因RNAi靶点序列慢病毒感染的TE-1细胞为实验组,阴性对照序列慢病毒感染的TE-1细胞为对照组,进行细胞计数、克隆形成和细胞凋亡实验;Stress and Apoptosis Signaling Antibody Array Kit检测DDX46基因靶向沉默前后信号通路关键分子的变化。结果与对照组相比,靶向沉默DDX46基因后细胞计数显示TE-1细胞生长被显著抑制(P<0.01),克隆形成实验显示细胞克隆形成能力被显著抑制(P<0.001),Annexin V-APC单染法流式细胞仪凋亡检测显示细胞凋亡显著增加(P<0.001)。Path Scan?Antibody Array检测发现Akt(Ser473,phosphorylation)和IκBα(total,N/A)表达水平显著下降(P<0.01),Caspase-3(Asp175,cleaved)表达水平升高(P<0.05)。结论 DDX46基因在食管鳞癌TE-1细胞中高表达;RNAi靶向沉默DDX46基因可抑制TE-1细胞增殖、诱导细胞凋亡;DDX46基因沉默可能是通过下调Akt/NF-κB信号转导通路,进而发挥抑制肿瘤细胞增殖、诱导细胞凋亡的作用。
Objective To investigate the effect of target-silenced RNA helicase DDX46 gene on the proliferation and apoptosis of esophageal squamous cell carcinoma TE-1 cells by sh RNA interference technique. Methods Human immortalized esophageal squamous cell Het-1A was used as a control. The relative expression of DDX46 m RNA in TE-1 cells was detected by real-time quantitative polymerase chain reaction (q RT-PCR). The target sequence of DDX46 RNAi The virus infected TE-1 cells were in the experimental group, and the negative control sequence lentivirus-infected TE-1 cells in the control group were used for cell counting, colony formation and apoptosis assay. Changes of Key Molecules in Signal Pathway Before and After Silencing. Results Compared with the control group, the cell counts of target-silenced DDX46 gene showed that the growth of TE-1 cells was significantly inhibited (P <0.01). Clone formation assay showed that the ability of cloning ability was significantly inhibited (P <0.001) Apoptosis was significantly increased by APC single-stained flow cytometry (P <0.001). The results of Path Scan? Antibody Array showed that the expressions of Akt (Ser473, phosphorylation) and IκBα (total, N / A) were significantly decreased (P <0.01) and Caspase-3 (Asp175, cleaved) Conclusion DDX46 gene is overexpressed in esophageal squamous cell carcinoma TE-1 cells. The silencing of DDX46 gene by RNAi can inhibit the proliferation of TE-1 cells and induce apoptosis. DDX46 gene silencing may be through the down-regulation of Akt / NF-κB signal transduction pathway, And then play a role in inhibiting tumor cell proliferation, induce apoptosis.