AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colo...AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum- free medium. A miRNA microarray was used to detect differential expression profiles of rniRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction （PCR） was performed to verify the dif- ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic- tion tools, miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription （RT）-PCR and western blotting.RESULTS： Compared with expression in SW1116 cells, 35 miRNAs （including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa- miR-32） were upregulated more than 1.5-fold, and 11 miRNAs （including hsa-miR-93, hsa-miR-1231, hsa- miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561） were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKNIA, HDACS, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHCl. Overexpressed miR-93 sig- nificantly inhibited cell proliferation and colony forma- tion by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION： Some miRNAs were differentially ex- pressed during differentiation of SW1116csc into SW1116 cells, miR-93 may inhibit SW1116csc proliferation and colony formation.展开更多
AIM： To identify cancer stern cells （CSCs） in human gallbladder carcinomas （GBCs）. METHODS： Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of th...AIM： To identify cancer stern cells （CSCs） in human gallbladder carcinomas （GBCs）. METHODS： Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133＋ cells were assayed.floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin （P 〈 0.05）. The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells （P 〈 0.05）. Spheroid ceils were more resistant to chemotherapeutic reagents than the congenetic adherent cells （P 〈 0.05）. Flow cytometry showed enriched CD133＋ population in sphereforming cells （P 〈 0.05）. CD133＋ cells possessed high colony-formation ability than the CD133 population （P 〈 0.01）. CD133＋ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts （P 〈 0.05）. CONCLUSION： CD133 may be a cell surface marker for CSCs in GBC.展开更多
AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medi...AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.展开更多
In vitro, mouse embryonic stem （ES） cells can differentiate into many somatic cell types, including neurons and glial cells. When cultured in serum-free medium, ES cells convert spontaneously and efficiently to a ne...In vitro, mouse embryonic stem （ES） cells can differentiate into many somatic cell types, including neurons and glial cells. When cultured in serum-free medium, ES cells convert spontaneously and efficiently to a neural fate. Previous studies have shown that the neural conversion of mouse ES cells includes both the participation of neural-specific transcription factors and the regulation of epigenetic modifications. However, the intracellular mechanism underlying this intrinsic transition still re- mains to be further elucidated. Herein, we describe a long intergenic non-coding RNA, LincRNA1230, which participates in the regulation of the neural lineage specification of mouse ES cells. The ectopic forced expression of LincRNAI230 dramatically inhibited mouse ES cells from adopting a neural cell fate, while LincRNA1230 knockdown promoted the conversion of mouse ES cells towards neural progenitors. Mechanistic studies have shown that LincRNA1230 inhibits the activation of early neural genes, such as Pax6 and Soxl, through the modulation of bivalent modifications （tri-methylation of histone3 lysine4 and his- tone3 lysine27） at the promoters of these genes. The interaction of LincRNA1230 with Wdr5 blocked the localization of Wdr5 at the promoters of early neural genes, thereby inhibiting the enrichment of H3K4me3 modifications at these loci. Collectively, these findings revealed a crucial role for LincRNA1230 in the regulation of the neural differentiation of mouse ES cells.展开更多
基金Supported by Medical guidance projects of Shanghai Science Committee,No.10411961800Youth Science Foundation of Fudan University,No.08FQ49
文摘AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum- free medium. A miRNA microarray was used to detect differential expression profiles of rniRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction （PCR） was performed to verify the dif- ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic- tion tools, miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription （RT）-PCR and western blotting.RESULTS： Compared with expression in SW1116 cells, 35 miRNAs （including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa- miR-32） were upregulated more than 1.5-fold, and 11 miRNAs （including hsa-miR-93, hsa-miR-1231, hsa- miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561） were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKNIA, HDACS, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHCl. Overexpressed miR-93 sig- nificantly inhibited cell proliferation and colony forma- tion by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION： Some miRNAs were differentially ex- pressed during differentiation of SW1116csc into SW1116 cells, miR-93 may inhibit SW1116csc proliferation and colony formation.
文摘AIM： To identify cancer stern cells （CSCs） in human gallbladder carcinomas （GBCs）. METHODS： Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133＋ cells were assayed.floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin （P 〈 0.05）. The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells （P 〈 0.05）. Spheroid ceils were more resistant to chemotherapeutic reagents than the congenetic adherent cells （P 〈 0.05）. Flow cytometry showed enriched CD133＋ population in sphereforming cells （P 〈 0.05）. CD133＋ cells possessed high colony-formation ability than the CD133 population （P 〈 0.01）. CD133＋ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts （P 〈 0.05）. CONCLUSION： CD133 may be a cell surface marker for CSCs in GBC.
基金Supported by Medical Guidance Project of Shanghai Science Committee (No. 10411961800)Youth Science Fund of Fudan University (No. 08FQ49)
文摘AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.
基金supported by National Natural Science Foundation of China (81530042, 31571529, 31210103905, 31571519, 31571390, 31371510, 31301208, 31471250, 31401257)the Ministry of Science and Technology (2012CB966603, 2013CB967600, 2013CB967401)+2 种基金Science and Technology Commission of Shanghai Municipality (15JC1403200, 15JC1403201)Shanghai Rising-Star Program (14QA1403900)the Fundamental Research Funds for the Central Universities (2000219099)
文摘In vitro, mouse embryonic stem （ES） cells can differentiate into many somatic cell types, including neurons and glial cells. When cultured in serum-free medium, ES cells convert spontaneously and efficiently to a neural fate. Previous studies have shown that the neural conversion of mouse ES cells includes both the participation of neural-specific transcription factors and the regulation of epigenetic modifications. However, the intracellular mechanism underlying this intrinsic transition still re- mains to be further elucidated. Herein, we describe a long intergenic non-coding RNA, LincRNA1230, which participates in the regulation of the neural lineage specification of mouse ES cells. The ectopic forced expression of LincRNAI230 dramatically inhibited mouse ES cells from adopting a neural cell fate, while LincRNA1230 knockdown promoted the conversion of mouse ES cells towards neural progenitors. Mechanistic studies have shown that LincRNA1230 inhibits the activation of early neural genes, such as Pax6 and Soxl, through the modulation of bivalent modifications （tri-methylation of histone3 lysine4 and his- tone3 lysine27） at the promoters of these genes. The interaction of LincRNA1230 with Wdr5 blocked the localization of Wdr5 at the promoters of early neural genes, thereby inhibiting the enrichment of H3K4me3 modifications at these loci. Collectively, these findings revealed a crucial role for LincRNA1230 in the regulation of the neural differentiation of mouse ES cells.
