摘要
目的 构建γ干扰素诱导蛋白30(IFI30)表达抑制的U251细胞,探讨IFI30表达受到抑制后对细胞生物学功能的影响及机制。方法 在线设计敲低靶点并合成3条小干扰RNA(siRNA),转染后通过实时定量PCR技术检测抑制效率,选取抑制效率最高的siRNA序列构建短发夹RNA (shRNA)质粒。重组质粒与包装质粒共转染HEK293T细胞制备慢病毒。用慢病毒感染胶质瘤U251细胞,经嘌呤霉素筛选得到阳性细胞。采用CCK-8实验、5-乙炔基-2′-脱氧尿苷(EdU)实验和集落形成实验分析细胞增殖活性,流式细胞术分析细胞周期和细胞凋亡,Transwell^(TM)实验检测细胞侵袭、划痕实验检测细胞迁移。Western blot法检测细胞周期蛋白D1(cyclin D1)、B细胞淋巴瘤因子2(Bcl2)、上皮钙黏蛋白(E-cadherin)和神经钙黏蛋白(N-cadherin)、信号转导子与转录激活子1(STAT1)。结果 筛选到有效的靶点序列并成功建立IFI30表达抑制的U251细胞。敲低IFI30的表达抑制U251细胞增殖,G0/G1期细胞比例增加,S期减少,细胞凋亡明显增加,细胞的侵袭和迁移能力降低。同时,U251细胞cyclin D1、Bcl2和N-cadherin的表达降低,E-cadherin, STAT1磷酸化表达增加。结论 敲低IFI30通过促进STAT1活化抑制U251细胞增殖、侵袭和迁移并促进其凋亡。
Objective To establish U251 cells with inhibited expression of interferon-γinducible protein 30(IFI30),and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism.Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs(siRNA)were synthesized.After transfection,the inhibition efficiency was detected by real-time quantitative PCR.The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA(shRNA)plasmids.The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus.The glioma U251 cells were transfected with lentivirus,and the positive cells were screened by puromycin.CCK-8 assay,5-ethyl-2'-deoxyuridine(EdU)and colony formation assays were used to analyze cell proliferation;the flow cytometry was used to analyze cell cycle and apoptosis;the Transwell^(TM) assay was used to detect cell invasion;the wound-healing assay was employed to detect cell migration,and western blot analysis to detect the protein expresison of cyclin D1,B-cell lymphoma factor 2(Bcl2),epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),signal transducer and activator of transcription 1(STAT1).Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established.When IFI30 expression was knocked down,the proliferation of U251 cells was inhibited,along with increased ratio of cells in the phase G0/G1,the decreased phase S,the increased rate of cell apoptosis.The cell invasion and migration capabilities was also reduced.The decreased expression of cyclin D1,Bcl2 and N-cadherin were observed in U251 cells,and the expression of E-cadherin and the phosphorylation of STAT1 were found increased.Conclusion Knockdown of IFI30 inhibits the proliferation,invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
作者
叶静静
徐文琴
陈天兵
YE Jingjing;XU Wenqin;CHEN Tianbing(Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution,Yijishan Hospital,Wannan Medical College,Wuhu 241001,China;Central Laboratory,Yijishan Hospital,Wannan Medical College,Wuhu 241001,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2024年第1期33-42,共10页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81901519)
安徽省自然科学基金(1908085QH380)。
