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OBJECTIVE@#To investigate the regulatory role of Musashi-1 (MSI1) in the proliferation and growth of hepatocellular carcinoma (HCC) cells.@*METHODS@#We examined the expression of MSI1 in HCC and paired adjacent tissues from 24 patients using immunohistochemistry and Western blotting. A MSI1-expressing vector was constructed and stably transfected into HepG2 cells, and short hairpin RNAs (shRNAs) that targeted MSI1 mRNA were ligated into the vector and stably transfected in Huh7 cells. The effects of MSI1 overexpression and silencing on the proliferation, viability and cell cycle of HepG2 cells were investigated using flow cytometry or MTT assay. The expressions of PCNA, cyclin D1, APC and β-catenin in the HCC cells were detected with Western blotting.@*RESULTS@#MSI1 expression was significantly up-regulated in HCC tissues as compared with that in the adjacent tissues. Overexpression of MSI1 in HepG2 cells resulted in significantly enhanced cell growth ( < 0.01) and significantly reduced G0/G1 phase cells from (58.42±3.18)% to (40.67±1.22)% and increased S phase cells from (28.51± 1.93)% to (40.06±1.92)% ( < 0.01), causing also increases in the expressions of PCNA and Cyclin D1. Knockdown of MSI1 in Huh7 cells obviously inhibited the cell growth and caused cell cycle arrest at the G1/S phase ( < 0.01) with reduced protein expressions of PCNA and cyclin D1. Overexpression of MSI1 in HepG2 cells also down-regulated the expression of APC and up-regulated the expression of β-catenin protein, while MSI1 knockdown caused reverse changes in Huh7 cells.@*CONCLUSIONS@#MSI1 promotes the progression of HCC through positive modulation of cell growth and cell cycle the Wnt/β-catenin pathway.
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Humans , Carcinoma, Hepatocellular , Cell Cycle , Cell Proliferation , Hep G2 Cells , Liver Neoplasms , Nerve Tissue Proteins , Metabolism , RNA-Binding Proteins , MetabolismABSTRACT
RNA binding proteins (RBPs) play a key role in gene regulation and participate in life activities such as RNA synthesis, alternative splicing, modification, transport and translation. It is necessary to study the interaction between RNA and RBP in order to explore RNA functions. The expression changes of RBPs are related to a variety of diseases. Musashi (MSI) family is a class of evolutionarily conserved RBPs including MSI1 and MSI2, which play an important role in many key processes such as tumorigenesis, progression and drug resistance. They were found to be overexpressed in many tumors and associated with prognosis in the blood system, nervous system, digestive system, respiratory system, etc. MSI binds to mRNA to regulate translation and mRNA stability. MSI maintains the number of cancer stem cells and affects tumor proliferation, invasion, metastasis and drug resistance. The preliminary research of MSI gene as a target to guide tumor therapy has achieved some results. This article describes the physiological functions of MSI family and its roles in tumorigenesis and development, and provides an overview of the latest research progress of MSI family as a diagnostic marker or a therapeutic target.
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Objective To disclose whether the down-regulation of Musashi1 gene can sensitize human colon carcinoma cell line HCT116 to radiation. Methods Lentviral vectors were used to knockdown the expression of Musashi1 gene in HCT116 cell line ( HCT116-Musashi1 ) and its negative control ( NC) . Cell survival was measured by the colony formation assay, cell apoptosis and cell cycle distribution were measured by a flow cytometry. Results HCT116-Musashi1 silence and its negative control cells were established successfully. The result of cell survival assay showed that D0 , Dq , N, SF2 were 1.55, 0.88, 1.76 Gy and 0.43 for the Musashil silence group, 2.17, 1.51, 2.01 Gy and 0.64 for control cells, and 1.99, 1.45, 2.07 Gy and 0.62 for siRN NC, respectively. The radiosensitivity of Musashi1 silence group was significantly higher than that of control and siRNA NC, and SER was 1.40 and 1.28 respectively. After 8 Gy irradiation, the apoptosis rate of silence group was always higher than other two groups at 24, 48, and 72 h after irradiation(F =65.16, P <0.05), but there was no statistically significant difference between control and NC (P>0.05). After 12 Gy irradiation, the percentage of cells in G2/M phase decreased significantly in the silence group compared with control group and NC group( F=65. 398,P<0. 05). Conclusions Knockdown of Musashi1 in HCT116 cells increases the radiosensitivity through promoting cell apoptosis and reversing G2/M arrest, indicating that Musashi1 may be a new target of radiotherapy.
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Objective To study the expression of Musashi-1 in tumor tissues and adjacent non-tumor tissues of patients with hepatocellular carcinoma (HCC), and to explore the relationship between Musashi-1 and clinical prognosis and the effect of Musashi-1 on the metastasis and invasionof HCC cells. Methods We collected HCC tissues and adjacent non-tumor tissues from 138 patients undergoing radical resection of HCC in our hospital from June 2000 to June 2010. Theexpression of Musashi-1 in all tissues was detected by immune histochemical method. The relationship between Musashi-1 expression and clinical characteristics and prognosis was analyzed by Pearson X test and Fisher exact test The expression of CD133 in HCC tissues and adjacent non-tumor tissues was detected by qPCR and the correlation between Musashi-1 and CE133 in tumor tissues was analyzed. Finally, we constructed SMMC-7721 cells with stable over expression of Musashi-1,determined the effect of Musashi-1 on the sphere-forming potential of SMMC-7721 cells using Sphere formation assay, and detected the effect of Musashi-1 on the ability of cell invasion by tumor invasion experiment Results Musashi-1 was highly expressed in 105 (76. 1%) HCC tissuescompared with the adjacent non-tumor tissues (P0. 05). Spearman analysis indicated that HCC patients with higherMusashi-1 expression have significantly worse overall survival rate (P<0. 05) and disease-free survival (P<0. 05) compared with those with low Musashi-1 expression. The expression of CD133mRNA in HCC tissues was significantly higher than that in the adjacent non-tumor tissues (P〈0. 05),and CD133 expression was positively correlated with Musashi-1 expression (R2 =0. 78, P<0. 001). In vitro experiments confirmed that overexpression of Musashi-1 enhanced the sphere-forming potential and invasive ability of SMMC-7721 cells. Conclusion Musashi-1 has higher expression in HCC tissues and may promote HCC progression and metastasis. High level of Musashi-1 may indicate poor prognosis,providing a reference for prognosis judgment in HCC patients.
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Musashi1 (Msi1) is an evolutionary conservative RNA-binding protein (RBP),and it is a stem marker in a variety of organizations,including intestinal,neural system.Msi1 maintains the balance between self-renewal and differentiation.Recently,many researchers report that Msi1 is overexpressed in many types of tumors,especially in colorectal neoplasms,participating in the regulation of cell cycle,proliferation,apoptosis and so on.Msi1 becomes a key regulator of many cancers,which is expected to turn into a new target for cancer therapy.
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Objective:To investigate the Musashi 1 expression, and its correlation with clinical variables, such as clinical pathologic factors,angiogenesis and prognosis in colon cancer.Methods: Musashi 1 mRNA level was determined by quantitative real-time PCR( qRT-PCR) in 36 pairs of matched colon cancer tissues and adjacent non-tumorous tissues.The expression of Musashi 1 and mi-crovascular density ( MVD) were tested by immunohistochemical in 96 cases of colon cancer.Combined with postoperative follow-up survival situation, the correlation between the expression of Musashi 1, pathological parameters, prognosis and MVD were statistical analylised.Results:Musashi 1 mRNA level of colon cancer tissues determined by PCR was significantly higher than adjacent non-tumorous tissues( P<0.05).Positive Musashi 1 immunoreactivity was seen in 63.5%of colon cancer specimens,which was correlated with the depth of invasion,Duckes stage,lymph node metastasis,and TNM stage (P<0.05).Compared with Musashi 1 negative patients, Musashi 1 positive patients had significantly shorter survival time ( 23.0% vs 60.0%, P<0.01 ) .High intratumor blood microvessel density patients had significantly shorter survival times than low intratumor blood microvessel density patients(P<0.05).Intratumor blood microvessel density correlated with Musashi 1 expression (P<0.01).Conclusion:Musashi 1 mRNA level of colon cancer tissues was higher than adjacent non-tumorous tissues,the difference has statistically significant.We conclude that Musashi 1 expression correlates with the depth of invasion,Dukes stage,lymph node metastasis,and TNM stage and microvascular density ( MVD) in colon cancer.We propose that synthesized Musashi 1 increases intratumor angiogenesis,thus promotes tumor progression.Musashi 1 expression may be a good biomarker for poor prognosis in colon cancer.
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Musashi is a family of RNA binding proteins with a conservative evolution. This protein family is selectively ex-pressed in the nervous system and comprises two members, namely, Musashi-1 and Musashi-2. Musashi-1 and Musashi-2 are transla-tional suppressors of Numb mRNA and can synergistically regulate the Notch signaling pathway;as a result, an asymmetric division of stem cells occurs. Musashi-1 is the first member of the family and was originally isolated from Drosophila. As a candidate stem gene, Musashi-1 participates in disease progression in stem cells. Musashi-1 is also an important protein that maintains the functions of stem cells, participates in tumor-related signaling pathways, and participates in cell proliferation and apoptosis. Furthermore, Musashi-1 is overexpressed in many solid tumors, such as neuroglioma, esophagus, gastric, colorectal, and breast cancers. Studies on Musashi-1 can provide new insights into genetic diagnosis and cancer treatments. In this study, the structure and function of Musashi-1 and the re-search progress of tumor mechanisms were summarized and reviewed.
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CONTEXT: The stem cell model for cancer assumes that a key event in tumorigenesis is the deregulation of genes involved in the regulation of stem cell self-renewal. The Musashi family is an evolutionarily conserved group of neural RNA-binding proteins. In mammals, the family consists of two individual genes, Musashi 1 (MSI1) and MSI2, encoding the Musashi 1 and Musashi 2 proteins. Musashi 1 is involved in the regulation of self-renewal of stem cells. Recently, its over-expression has also been reported in a variety of human tumors. AIMS: To investigate a potential expression of the stem cell self-renewal gene, Musashi 1, in human bladder cancer, we examined its gene expression in a series of tumor and non-tumor tissue samples of bladder. MATERIALS AND METHODS: Relative expression of MSI1 was determined by the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 70 surgical samples of bladder. RESULTS: Using specific primers for MSI1 and TBP (as an internal control) for qRT-PCR technique, we found a relatively high expression level of MSI1 in all examined tumor and non-tumor bladder tissue specimens. However, our data did not show any correlation between the level of gene expression and tumor/non-tumor states of the samples (P>0.05). CONCLUSIONS: All together, our data demonstrated that Musashi 1 is highly and un-differentially expressed in both examined tumoral and apparently normal bladder tissues.
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Objective To discuss the technology of modest isolating and enriching the intestinal epithelial stem cells. Methods Mouse intestinal epithelial cells were stained by Rhodamine 123 (Rho), sorting the Rhodamine 123 low staining cell population ( Rholow ) and Rhodamine 123 strong straining cell population ( Rhobri ) by fluorescence activated cell sorting(FACS) in flow cytometer; Detecting the musaashi-1 and p-glycoprotein 1 (p-g-p1) mRNA expression of two groups by RT-PCR; Analyzing the cell cycle and the percentage of the musashi-1 positive cells by flow cytometry. Results The intestinal epithelial cells were divided into three groups, Rhodamine 123 low staining cells( 12. 34% of total cells), Rhodamine 123 middle staining cells (45.26% of total cells) and Rhodamine 123 strong staining cells ( 41. 40% of total cells). The Rholow cell fraction and Rhobri cell fraction were isolated successfully. Both of musashi-1 and p-g-p1 mRNA were strongly expressed in Rho1ow cell fraction, and Rhobri cell fraction little expressed p-g-p1 mRNA. Most of Rho1ow cells were in G0/G1 phase, and the musashi-1 positive cells were about 10.37% of total cells in this fraction. Conclusion The intestinal epithelial stem cells can be modestly isolated and enriched by Rhodamine 123 staining.
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@#ObjectiveTo characterize the neural progenitor cell in the human amnion mesenchyme and epithelial layer with specific mark proteins of neural stem cell.MethodsExpressions of specific mark proteins of neural stem cell including nestin, glial fibrillary acidic protein (GFAP), musashi-1, vimentin and PSA-NCAM in human amnion tissue and cultured amniotic cells were determined by immunohistochemistry and immunofluorescence staining.ResultsExpressions of pluripotent neural stem cell specific makers (nestin, musashi-1, vimentin and PSA-NCAM) were detected in the human amnion mesenchyme and epithelial layer. In addition, cultured amniotic cells were expressed several neural stem cell specific markers including nestin, GFAP and PSA-NCAM. Nestin+ and GFAP+ double positive cells were identified in the human amnion tissue and cultured amniotic cells by immunohistochemistry and immunofluorescence staining.ConclusionSpecific mark proteins of neural stem cell are expressed in human amnion tissue and cultured amniotic cells.
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Objective:To clone and functionally analyze the promoter region of an intestinal specific expressed gene,Musashi-1.Methods:The 5' flanking region of MSI-1 gene was cloned from C57BL/6J mouse genomic DNA using PCR-mediated recombination.Expression of MSI-1 mRNA was determined in Colon26 and B16 cell lines using Northern Blotting.Various 5′-deletion recombination plasmids were constructed and transfected transiently into the Colon26 cell line.Luciferase reporter assay was performed to determine the relative transcriptional activities of various 5′-deltion fragments.Results:MSI-1 mRNA was expressed in both Colon26 and B16 cell lines,but much higher in Colon26 cell line.The transcriptional activity of DNA fragment from 73bp downstream to 4 939bp upstream the(MSI-1) gene transcriptional start site was 29.9 fold of the pGL3-basic empty vector.Conclusion:(pSMI+73~)-4 939 has the transcriptional activity and can be regarded as the promoter of MSI-1 gene.A cis-acting element lies between 4 011bp to 4 939 bp upstream the transcriptional start site of(MSI-1) gene,which may be responsible for the tissue specific expression of MSI-1 gene.The cloning of MSI-1 gene promoter is a precondition for the isolation and purification of intestinal stem cells using this promoter.
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Objective To investigate the expression of musashi-1(msi-1)and its significances in small intestinal mucous severely damaged by high close 5-FU.Methods Total 40 adult C57BL/6J mice were divided into control group(n= 8,group D)and experimental group(n=32).Mice in control group received intraperitoneal injection of PBS as control,and mice in experimental group were intraperitoneally injected high dose 5-FU(150 mg per kg of body weigh for five days).After treatment 1 day(group A),3 days(group B)and 5 days( group C),the dying mice were killed,HE straining and immunohistochemical technique were carried out for detecting the expression of putative marker of intestinal epithelial stem cells——musashi-1(msi-1)in the samples of the meddle intestine,and the percentage of the msi-1 positive cells from the intestinal mucosal cells of the mice in group A was detected by flow cytometry.Results After the treatment of high dose 5-FU,the intestinal mucous was damaged severely;the number of msi-1 positive cells increased markedly;The intestinal mucosal cells can be divided into two groups by flow cytometry,and in the group in which the value of FSC was higher,the percentage of msi-1 positive cells increased to 67.75 %.Conclusions After the treatment of high dose of 5-FU,the percentage of intestinal stem cells increased significantly;this model may be useful for further isolation and enrichment of intestinal epithelial stem cells.
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Objective:To investigate the configuration changes of human cord blood monocytes (HCMNCs)under specific culture conditions and the expression of NSCs marker under induction.Methods:HCMNCs were prepared from normal placenta after full term normal delivery. Observe the shape by microscope and reverse transcriptase polymerase chain reaction (RT-PCR) for nestin and musashi-1 antigen were performed to confirm nestin and musashi-1 antigen expression in HCMNCs.Results:RT-PCR demonstrated small amount of nestin expressed in HCMNCs but no musashi-1 antigen before induction, and both showed the strongest expression in 48h,then faded away gradually. The uncultivated MNCs were small and round.After culturing,the cells became larger,some cells change their shape into rhombic.Conclusion:The newly HCMNCs isolated by centrifugation over Lymphoprep densitgradient expressed nestin mRNA in low level and no musashi-1 show up,after induction,the former and musashi-1 increased intermittently and then decreased with the time of culturing.