目的人分化抑制因子3(inhibitor of differentination 3,Id3)基因在A549细胞中的外源性表达能抑制细胞生长并诱导细胞凋亡。文中旨在构建靶向人Id3基因的微小分子RNA(miRNA)表达载体,观察miRNA对A549细胞中Id3表达的下调作用,为进一步探...目的人分化抑制因子3(inhibitor of differentination 3,Id3)基因在A549细胞中的外源性表达能抑制细胞生长并诱导细胞凋亡。文中旨在构建靶向人Id3基因的微小分子RNA(miRNA)表达载体,观察miRNA对A549细胞中Id3表达的下调作用,为进一步探讨Id3基因在A549细胞生长调控中的作用机制提供一定实验依据。方法依据miRNA设计原则,针对人Id3基因的mRNA序列,设计并合成编码miRNA的2条寡核苷酸序列,经退火成互补双链,再克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体中,构建含靶向人Id3基因的miRNA表达质粒(pcDNA6.2-GW/EmGFP-miR-Id3),DNA测序验证后,采用Lipofectamine2000TM脂质体转染技术将该质粒导入A549细胞。荧光倒置显微镜下观察miRNA干扰质粒转染A549细胞后EGFP的表达情况,应用RT-PCR和Western blot方法分别检测miRNA干扰后Id3mRNA及蛋白水平的表达。结果DNA测序表明重组质粒pcDNA6.2-GW/EmGFP-miR-Id3构建正确,将其成功导入A549细胞后,在Id3mRNA水平和蛋白表达水平均有不同程度的抑制作用。结论成功构建了针对人Id3基因的4组pcDNA6.2-GW/EmGFP-miR-Id3miRNA表达载体,其中,SRId3-1干扰组在mRNA水平和蛋白表达水平均能有效抑制A549细胞Id3的表达。展开更多
Colorectal cancer(CRC) is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which malignant cells dissemin...Colorectal cancer(CRC) is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which malignant cells disseminate from the primary tumor to seeding other organs, a multistep process is involved. Cancer cells proliferate, invade microenvironment, enter into the blood circulation, then survive and colonize into distant organs. Micro RNAs(mi RNAs) and epithelial-mesenchymal transition(EMT) are key regulators and mechanism in tumorigenesis and cancer metastasis. We review the roles of EMT and micro RNAs, especially EMT related micro RNAs in the metastatic pathway of CRC. Micro RNAs provide us a set of potential therapeutic applications and molecular target for CRC.展开更多
MicroRNAs, a class of small noncoding RNAs, play key roles in diverse biological and pathological processes. ER stress, resulting from the accumulation of unfolded or misfolded proteins in the ER lumen, is triggered b...MicroRNAs, a class of small noncoding RNAs, play key roles in diverse biological and pathological processes. ER stress, resulting from the accumulation of unfolded or misfolded proteins in the ER lumen, is triggered by various physiological events and pathological insults. Here, using RNA deep sequencing analysis, we found that the expression of some microRNAs was altered in HeLa and HEK293 cells under ER stress. Protein and RNA levels of DGCR8, Drosha, Exportin-5, Dicer, and Ago2 showed no significant alteration in ER-stressed cells, which suggested that the change in microRNA expression might not be caused by the microRNA biogenesis pathway but by other, unknown factors. Real-time PCR assays confirmed that hsa-miR-423-5p was up-regulated, whereas hsa-miR-221-3p and hsa-miR-452-5p were down-regulated, in both HeLa and HEK293 cells under ER stress. Luciferase activity and Western blot assays verified that CDKN1A was a direct target of hsa-miR-423-5p and that CDKN1B was a direct target of hsa-miR-221-3p and hsamiR-452-5p. We speculated that by regulating their targets, microRNAs might function cooperatively as regulators in the adaptive response to ER stress.展开更多
文摘目的人分化抑制因子3(inhibitor of differentination 3,Id3)基因在A549细胞中的外源性表达能抑制细胞生长并诱导细胞凋亡。文中旨在构建靶向人Id3基因的微小分子RNA(miRNA)表达载体,观察miRNA对A549细胞中Id3表达的下调作用,为进一步探讨Id3基因在A549细胞生长调控中的作用机制提供一定实验依据。方法依据miRNA设计原则,针对人Id3基因的mRNA序列,设计并合成编码miRNA的2条寡核苷酸序列,经退火成互补双链,再克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体中,构建含靶向人Id3基因的miRNA表达质粒(pcDNA6.2-GW/EmGFP-miR-Id3),DNA测序验证后,采用Lipofectamine2000TM脂质体转染技术将该质粒导入A549细胞。荧光倒置显微镜下观察miRNA干扰质粒转染A549细胞后EGFP的表达情况,应用RT-PCR和Western blot方法分别检测miRNA干扰后Id3mRNA及蛋白水平的表达。结果DNA测序表明重组质粒pcDNA6.2-GW/EmGFP-miR-Id3构建正确,将其成功导入A549细胞后,在Id3mRNA水平和蛋白表达水平均有不同程度的抑制作用。结论成功构建了针对人Id3基因的4组pcDNA6.2-GW/EmGFP-miR-Id3miRNA表达载体,其中,SRId3-1干扰组在mRNA水平和蛋白表达水平均能有效抑制A549细胞Id3的表达。
基金Supported by a grant from the National Natural Sciences Foundation of China(No.81302131)Natural Science Foundation of Hubei Province,China(No.2012FKB04432)
文摘Colorectal cancer(CRC) is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which malignant cells disseminate from the primary tumor to seeding other organs, a multistep process is involved. Cancer cells proliferate, invade microenvironment, enter into the blood circulation, then survive and colonize into distant organs. Micro RNAs(mi RNAs) and epithelial-mesenchymal transition(EMT) are key regulators and mechanism in tumorigenesis and cancer metastasis. We review the roles of EMT and micro RNAs, especially EMT related micro RNAs in the metastatic pathway of CRC. Micro RNAs provide us a set of potential therapeutic applications and molecular target for CRC.
基金supported by the National Basic Research Program of China (2011CBA01103)the National Natural Science Foundation of China (81372215, 31301069, 81171074, and 91232702)+2 种基金the Science Foundation of the Chinese Academy of Sciences (KJZD-EW-L01-2)Anhui Provincial Natural Science Foundation (1408085MC42)the Fundamental Research Funds for the Central Universities (WK2070000034)
文摘MicroRNAs, a class of small noncoding RNAs, play key roles in diverse biological and pathological processes. ER stress, resulting from the accumulation of unfolded or misfolded proteins in the ER lumen, is triggered by various physiological events and pathological insults. Here, using RNA deep sequencing analysis, we found that the expression of some microRNAs was altered in HeLa and HEK293 cells under ER stress. Protein and RNA levels of DGCR8, Drosha, Exportin-5, Dicer, and Ago2 showed no significant alteration in ER-stressed cells, which suggested that the change in microRNA expression might not be caused by the microRNA biogenesis pathway but by other, unknown factors. Real-time PCR assays confirmed that hsa-miR-423-5p was up-regulated, whereas hsa-miR-221-3p and hsa-miR-452-5p were down-regulated, in both HeLa and HEK293 cells under ER stress. Luciferase activity and Western blot assays verified that CDKN1A was a direct target of hsa-miR-423-5p and that CDKN1B was a direct target of hsa-miR-221-3p and hsamiR-452-5p. We speculated that by regulating their targets, microRNAs might function cooperatively as regulators in the adaptive response to ER stress.
